InteractiveFly: GeneBrief

fat facets : Biological Overview | Regulation | Developmental Biology | Effects of Mutation | Evolutionary Homologs | References


Gene name - fat facets

Synonyms -

Cytological map position - 100E1--100E3

Function - ubiquitin-specific protease

Keywords - eye, oogenesis, protein degradation

Symbol - faf

FlyBase ID: FBgn0005632

Genetic map position - 3-[105]

Classification - conserved Cys and His domains ubiquitin-specific protease

Cellular location - cytoplasmic



NCBI link: Entrez Gene
faf orthologs: Biolitmine
Recent literature
Koch, M., Nicolas, M., Zschaetzsch, M., de Geest, N., Claeys, A., Yan, J., Morgan, M. J., Erfurth, M. L., Holt, M., Schmucker, D. and Hassan, B. A. (2017). A Fat-facets-Dscam1-JNK Pathway enhances axonal growth in development and after injury. Front Cell Neurosci 11: 416. PubMed ID: 29472843
Summary:
Injury to the adult central nervous systems (CNS) can result in severe long-term disability because damaged CNS connections fail to regenerate after trauma. Identification of regulators that enhance the intrinsic growth capacity of severed axons is a first step to restore function. A gain-of-function genetic screen was constructed in Drosophila to identify strong inducers of axonal growth after injury. Focus was placed on a novel axis the Down Syndrome Cell Adhesion Molecule (Dscam1), the de-ubiquitinating enzyme Fat Facets (Faf)/Usp9x and the Jun N-Terminal Kinase (JNK) pathway transcription factor Kayak (Kay)/Fos. Genetic and biochemical analyses link these genes in a common signaling pathway whereby Faf stabilizes Dscam1 protein levels, by acting on the 3'-UTR of its mRNA, and Dscam1 acts upstream of the growth-promoting JNK signal. The mammalian homolog of Faf, Usp9x/FAM, shares both the regenerative and Dscam1 stabilizing activities, suggesting a conserved mechanism.
BIOLOGICAL OVERVIEW

Ubiquitin is a 76 amino acid polypeptide, whose main function is to target proteins for degradation by a multi-subunit proteolytic complex called the proteasome. Ubiquitin can be covalently bound to an internal lysine of a target protein. This process is mediated by a complex and highly selective enzymatic machinery. Ubiquitin conjugates take the form of one or more multimeric chains. The Drosophila fat facets gene, encodes a deubiquitinating enzyme (Huang, 1995), one member of a family of proteins that cleave ubiquitin-protein bonds (Hochstrasser, 1995). Faf is required to regulate the number of photoreceptors during eye development. Mutants lacking zygotic faf function develop to adulthood, but have rough eyes caused by the presence of one to two ectopic outer photoreceptors per ommatidium. faf is also required during oogenesis perhaps playing a role in pole cell determination, development or function (Fischer-Vize, 1992).

The role of deubiquitination enzymes in the ubiquitin pathway has best been characterized in Saccharomyces cerevisiae where 15 genes have been identified. The deubiquitination enzymes can broadly be divided into two groups, one promoting and the other inhibiting ubiquitin-dependent proteolysis. Sequence alignments indicate that Faf may be functionally homologous to Doa4, which promotes efficient ubiquitin-dependent degradation. Doa4 is associated with the 26S proteasome and appears to be required for removing the ubiquitin tail from substrate proteins (Hochstrasser, 1995). Lack of Doa4 function generally impairs ubiquitin-dependent degradation, since both natural and artificial substrates are degraded less efficiently. The resulting accumulation of several endogenous proteins in Doa4 mutants is thought to be the cause of the pleotropic phenotype, characterized by slow growth, radiation sensitivity and defects in the initiation of DNA replication (Singer, 1996).

Gain-of-function alleles of sevenless, Ras1, D-raf and other Ras pathway components can cause the differentiation of supernumerary photoreceptors. A similar ectopic photoreceptor phenotype is observed in animals carrying mutations in the fat facets gene (Fischer-Vize, 1992). The homology between DOA4 and Faf suggests that the faf phenotype might also be caused by stabilization and accumulation of proteins, which are normally subject to ubiquitin dependent degradation. Genetic analysis was undertaken to discover whether faf might interact with components of the Ras pathway (Isaksson, 1997).

faf interacts genetically with the receptor tyrosine kinase (RTK)/Ras pathway, which induces photoreceptor differentiation in the developing eye. faf also interacts with pointed: the extra-photoreceptor phenotype observed in faf mutants is clearly suppressed by pointed mutation; many more ommatidia have six outer photoreceptors in a trapezoidal arrangement characteristic of wildtype ommatidia. yan mutation in combination with faf strongly enhances the faf phenotype. Reducing the D-Jun activity suppresses the faf mutant phenotype. In sevenless;faf double mutants, R7 cells, normally absent in sevenless mutants, form in 60% of the ommatidia. Thus, faf can alleviate the requirement for sev in the R7 precursor. These results indicate that RTK/Ras signaling is increased in faf mutants, causing normally non-neuronal cells to adopt photoreceptor fate. Consistently, the protein level of at least one component of the Ras signal transduction pathway, the transcription factor D-Jun, is elevated in faf mutant eye discs when the ectopic photoreceptors are induced. It is proposed that defective ubiquitin-dependent proteolysis leads to increased and prolonged D-Jun expression, which together with other factors contributes to the induction of ectopic photoreceptors in faf mutants (Isaksson, 1997).

Stabilization of D-Jun is not likely to be the only cause for the faf phenotype, because elevated levels of Jun per se do not elicit a gain-of-function effect as shown by transgenic expression of Jun in a wild-type background (Bohmann, 1994 and Treier, 1995). Nevertheless, in combination with even small disturbances in the ras pathway D-Jun overexpression causes marked differentiation of extra photoreceptors (Bohmann, 1994).

Examination of faf mutant clones reveals a potential non-autonomy to Faf function. Genotypes of different photoreceptors in phenotypically wild-type mosaic ommatidia were scored to determine if there is a tendency for pharticular photoreceptors to be faf+. None of the eight photoreceptors of normal facets is nearly as frequently as expected if faf+ function is required cell autonomously in a particular photoreceptor cell. Second, in the phenotypically mutant facets, ectopic photoreceptor cells are not always faf-, and the ectopic neurons that they contain (R3, R4 and R8) are also not always faf-. Thus, it is not the absence of faf+ function in the ectopic cells, or the cells they contact, that results in their misdetermination as photoreceptors. In summary, these observations indicate that cells near to, but outside the normal or ectopic photoreceptors in a particular facet must be faf+ in order to prevent the neuralization of extra photoreceptor cells (Fischer-Vize, 1992).

Other evidence points to a more complex role for Faf in eye differentiation. Faf expression behind the furrow in precluster cells, where D-Jun is thought to function, is not sufficient to rescue the Faf function in null flies. Faf must be expressed in front of the morphogenic furrow or within the furrow for reversion of the faf phenotype (Huang, 1996). In a screen for mutations that act as dominant enhancers of fat facets phenotype, it was expected that enhancers of faf would increase the number of facets that are faf-like, that is, give rise to ectopic photoreceptors. Surprisingly, each enhancer of faf fell into one of three groups based on its dominant phenotype in a hypomorphic faf mutant background; retinas of the "faf" group displayed a faf-like phenotype, retinas of the "sevenless" group had facets that were often missing the R7 photoreceptor (resembling the sevenless mutation and were also often missing other photoreceptor cells, and mutants of the 'wild-type" group had a wild-type photoreceptor arrangement. Since the wild-type group has roughened external eyes in a hypomorphic faf background, the defects must be in later cone and/or pigment cell development. This study suggests that Ffaf may have multiple roles in eye development (Fischer, 1997).

Novel gain-of-function mutations in the Drosophila Rap1 and Ras1 genes are described that interact genetically with fat facets mutations. Analysis of these genetic interactions reveals that Fat facets has an additional function later in eye development involving Rap1 and Ras1 proteins. faf expressed from a rough promoter (engendering faf expression in the furrow and R2/5 and R3/4 photoreceptors) has no ability to complement the mutant phenotypes of Rap1 or Ras1 combined with mutant faf. In contrast faf expressed from a glass promoter (engendering faf expression in all cells posterior to the furrow) complements extremely well. The results suggest that undifferentiated cells outside the facet play a role in recruiting photoreceptors into the facet. This is remarkble, as there is no other evidence that the undifferentiated cells surrounding the facets send any inductive signals. The results also suggest that undifferentiated cells outside the facet continue to influence facet assembly later in eye development (Li, 1997).

Fat facets and Liquid facets promote Delta endocytosis and Delta signaling in the signaling cells

Endocytosis modulates the Notch signaling pathway in both the signaling and receiving cells. One recent hypothesis is that endocytosis of the ligand Delta by the signaling cells is essential for Notch activation in the receiving cells. Evidence is presented in strong support of this model. In the developing Drosophila eye Fat facets (Faf), a deubiquitinating enzyme, and its substrate Liquid facets (Lqf), an endocytic epsin, promote Delta internalization and Delta signaling in the signaling cells. While Lqf is necessary for three different Notch/Delta signaling events at the morphogenetic furrow, Faf is essential only for one: Delta signaling by photoreceptor precluster cells, which prevents recruitment of ectopic neurons. In addition, the ubiquitin-ligase Neuralized (Neur), which ubiquitinates Delta, is shown to function in the signaling cells with Faf and Lqf. The results presented bolster one model for Neur function in which Neur enhances Delta signaling by stimulating Delta internalization in the signaling cells. It is proposed that Faf plays a role similar to that of Neur in the Delta signaling cells. By deubiquitinating Lqf, which enhances the efficiency of Delta internalization, Faf stimulates Delta signaling (Overstreet, 2004).

Cells with decreased lqf+ activity accumulate Delta on apical membranes and fail to signal to neighboring cells. Three Notch/Delta signaling events were examined in the eye: proneural enhancement, lateral inhibition and R-cell restriction. Loss of lqf+-dependent endocytosis during all three events has identical consequences to loss of Delta function in the signaling cells. It is concluded that lqf+-dependent endocytosis of Delta is required for signaling, supporting the notion that endocytosis in the signaling cells activates Notch in the receiving cells. However, Lqf is not required absolutely for all Delta internalization in the eye. Even in lqf-null cells, which are incapable of Delta signaling, some vesicular Delta is present. Perhaps not all of the vesicular Delta present in wild-type discs results from signaling (Overstreet, 2004).

Genetic studies in Drosophila indicate clearly that deubiquitination of Lqf by Faf activates Lqf activity. Moreover, genetic and biochemical evidence in Drosophila suggests that Faf prevents proteasomal degradation of Lqf. In vertebrates, however, it is thought that epsin is mono-ubiquitinated to modulate its activity rather than poly-ubiquitinated to target it for degradation. If Lqf regulation by ubiquitin also occurs this way in the Drosophila eye, the removal of mono-ubiquitin from Lqf by Faf would activate Lqf activity (Overstreet, 2004).

Whatever the precise mechanism, given that both Faf and Lqf are expressed ubiquitously in the eye, two related questions arise. First, why is Lqf ubiquitinated at all if Faf simply deubiquitinates it everywhere? One possibility is that Faf is one of many deubiquitinating enzymes that regulate Lqf, and expression of the others is restricted spatially. This could also explain why Faf is required only for R-cell restriction. Another possibility is that Faf activity is itself regulated in a spatial-specific manner in the eye disc. Alternatively, Lqf ubiquitination may be so efficient that Faf is needed to provide a pool of non-ubiquitinated, active Lqf. Similarly, Faf could be part of a subtle mechanism for timing Lqf activation. Second, why is Faf essential only for R-cell restriction? One possibility is that there is a graded requirement for Lqf in the eye disc, such that proneural enhancement requires the least Lqf, lateral inhibition somewhat more and neural inhibitory signaling by R2/3/4/5 the most. The mutant phenotype of homozygotes for the weak allele lqfFDD9 supports this idea, as R-cell restriction is most severely affected. Alternatively, Lqf may be expressed or ubiquitinated with dissimilar efficiencies in different regions of the eye disc. More experiments are needed to understand the precise mechanism by which the Faf/Lqf interaction enhances Delta signaling (Overstreet, 2004).

In neur mutants, Delta accumulates on the membranes of signaling cells and Notch activation in neighboring cells is reduced. These results support a role for Neur in endocytosis of Delta in the signaling cells to achieve Notch activation in the neighboring receiving cells, rather than in downregulation of Delta in the receiving cells. Because neur shows strong genetic interactions with lqf and both function in R-cells, Neur and Lqf might work together to stimulate Delta endocytosis. Lqf has ubiquitin interaction motifs (UIMs) that bind ubiquitin. One explanation for how Neur and Faf/Lqf could function together is that Lqf facilitates Delta endocytosis by binding to Delta after its ubiquitination by Neur. This is anattractive model that will stimulate further experiments (Overstreet, 2004).

One exciting observation is that the endocytic adapter Lqf may be essential specifically for Delta internalization. Although, hedgehog, decapentaplegic and wingless signaling pathways have not been examined directly, they appear to be functioning in the absence of Lqf. These three signaling pathways regulate movement of the morphogenetic furrow and are thought to require endocytosis. The furrow moves through lqf-null clones and at the same pace as the surrounding wild-type cells. Moreover, all mutant phenotypes of lqf-null clones can be accounted for by loss of Delta function. Further experiments will clarify whether this apparent specificity means that Lqf functions only in internalization of Delta, or if the process of Delta endocytosis is particularly sensitive to the levels of Lqf (Overstreet, 2004).

Lqf expands the small repertoire of endocytic proteins that are known targets for regulation of cell signaling. In addition to Lqf, the endocytic proteins Numb and Eps15 (EGFR phosphorylated substrate 15) are objects of regulation. In vertebrates, asymmetrical distribution into daughter cells of the alpha-adaptin binding protein Numb may be achieved through ubiquitination of Numb by the ubiquitin-ligase LNX (Ligand of Numb-protein X) and subsequent Numb degradation. In addition, in vertebrate cells, Eps15 is phosphorylated and recruited to the membrane in response to EGFR activation and is required for ligand-induced EGFR internalization. Given that endocytosis is so widely used in cell signaling, endocytic proteins are likely to provide an abundance of targets for its regulation (Overstreet, 2004).


REGULATION

Targets of Activity

Eukaryotic genomes encode large families of deubiquitinating enzymes (DUBs). Genetic data suggest that Fat facets (Faf), a Drosophila DUB essential for patterning the compound eye, might have a novel regulatory function; Faf might reverse the ubiquitination of a specific substrate, thereby preventing proteasomal degradation of that protein. Additional genetic data implicate Liquid facets (Lqf), a homolog of the vertebrate endocytic protein epsin, as a candidate for the key substrate of Faf. Here, biochemical experiments critical to testing this model were performed. The results show definitively that Lqf is the key substrate of Faf in the eye; Lqf concentration is Faf-dependent, Lqf is ubiquitinated in vivo and deubiquitinated by Faf, and Lqf and Faf interact physically (Chen, 2002).

To detect Lqf protein levels in developing eyes, an antibody was generated to Lqf. Eye discs were double-labeled with anti-Lqf and antibodies to the endocytic protein Shibire. This shows that Lqf and Shi colocalize at cell membranes: Lqf and Shi are concentrated apically in cells within the morphogenetic furrow, an indentation that marks the onset of differentiation, and also in developing photoreceptors where their membranes meet. Similar results were obtained with antibodies to two other endocytic proteins (Dap160 and alpha-Adaptin [alpha-Ada]), and with phalloidin, which labels f-actin at cell membranes (Chen, 2002).

One prediction of the hypothesis that Faf activity prevents the degradation of Lqf is that in the developing eyes (larval eye discs) of faf null mutant flies, there should be less Lqf protein than in wild-type eyes. It was expected there would be less Lqf protein, as opposed to no Lqf protein, because the lqf null mutant eye phenotype is much more severe than the faf null mutant eye phenotype (Chen, 2002).

To test whether the level of Lqf is affected by faf+ gene activity, first the levels of Lqf were compared in adjacent groups of faf+ and faf- cells in the eye disc, using confocal microscopy. Clones of homozygous faf- cells were generated in faf+/faf- heterozygous eye discs, marked by the absence of ß-galactosidase (ß-gal) expression. The eye discs containing clones were triple-labeled with antibodies to ß-gal (to outline the clones), to Lqf (to detect the level of Lqf protein), and to Shi (as a negative control). It was found that throughout the eye disc, the level of Lqf protein, reflected in the strength of the signal from antibody labeling, is lower within the faf- clones than in the faf+/faf- heterozygous cells surrounding them. In contrast, the levels of Shi protein are the same within and outside the clone boundaries (Chen, 2002).

To quantify the difference in Lqf protein levels in faf+ and faf- cells, the levels of Lqf were assayed in eye disc protein extracts prepared from wild-type and faf- flies in Western blot experiments. Homozygotes for two different mutant faf alleles that behave genetically as strong loss-of-function mutations were used: fafBX4 is an inversion that makes no functional Faf protein, and fafFO8 encodes an Faf protein with histidine residue 1986, which is critical for DUB catalytic activity, changed to tyrosine. There is two- to three-fold less Lqf in eye disc protein extracts of faf- homozygotes than in wild-type extracts. faf+ transgene restores function back to faf- flies. The transgene containing faf+ genomic DNA, which when introduced into faf- homozygotes complements the mutant eye phenotype, results in a two- to three-fold increase in Lqf protein level in eye disc extracts. A nearly identical transgene that fails to complement the faf- mutant phenotype because it has a point mutation in the codon for cysteine 1677, which is critical to the DUB activity of Faf, fails also to increase the level of Lqf protein in eye disc extracts. It is concluded that faf+ activity results in an increase in the level of Lqf protein (Chen, 2002).

A second prediction of the model wherein Faf prevents proteolysis of Lqf by deubiquitinating it, is that there should be Lqf protein linked to Ub chains present in eye discs. Ubiquitinated proteins are usually detected on Western blots as ladders of protein bands of higher molecular weight than the protein in question, in increments of ~8 kD; each 'rung' on the ladder represents a protein species with a Ub chain that is one Ub residue longer than the previous rung. Proteins with Ub chains are rapidly degraded, and thus difficult to detect; usually, inhibition of proteasome and/or DUB activity is required to detect them. It this study, inhibition of the DUB activity of Faf, genetically, stabilizes ubiquitinated forms of Lqf (Chen, 2002).

It is concluded that in eye discs, Lqf is ubiquitinated, and subsequently either deubiquitinated by Faf or degraded. The observation that considerable amounts of nonubiquitinated Lqf protein remain in faf- eye discs indicates either that only a fraction of the Lqf protein in the eye disc is ubiquitinated, and/or that DUBs other than Faf also deubiquitinate some Lqf protein (Chen, 2002).

A third prediction of the model wherein Lqf is the substrate of Faf is that the proteins should, either directly or indirectly, interact. Anti-Lqf was used to immunoprecipitate Lqf from protein extracts prepared from embryos, and tested for the presence of Faf in the immunoprecipitates on Western blots. Embryos were used because sufficient protein could not be obtained from eye discs. In addition, to facilitate detection of Faf, the embryos were transformed with a P{hs-myc-faf+} transgene, which expresses a fully functional, myc-tagged Faf protein upon heat shock, that can be detected on Western blots with anti-myc. myc-Faf was detected in the anti-Lqf immunoprecipitate of the protein extract from heat-shocked transformant embryos (Chen, 2002).

It is concluded that myc-Faf and endogenous Lqf proteins interact physically in Drosophila embryos. Bacterially produced or in vitro translated partial Faf and full-length Lqf proteins do not bind to each other in GST pull-down assays. One possible explanation is that only full-length Faf can bind to Lqf in these assays. Alternatively, Faf and Lqf may require other proteins for their interaction (Chen, 2002).

These experiments provide critical biochemical evidence for a model in which a DUB called Faf specifically deubiquitinates Lqf protein, thereby preventing its proteolysis. There is less Lqf protein in the developing eye in the absence of catalytically functional Faf protein, that Lqf is ubiquitinated and subsequently deubiquitinated by Faf, and that Faf and Lqf interact physically. Taken together with previous genetic evidence that provides strong support for the model, it is concluded that Faf is a substrate-specific regulator of ubiquitination, a novel function for a DUB (Chen, 2002).

The eyes of faf null or lqf hypomorphic mutants have more than the normal complement of eight photoreceptor cells in each facet, owing to the failure of a cell communication pathway early in eye development. The Faf/Lqf interaction is essential in only a small number of cells in the eye disc, which must be particularly sensitive to the levels of Lqf, and in these cells, Lqf presumably controls the frequency or specificity of endocytosis. Although the precise mechanism of epsin function is unknown, vertebrate epsin binds to the endocytosis complex and also to PIP2 at the cell membrane, and is required for endocytosis (Chen, 1998; Itoh, 2001). Apparently, appropriate endocytosis in this small group of cells is essential for successful communication with their neighbors; increased Lqf levels either enables these cells to send a signal to their neighbors that inhibits neural determination, or else prevents them from sending their neighbors a positive differentiation signal (Chen, 2002).

Through a variety of mechanisms, endocytosis is proposed to regulate ligand/receptor interactions during development. How Lqf and endocytosis regulate faf+-dependent cell signaling remains to be determined. Since faf has vertebrate homologs, this mode of regulation is likely to be conserved. The finding that Lqf is the key substrate of Faf in the Drosophila eye shows not only that a DUB can regulate ubiquitination and thus proteolysis, but also that an endocytosis complex protein can be a target for the control of a cell communication event critical to cell determination (Chen, 2002).

Protein Interactions

The Fat facets protein (FAF) acts as a regulatory Ubiquitin-specific protease (Ubp) that prevents degradation of its substrate by the proteasome. Flies bearing fat facets gene mutations have been used to show that a Ubp is a cell type- and substrate-specific regulator of cell fate decisions. Ubiquitin conjugates are cleaved by a 744-amino acid partial Faf protein that includes the Cys and His domains. Four mutant faf genes that encode proteins with either an altered key Cys residue or with changes in one or both of the two conserved His residues abolish the Udps activity of Fas (Huang, 1995).

Anterior-posterior patterning and germ cell specification in Drosophila requires the establishment, during oogenesis, of a specialized cytoplasmic region termed the pole plasm. Numerous RNAs and proteins accumulate to the pole plasm and assemble in polar granules. Translation of some of these RNAs is generally repressed and active only in pole plasm. Vasa (Vas) protein, an RNA helicase and a component of polar granules, is essential maternally for posterior patterning and germ cell specification, and Vas is a candidate translational activator in the pole plasm. Vas is stabilized within the pole plasm in that it is initially present throughout the entire embryo but strictly limited to the pole cells by the cellular blastoderm stage. hsp83 mRNA, which accumulates in the pole plasm through a stabilization-degradation mechanism, is another example. A biochemical approach has been used to identify proteins that copurify with Vas in crosslinked extracts. Prominent among these proteins was the ubiquitin-specific protease Fat facets (Faf), a pole plasm component, but one whose roles in posterior patterning and germ line specification have remained unclear. Evidence suggests that Faf interacts with Vas physically and reverses Vas ubiquitination, thereby stabilizing Vas in the pole plasm (Liu, 2003).

Vas and Faf can be copurified from chemically crosslinked embryonic and ovarian extracts; in faf mutants, the ubiquitination of Vas is increased and its levels are decreased in ovaries and more strikingly in progeny embryos. The simplest interpretation of these data is that Vas is a specific substrate for the deubiquinating enzyme Faf. It is believed the reduction of localized Osk observed in faf mutant ovaries is an indirect result of the reduced stability of Vas, since vas function is required for the stable accumulation of Osk in the pole plasm (Liu, 2003).

Heterozygotes for a vas null mutation produce embryos with a 20%-25% reduction in pole cell number, yet such embryos, unlike progeny of faf mutant mothers, show prominent posterior Vas staining. The severe phenotype of embryos produced by homozygous faf mothers renders difficult a direct analysis of its requirement in the pole plasm. However, based on the vas heterozygous phenotype, the obvious reduction in posterior Vas accumulation in faf mutants should affect development, and therefore Faf-mediated stabilization of Vas must contribute to pole plasm function. Faf-mediated protection of Vas is mostly restricted to the pole plasm, perhaps because during oogenesis, Faf itself becomes localized to the posterior pole and is incorporated into pole cells. As is the case for other pole plasm components, Faf localization depends on osk function, and a role for Faf in germ cell differentiation and development has been proposed. Interestingly, Usp9x, a bona fide mouse ortholog of Faf, is predominantly expressed in both germ cell and supporting cell lineages during gonadal development, suggesting a conserved role for Faf in mammalian germ line development (Liu, 2003).

Control of a Kinesin-Cargo linkage mechanism by JNK pathway kinases

Long-distance organelle transport toward axon terminals, critical for neuron development and function, is driven along microtubules by kinesins . The biophysics of force production by various kinesins is known in detail. However, the mechanisms of in vivo transport processes are poorly understood because little is known about how motor-cargo linkages are controlled. A c-Jun N-terminal kinase (JNK)-interacting protein (JIP1) has been identified previously as a linker between kinesin-1 and certain vesicle membrane proteins, such as Alzheimer's APP protein and a reelin receptor ApoER2. JIPs are also known to be scaffolding proteins for JNK pathway kinases. Evidence is presented that a Drosophila ubiquitin-specific hydrolase (Fat facets) and a JNK signaling pathway that it modulates can regulate a JIP1-kinesin linkage. The JNK pathway includes a MAPKKK (Wallenda/DLK), a MAPKK (Hemipterous/MKK7), and the Drosophila JNK homolog Basket. Genetic tests indicate that those kinases are required for normal axonal transport. Biochemical tests show that activation of Wallenda (DLK) and Hemipterous (MKK7) disrupts binding between kinesin-1 and APLIP1, which is the Drosophila JIP1 homolog. This suggests a control mechanism in which an activated JNK pathway influences axonal transport by functioning as a kinesin-cargo dissociation factor (Horiuchi, 2007).

Maintaining proper distributions of protein complexes, RNAs, vesicles, and other organelles in axons is critical for the development, function, and survival of neurons. The primary distribution mechanism relies on long-distance transport driven by microtubule motor proteins. Components newly synthesized in the cell body, but needed in the axon, bind kinesin motors that carry them toward microtubule plus ends and the axon terminal (anterograde transport). Neurotrophic signals and endosomes, examples of axonal components that require transport to the cell body, bind dynein motors that carry them toward minus ends (retrograde transport). The importance of these processes is highlighted by the observation that mutation of motors and other transport machinery components can cause neurodegenerative diseases in humans and analogous phenotypes in model organisms (Horiuchi, 2007).

Two key questions are (1) how do cargoes link to particular motors, and (2) how are such linkages regulated to ensure appropriate pickup and dropoff dynamics? For kinesin-vesicle linkages, scaffolding proteins have emerged as key connectors. For example, the cargo-binding kinesin light chain (Klc) subunit of kinesin-1 binds not only the kinesin-1 heavy chain (Khc) but also JNK-interacting proteins (JIPs). Vertebrate JIPs can bind multiple components of the JNK signaling pathway, e.g., JNK itself, upstream activating kinases (MAPKKs), and regulatory kinases (MAPKKKs). JIPs can also bind vesicle-associated membrane proteins, such as ApoER2, which is a reelin receptor, and APP, a key factor in Alzheimer's disease. Therefore, JIP scaffolding proteins are likely to link JNK pathway kinases and kinesin-1 to vesicles carrying these membrane proteins. This raises an interesting question: Are the JNK pathway kinases simply passive hitchhikers on the kinesin-1/JIP/vesicle complex, or can they actively regulate its transport (Horiuchi, 2007)?

A genetic screen was conducted for factors that control kinesin-JIP linkage during axonal transport. The screen was based on the previous observation that neuron-specific overexpression of Aplip1, which encodes the Drosophila JIP1, causes synaptic protein accumulation in axons, larval paralysis, and larval-pupal lethality, the classic axonal-transport-disruption phenotypes caused by Khc and Klc mutations. Why might overexpression of the JIP1 cargo linker for kinesin-1 disrupt axonal transport? The disruptive effect requires APLIP1 (JIP1)-Klc binding. It may be that excess APLIP1 (JIP1) competes with other Klc-binding proteins, for example, different linkers that may attach kinesin-1 to other cargoes. In search of factors that can disrupt or antagonize APLIP1 (JIP1)-Klc binding, a screen was performed for genes that can suppress the axonal-transport phenotypes when co-overexpressed with Aplip1. An 'EP' collection of fly strains capable of the targeted overexpression of endogenous Drosophila genes was screened and P{EP}fafEP381, a line that overexpresses fat facets (faf), was identified as a strong suppressor of the APLIP1 (JIP1)-induced lethality and other neuronal overexpression phenotypes (Horiuchi, 2007).

Faf protein antagonizes ubiquitination and proteasome-mediated degradation of its target proteins. Interestingly, Faf was recently reported to stimulate a Drosophila neuronal JNK signaling pathway that is regulated by the MAPKKK Wallenda (Wnd), a homolog of dual leucine zipper-bearing kinase (DLK) that is known to bind JIP1. Overexpression of faf leads to increased levels of Wnd (MAPKKK) protein and thereby causes excessive synaptic sprouting through a pathway that requires the Drosophila JNK homolog Basket. It was found that mutating just one copy of wnd blocked the suppression of Aplip1 overexpression by P{EP}fafEP381. This suggests that faf overexpression suppresses APLIP1 (JIP1)-Klc interaction by elevating the level of Wnd (MAPKKK). Consistent with this, direct overexpression of wnd in neurons with a wild-type transgene (UAS-wnd) was as effective as P{EP}fafEP381 in suppressing UAS-Aplip1-induced axonal accumulation of synaptic proteins. Equivalent expression of a 'kinase-dead' mutant transgene (UAS-wndKD) did not suppress the defects. Thus, Wnd (MAPKKK) and its downstream phosphorylation targets may actively regulate APLIP1 (JIP1)-Klc binding in neurons (Horiuchi, 2007).


DEVELOPMENTAL BIOLOGY

Larval

Staining in the eye disc reveals protein within the entire region ahead of the morphogenetic furrow, including the antennal disc, and lower levels behind the furrow. Protein is also consistently detected in other discs and larval and adult tissues (i.e. fat body, gut, larval ovary and testes and adult male sex organs) (Fischer-Vize, 1992).

Adult

Faf protein is seen throughout oogenesis within the nurse cell-oocyte cluster. When the oocyte becomes distinct from the nurse cells, cytoplasmic stain appears to be confined to the nurse cells. Later, high levels of stain are seen at the posterior pole of oocytes. The posterior staining persists throughout oogenesis, and after the egg is laid, maternal protein at the posterior becomes incorporated into the pole cells. FAF mRNA is not posteriorly localized. Faf protein is also found in testis (Fischer-Vize, 1992).


EFFECTS OF MUTATION

Localization of Faf protein depends on Oskar. As Staufen protein and OSK mRNA are posteriorly localized earlier than Faf protein, and Vasa protein is localized at about the same time as Faf, faf appears to fit into the posterior group hierarchy. Pole plasm components are posteriorly localized in a normal fashion in faf mutants and Nanos protein function is unimpaired in faf mutant embryos (Fischer-Vize, 1992).

All the strong faf alleles cause female sterility. Females homozygous for any strong allele have apparently normal ovaries, but lay eggs that never form cuticle and never hatch. In embryos from faf mutant mothers, no normal syncytial blastoderm embryos are observed. Most of the nuclei fail to migrate to the periphery. Also commonly observed are embryos in which patches of asynchronously dividing nuclei have migrated to the periphery. Except for the pole cells (which are fewer in number and spread out, instead of grouped together as in wild type), no cellularization of faf mutant embryos is ever observed (Fischer-Vize, 1992).

Many defects are present in strong faf mutant retinas, the most striking of which is the appearance of one, two or occasionally three outer photoreceptor cells in most facets (Fischer-Vize, 1992).

In order to identify the genes encoding the substrate of Fat facets and other components of the neural inhibition pathway, a mutagenesis screen for dominant enhancers of the fat facets mutant eye phenotype was performed. Several genes were identified, one of which is an excellent candidate for encoding a component of the pathway regulated by Fat facets. Three different eye phenotypes were observed when the fat facets mutants were dominantly enhanced by different mutations, suggesting that fat facets has other functions in addition to its critical role early in eye development (Fischer, 1997).

The Drosophila fat facets (faf) gene encodes a deubiquitination enzyme with a putative function in proteasomal protein degradation. Mutants lacking zygotic faf function develop to adulthood, but have rough eyes caused by the presence of one to two ectopic outer photoreceptors per ommatidium. faf interacts genetically with the receptor tyrosine kinase (RTK)/Ras pathway, which induces photoreceptor differentiation in the developing eye.

faf also interacts with pointed. The extra-photoreceptor phenotype observed in faf mutants is clearly suppressed by pointed mutation; many more ommatidia have six outer photoreceptors in a trapezoidal arrangement characteristic of wildtype ommatidia. yan mutation in combination with faf strongly enhances the faf phenotype. Reducing the D-Jun activity suppresses the faf mutant phenotype. In sevenless;faf double mutants, R7 cells, normally absent in sevenless mutants, form in 60% of the ommatidia. Thus, faf can alleviate the requirement for sev in the R7 precursor. These results indicate that RTK/Ras signaling is increased in faf mutants, causing normally non-neuronal cells to adopt photoreceptor fate. Consistently, the protein level of at least one component of the Ras signal transduction pathway, the transcription factor D-Jun, is elevated in faf mutant eye discs when the ectopic photoreceptors are induced. It is proposed that defective ubiquitin-dependent proteolysis leads to increased and prolonged D-Jun expression, which together with other factors contribute to the induction of ectopic photoreceptors in faf mutants. These studies demonstrate the relevance of ubiquitin-dependent protein degradation in the regulation of RTK/Ras signal transduction in an intact organism (Isaksson, 1997).

The Drosophila fat facets gene encodes a deubiquitinating enzyme required during eye development to limit to eight the number of photoreceptors in each facet. Ubiquitin is a small polypeptide that targets proteins for degradation by the proteasome. Deubiquitinating enzymes cleave ubiquitin-protein bonds. In order to investigate the role of Fat facets in the ubiquitin pathway, genetic interactions between fat facets and the Drosophila UbcD1 gene were assessed. In addition, three yeast deubiquitinating enzyme genes were tested for their ability to substitute for fat facets in the developing Drosophila eye and for their effects on eye morphology. These experiments support the hypothesis that Fat facets activity antagonizes that of the proteolytic machinery (Wu, 1999).

Fat facets is a deubiquitinating enzyme required in a cell communication pathway that limits to eight the number of photoreceptor cells in each facet of the Drososphila compound eye. Genetic data support a model whereby Faf removes ubiquitin, a polypeptide tag for protein degradation, from a specific ubiquitinated protein, thus preventing its degradation. Mutations in the liquid facets gene have been identified as dominant enhancers of the fat facets mutant eye phenotype. The liquid facets locus encodes epsin, a vertebrate protein associated with the clathrin endocytosis complex. Genetic experiments reveal that fat facets and liquid facets facilitate endocytosis and function in common cells to generate an inhibitory signal that prevents ectopic photoreceptor determination. The fat facets mutant phenotype is extraordinarily sensitive to the level of liquid facets expression. It is proposed that Liquid facets is a candidate for the critical substrate of Fat facets in the eye (Cadavid, 2000).

There are three key components of the endocytosis complex: (1) clathrin, which forms a cage structure engulfing the cell membrane; (2) AP-2, the core adaptor complex, which binds to clathrin and brings it to the cell surface and (3) dynamin, a GTPase required for vesicle formation. Additional proteins associated with AP-2 have been identified, many of which contain protein-protein interaction domains called EH-domains and EH-domain-binding motifs. Epsin is an EH-domain-binding protein identified as a partner for Eps15, an EH-domain protein that also binds AP-2. The large number of AP-2-binding proteins identified suggests that many of them may have temporal and/or tissue-specific functions (Marsh and McMahon, 1999). The precise roles of Eps15 and epsin in endocytosis are unknown (Cadavid, 2000 and references therein).

The lqf gene itself is essential in Drosophila; in an otherwise wild-type background, lqf null mutants die as embryos. Clones of cells in the eye in which there is little or no lqf gene function have severely disrupted eye morphology indicating that lqf is required also after embryogenesis for eye development. The mutant phenotypes associated with two weak lqf mutant alleles reveal specific roles for lqf in eye, wing and leg development (Cadavid, 2000).

The eye defects in homozygous adults for a weak allele of lqf resemble those in faf null mutants. As in faf mutants, the additional photoreceptors in lqf mutants arise from specific precursor cells (M-cells) present early during eye development. In contrast to lqf null mutants, faf null mutants are viable, have normal wings and legs and have less severe eye defects. Thus, lqf functions more broadly than faf, but both the lqf and faf genes are required during eye development in order to prevent the M-cells from becoming photoreceptors (Cadavid, 2000).

The faf mutant eye phenotype is unusually sensitive to a decrease in the dose of the lqf gene, suggesting strongly that the two genes function in a common pathway. Genetic interactions with endocytosis and Ub pathway mutants show that faf and lqf facilitate endocytosis and antagonize ubiquitination. In addition, although lqf is more broadly required than faf in the eye and elsewhere in the fly, weak lqf mutations reveal that like faf, lqf is required to prevent the misdetermination of M-cells as photoreceptors. Moreover, when expressed only in the rough positive cells surrounding the facet preclusters, both faf and lqf genes rescue completely to wild-type their respective M-cell misdetermination mutant phenotypes in the eye. Finally, given the relationship between Faf and its substrate protein, it would be expected that increasing the dose of the substrate should suppress the faf mutant phenotype. Slight overexpression of lqf completely obviates the need for faf in eye development. The simplest model consistent with all of this genetic data is that Lqf is the substrate of Faf. Other more complicated explanations are, of course, possible (Cadavid, 2000).

There is biochemical evidence that AF-6, a scaffolding protein thought to modulate cell-cell junctions in response to Ras activation may be an in vivo substrate of Fam, the mouse homolog of Faf; AF-6 and Fam bind each other in vitro and ubiquitinated AF-6 can be detected and deubiquitinated by Fam in cultured cells. Like lqf, the Drosophila Af6 homolog, canoe, is required pleiotropically for Drosophila eye development. In contrast to lqf mutations, however, canoe mutations do not act as strong dominant enhancers of the faf mutant eye phenotype . Given the striking genetic interactions between faf and lqf, it seems that canoe is unlikely to play a significant role in the essential faf pathway in the eye (Cadavid, 2000 and references therein).

While only one Faf/substrate interaction may be essential to normal eye development in Drosophila, Faf and Fam may have several substrates in vivo. Normally non-essential roles for faf later in eye development have been revealed in particular mutant backgrounds and Faf could have different substrates for its critical role in M-cell fate determination than in its redundant roles. Moreover, in addition to its essential role in eye development, faf is required maternally for cellularization of embryos and the critical maternal substrate of Faf is unknown. Because faf has mouse and human homologs, the modes of regulation by Faf are likely to be conserved. However, it is possible that the critical substrate(s) of Faf in Drosophila may differ from those in vertebrates (Cadavid, 2000 and references therein).

If Lqf is the substrate of Faf, then epsin levels, determined by the balance between its ubiquitination and deubiquitination, could regulate endocytosis. Mono-ubiquitination, however, has been shown previously to regulate endocytosis in two different ways. (1) Mono-ubiquitination of cell surface receptors can act as a signal for receptor endocytosis, which leads to lysosomal degradation. Here, the Ub moiety is somehow recognized by the endocytosis machinery; this process has nothing to do with the proteasome. (2) Eps15, an endocytosis complex component in mammalian cells, is mono-ubiquitinated in response to EGF receptor activation and Eps15 may require this modification to stimulate receptor endocytosis. In addition, Pan1p a yeast protein similar to Eps15, is required for endocytosis in yeast. Although it is unknown whether Pan1p is mono-ubiquitinated in yeast, there is evidence that ubiquitination of an endocytic complex component is required for endocytosis in yeast; Rsp5p, a component of the ubiquitination machinery called a ubiquitin-ligase, may bind to Pan1p and is required generally for endocytosis in yeast, even for endocytosis of proteins with non-Ub endocytosis signals (Cadavid, 2000 and references therein).

Since Eps15 binds to epsin, could a mono-ubiquitinated Drosophila Eps15 homolog be the substrate of Faf? Two of experimental results are inconsistent with this model. (1) It has been shown previously that the activity of Faf antagonizes proteolysis, not just ubiquitination; mutations in a gene encoding a proteasome subunit act as strong suppressors of the faf mutant eye phenotype. This result strongly suggests that Faf activity antagonizes proteolysis and thus that Faf deubiquitinates a protein containing a Ub chain targeting it for degradation, rather than a mono-ubiquitinated protein. (2) If mono-ubiquitination of Eps15 activates it, as the available data suggests, then deubiquitination of Eps15 by Faf would render Eps15 inactive and thus the function of Faf would antagonize endocytosis. The data presented here clearly indicate the opposite; mutations in endocytosis complex genes (particularly lqf and Clathrin heavy chain) act as strong dominant enhancers of faf, suggesting that the normal function of Faf is to facilitate endocytosis (Cadavid, 2000 and references therein).

Elevated levels of Lqf obviate the need for Faf, presumably by stimulating epsin-dependent endocytosis generally or stimulating endocytosis of a specific cell surface protein. How can the observation that Lqf and Faf function outside the M-cells to determine M-cell fate be reconciled with a role for Lqf in endocytosis? Endocytosis is known to modulate ligand/receptor interactions by a variety of mechanisms. One possibility is that M-cell fate is affected by a diffusible ligand that, like Wingless, travels via endocytosis through several cell distances. Alternatively, regulation of a membrane-bound receptor by endocytosis in cells adjacent to the M-cells could affect M-cell fate indirectly. For example, EGF receptor activity is downregulated by endocytosis following ligand binding. By contrast, activity of the Notch receptor may be up-regulated by endocytosis of activated receptors whose intracellular domains have been cleaved off prior to their translocation into the nucleus. Membrane-bound Notch receptors lacking their intracellular domains display dominant negative activity and endocytosis of cleaved Notch receptors may be required normally for precise modulation of Notch activity. Patterning of the photoreceptor preclusters in the developing eye may require that both Notch and the EGF receptor are activated in the rough-expressing cells surrounding the facet preclusters. Thus, Faf could regulate the activity of one or both of these receptors (Cadavid, 2000 and references therein).

The Drosophila DNAprim gene encodes the large subunit (60 kD) of DNA primase, the part of DNA polymerase alpha that synthesizes RNA primers during DNA replication. The precise function of the 60-kD subunit is unknown. In a mutagenesis screen for suppressors of the fat facets (faf) mutant eye phenotype, mutations in DNAprim were identified. The faf gene encodes a deubiquitinating enzyme required specifically for patterning the compound eye. The DNA sequences of four DNAprim alleles have been determined and these define essential protein domains. While flies lacking DNAprim activity are lethal, flies with reduced DNAprim activity display morphological defects in their eyes, and unlike faf mutants, cell cycle abnormalities in larval eye discs (Chen, 2000b).

Eight of the 11 DNAprim alleles, including In(3L)78Cb1, are early lethal in trans to Dfr(3L)rdgC-co2 or S(faf)240. Since In(3L)78Cb1 breaks within the coding region of DNAprim, the other 7 alleles are also likely to be null mutants. A weaker allele, DNAprimT2, is pupal lethal in trans to Dfr(3L)rdgC-co2 or S(faf)240. There is cell death in the eye anterior of late-stage pupae of these genotypes. The weakest alleles are the homozygous viable group of imprecise excisions, exemplified by DNAprimj10B2Delta13-15. When homozygous or in trans to Df(3L)rdgC-co2, the DNAprimj10B2Delta13-15 alleles result in slightly roughened external eyes. Patterning defects, either in the accessory cells (cone and pigment cells) of pupal eyes, could not be detected in these flies. Thus, the external rough eyes are most likely due to subtle defects in cone cells, pigment cells, and/or bristles in the final stages of eye development; absent bristles and bristle misplacements are apparent in the adult eyes (Chen, 2000b).

To understand the basis for the genetic interactions observed between faf and DNAprim, it was asked whether the cell cycle is altered in these mutants. DNAprim mutants would be expected to have difficulty during S phase when DNA is replicated. If S phase is slower in these mutants, then more cells would be expected to be in S phase at any given time. During larval eye disc development, undifferentiated cells anterior to the morphogenetic furrow divide asynchronously and then arrest in G1 within the morphogenetic furrow prior to differentiation. No cell division occurs again until later in the eye disc, when cells that will give rise to three of the eight photoreceptor types and all of the other cell types undergo a single mitosis. By assaying BrdU incorporation with anti-BrdU antibodies, cells in S phase were visualized in larval eye discs of flies with a pupal lethal combination of DNAprim mutations, as well as wild-type and faf - eye discs. BrdU incorporation in the faf - eye discs resembles wild type. By contrast, in the DNAprim mutants, more cells are in S phase at a given time. Since faf mutants do not have defects in S phase, the genetic interactions between DNAprim and faf most likely reflect indirect interactions between the functions of the two proteins (Chen, 2000b).

Why do DNAprim mutations suppress the faf mutant eye phenotype? To answer this question, the origins of the ectopic photoreceptors in faf mutant eyes must be considered and also the time and place in the larval eye disc in which faf normally functions. The ommatidia of faf mutant eyes contain one or more photoreceptors in addition to the normal eight. The extra photoreceptors originate from the mystery cells, which are present in early facet preclusters. The precursors to the first five photoreceptors (R8, R2/5, and R3/4) and one or more mystery cells emerge posterior to the morphogenetic furrow as preclusters of six or more cells, each surrounded by undifferentiated cells. Normally, the mystery cells detach from the preclusters and go on to divide in the wave of mitosis posterior to the furrow, giving rise to the remainder of the photoreceptors (R1, R6, and R7) and to the accessory cells (cone, pigment, and bristle cells). In faf mutants, the mystery cells remain within the preclusters and become extra R3/4-like cells. Although faf is required for inhibition of mystery cell neurogenesis, mosaic analysis as well as faf transgene complementation experiments indicate that faf is not required within the mystery cells nor within any photoreceptor precursors in the precluster (Chen, 2000b).

Normally, faf is expressed at high levels anterior to the morphogenetic furrow, where cells are undergoing mitosis, and then its expression shuts off within the furrow, just prior to precluster formation. Although faf normally functions anterior to the furrow, later expression of faf in a subgroup of cells within the furrow is sufficient to rescue the faf mutant eye phenotype. The subgroup of cells that require faf expression are those cells that express the rough gene; these cells surround those that express the proneural gene atonal, which are destined to become the precluster cells. Thus, faf activity in the dividing cells anterior to the furrow facilitates a cell communication pathway that may occur anterior to or within the furrow. Perhaps when DNA primase levels are halved in heterozygous mutants, the cells anterior to the furrow remain in S phase longer, which changes subtly the output of the signaling pathway that faf modulates in such a way that the requirement for faf is largely overcome (Chen, 2000b).

Mutations in cell cycle proteins have been isolated previously in genetic screens for mutants affecting the Sevenless receptor tyrosine kinase signaling pathway in eye development. For example, mutations in the Drosophila homolog of Saccharomyces cerevisiae cell cycle regulator cdc37 were identified as dominant enhancers of weak sevenless mutants. Also, mutations in the peanut gene, which encodes a protein similar to yeast bud neck filaments and functions in cytokinesis, were identified as dominant enhancers of the phenotype of weak seven in absentia mutants; seven in absentia is a nuclear protein that functions downstream of Sevenless to target a transcriptional repressor for proteolysis. In these cases, the genetic interactions may also be an indirect consequence of cells that are receiving an inductive signal remaining longer in a particular phase of the cycle than they would normally. The results presented here underscore the connection between the cell cycle and signaling pathways that govern cell determination. A less likely possibility is that the Cdc37, Peanut, and/or the 60-kD subunit of DNA primase are bifunctional proteins, with distinct functions in the cell cycle and in signal transduction (Chen, 2000b).

The covalent attachment of ubiquitin to cellular proteins is a powerful mechanism for controlling protein activity and localization. Ubiquitination is a reversible modification promoted by ubiquitin ligases and antagonized by deubiquitinating proteases. Ubiquitin-dependent mechanisms regulate many important processes including cell-cycle progression, apoptosis and transcriptional regulation. Ubiquitin-dependent mechanisms regulate synaptic development at the Drosophila neuromuscular junction (NMJ). Neuronal overexpression of the deubiquitinating protease Fat facets leads to a profound disruption of synaptic growth control; there is a large increase in the number of synaptic boutons, an elaboration of the synaptic branching pattern, and a disruption of synaptic function. Antagonizing the ubiquitination pathway in neurons by expression of the yeast deubiquitinating protease UBP2 also produces synaptic overgrowth and dysfunction. Genetic interactions between fat facets and highwire, a negative regulator of synaptic growth that has structural homology to a family of ubiquitin ligases, suggest that synaptic development may be controlled by the balance between positive and negative regulators of ubiquitination (Diantonio, 2001).

Synaptic morphology is dynamic; once formed, synapses expand, retract, and remodel throughout life. This plasticity underlies the refinement of neuronal circuits during development and may be critical for plasticity in the adult brain. To identify molecular mechanisms regulating the morphological growth of synapses, a genetic screen was performed for molecules whose neuronal overexpression disrupts synaptic growth control at the Drosophila NMJ. A collection of flies capable of the targeted overexpression of endogenous Drosophila genes was screened and two lines, EP(3)381 and EP(3)3520, were identified whose overexpression in the nervous system leads to synaptic overgrowth. Both EP(3)381 and EP(3)3520 overexpress fat facets (faf), a deubiquitinating protease. Endogenous faf transcript is strongly and widely expressed in the developing central nervous system (CNS), demonstrating that neuronal expression of faf from the EP lines produces overexpression, not misexpression, of the transcript (Diantonio, 2001).

Anatomical analysis at the NMJ reveals that neuronal expression from both EP(3)381 and EP(3)3520 leads to an increase both in the number of synaptic boutons and in the synaptic span (the extent of the muscle covered by the synapse). This increase is not seen in flies that do not overexpress faf v or that overexpress a non-functional faf gene (elav-Gal4 crossed to EP(3)381faf-). Neuronal overexpression of faf also causes an increase in the number of synaptic branches as quantified by the number of branch points. Postsynaptic expression of faf does not affect synaptic morphology (Diantonio, 2001).

To assess the physiological consequence of neuronal faf overexpression, both spontaneous and evoked neurotransmitter release were analysed at muscle 6 of third instar larvae. Despite the greatly expanded size of the NMJ with faf overexpression, the amplitude of evoked excitatory junctional potentials (EJPs) is markedly reduced. Given that the amplitude of miniature EJPs (mEJPs) shows only a small, albeit significant, reduction, a large decrease was measured in quantal content (the number of vesicles released by the nerve) as measured by dividing the EJP amplitude by the mEJP amplitude. Neuronal overexpression of faf also leads to a reduction in the frequency of spontaneous mEJPs. The reduction in both quantal content and mEJP frequency indicates a presynaptic defect in neurotransmitter release. Other presynaptic mutants with even greater reductions in quantal content do not show a structural overgrowth, thus the anatomical phenotype described above is probably a direct consequence of faf overexpression and not a secondary consequence of this physiological phenotype (Diantonio, 2001).

faf antagonizes ubiquitin-dependent mechanisms by deubiquitinating target proteins. Alterations in synaptic structure and function owing to overexpression of faf suggest that ubiquitin-dependent mechanisms normally act to regulate the developing synapse. However, faf, a characterized deubiquitinating protease, might have other functions. To investigate the role of deubiquitination in the regulation of synaptic development, transgenic flies were generated capable of the targeted overexpression of the yeast deubiquitinating protease UBP2. This enzyme antagonizes ubiquitin-dependent mechanisms in yeast and has overlapping substrate specificity with FAF+9. Overexpression of yeast UBP2 in the nervous system of Drosophila leads to marked synaptic overgrowth and a severe reduction in presynaptic transmitter release. This phenotype is very similar to that seen with faf overexpression. Hence, antagonizing ubiquitin-dependent mechanisms by overexpression of deubiquitinating proteases markedly affects synaptic development (Diantonio, 2001).

To identify molecular pathways regulated by faf, a genetic interaction screen was performed to identify genes that enhance the faf overexpression phenotype. The X chromosome was screened for viable mutations that are lethal in combination with neuronal overexpession of faf. 7,000 chromosomes were screened and 15 lethal enhancers, 12 of which form one complementation group, were identifed. These 12 mutants are alleles of the highwire (hiw) gene and share the synaptic overgrowth phenotype described for loss-of-function hiw mutants. The hiw loss-of-function phenotypes are very similar to the faf gain-of-function phenotypes described here, with a large increase in the number of synaptic boutons, branches, and synaptic span, a small decrease in quantal size, and a large decrease in quantal content. The hiw transcript encodes a greater than 5,000 amino-acid protein that is localized to synapses and that contains a RING-H2 finger, a domain recently identified in a large family of E3 ubiquitin ligases. Hence, a potential synaptic E3 ligase was identified as a lethal enhancer of neuronal overexpression of faf. This genetic interaction provides further evidence that ubiquitination may have a central role in regulating synaptic growth and function (Diantonio, 2001).

To further investigate the genetic relationship between hiw and faf, double mutants were generated between loss-of-function alleles of hiw and faf. faf loss-of-function mutants have phenotypes in the developing eye and female germ line. In faf mutants, no defects were found in either synaptic morphology or function, possibly due to genetic redundancy between faf and one of the 17 other putative deubiquitinating proteases in the Drosophila genome. Although no phenotype was found for faf mutants in otherwise wild-type flies, in the sensitized background of a hiw mutant a requirement for faf is found. Two different loss-of-function alleles of faf both suppress the physiological phenotype of hiw, leading to a more than doubling of both quantal content and mEJP frequency. Hence endogenous faf activity acts to inhibit neurotransmitter release in a hiw background, much as increased faf activity inhibits neurotransmitter release in an otherwise wild-type background. Mutants of faf do not suppress the synaptic overgrowth seen in hiw, indicating that the physiological and morphological phenotypes in hiw are genetically separable. This suggests that either these phenotypes are mediated by different hiw substrates, or the anatomical phenotype is more sensitive to disruption of ubiquitin-dependent mechanisms. Finally, the inability to suppress the small quantal size defect suggests that this phenotype, seen in both hiw mutants and with overexpression of faf, may be a secondary consequence of synaptic overgrowth (Diantonio, 2001).

Decreases in postsynaptic activity induce a compensatory increase in presynaptic transmitter release, demonstrating that a homeostatic mechanism regulates synaptic strength during development. To assess the relationship between homeostatic and ubiquitin-dependent regulation, glutamate receptor function was disrupted in a hiw mutant. Postsynaptic expression of a dominant negative glutamate receptor (DgluRIIA-M/R) in a hiw mutant leads to an 18% decrease in quantal size. Since homeostatic compensation still occurs in a hiw mutant, it is suggested that homeostatic and ubiquitin-dependent regulation are mechanistically distinct (Diantonio, 2001).

The data presented here indicate that ubiquitin-dependent mechanisms regulate synaptic development at the Drosophila NMJ and suggest that a balance between positive and negative regulators of ubiquitination controls the structure and function of the synapse. Antagonizing ubiquitination by the neuronal overexpression of the deubiquitinating proteases faf or yeast UBP2 leads to synaptic overgrowth and defects in neurotransmitter release. This phenotype is very similar to the loss-of-function phenotype of hiw, a putative synaptic E3 ubiquitin ligase. Gain-of-function mutants of faf enhance hiw and loss-of-function alleles of faf suppress hiw. It is proposed that hiw-dependent ubiquitination controls the level or activity of critical regulatory molecules at the synapse, and that these molecules can be deubiquitinated by faf and other deubiquitinating proteases. Ubiquitinated proteins have been identified at mammalian synapses, and ubiquitin-processing enzymes can regulate long-term potentiation and facilitation, therefore control of ubiquitination by molecules such as HIW and FAF could be a widely used mechanism for regulating synaptic growth and function (Diantonio, 2001).

The Drosophila epsin 1 is required for ubiquitin-dependent synaptic growth and function but not for synaptic vesicle recycling

The ubiquitin-proteasome system plays an important role in synaptic development and function. However, many components of this system, and how they act to affect synapses, are still not well understood. This study used the Drosophila neuromuscular junction to study the in vivo function of Liquid facets (Lqf), a homolog of mammalian epsin 1. The data show that Lqf plays a novel role in synapse development and function. Contrary to prior models, Lqf is not required for clathrin-mediated endocytosis of synaptic vesicles. Lqf is required to maintain bouton size and shape and to sustain synapse growth by acting as a specific substrate of the deubiquitinating enzyme Fat facets. However, Lqf is not a substrate of the Highwire (Hiw) E3 ubiquitin ligase; neither is it required for synapse overgrowth in hiw mutants. Interestingly, Lqf converges on the Hiw pathway by negatively regulating transmitter release in the hiw mutant. These observations demonstrate that Lqf plays distinct roles in two ubiquitin pathways to regulate structural and functional plasticity of the synapse (Bao, 2008).

One important finding from this study is that Lqf does not play a detectable role in SV endocytosis. Multiple lines of evidence obtained from electrophysiological, ultrastructural and optical imaging studies support this conclusion. This is the first in vivo study of Lqf or epsin 1 on SV recycling. The finding is also clearly surprising given that epsin 1 has been highly implicated to play a key role in the initiation of clathrin-coated vesicle formation and endocytosis. Does the observation reflect the special property of the fly NMJ? Lqf lacking either the ENTH domain or the clathrin-interacting C-terminus has been shown to rescue the mutant phenotype in the developing eye. These rescue results are intriguing, but they do not readily support a specific role for Lqf in CME. In particular, there are no clear mechanisms on how these truncated fragments could fulfill Lqf's clathrin-dependent functions. Interestingly, RNA interference and small interfering RNA-induced knockdown of epsin 1 fails to block the internalization of EGF receptors in HeLa cells. There is also evidence that epsin 1 functions only in clathrin-independent endocytosis. Furthermore, Lqf has been shown to be required for endocytosis of select receptors but not of all receptors. More importantly, Lqf itself is not required for receptor-mediated endocytosis. Rather, Lqf appears to signal select ligands (such as Delta/Serrate/Lag2) for internalization or recycling. Hence, these studies lend strong support to observations that Lqf does not play a significant role in CME of SVs (Bao, 2008).

It should be noted that recent studies reveal that the epsin 1-interacting protein Eps15 is required for SV recycling in both C. elegans and Drosophila. However, Eps15 is required to maintain the level of endocytotic proteins in nerve terminals. Strikingly, key endocytotic proteins such as Dynamin and Dap160 are reduced in synaptic boutons by ∼90% and ∼80%, respectively, in eps15 mutants. These observations make it difficult, if not impossible, to assign a direct role for Eps15 in CME (Bao, 2008).

Synapse development is a highly regulated process involving a large number of molecules. The first suggestion that Lqf could have a potential role in synapse development came from studies of its deubiquitinating enzyme Faf. This notion was further supported by a direct biochemical demonstration that Lqf is a specific substrate of Faf. The current studies provide the first experimental test of this hypothesis by showing that Lqf acts downstream of Faf in promoting synaptic overgrowth. This effect on NMJ growth appears to be Faf dependent as lqf mutations alone do not dramatically affect bouton numbers. It is interesting to note that neuronal overexpression of Lqf promotes bouton budding but does not mimic the exuberant synaptic overgrowth induced by overexpression of Faf. Hence, it is suggested that Lqf is necessary but insufficient for synaptic overgrowth, raising the possibility that Lqf is not the only substrate of Faf in motoneurons (Bao, 2008).

Another important finding emerging from this study is that two distinct UPS pathways may be employed at the Drosophila larval NMJ to regulate synapse growth. The Hiw/RPM-1/Phr1 proteins have a conserved role in inhibiting presynaptic development in Drosophila, C. elegans and mammals. In C. elegans and Drosophila, the substrates of RPM-1/Hiw have been shown to be MAP kinases and MAPKKK. The current study indicates that Lqf is unlikely a substrate of Hiw in conditioning synaptic growth. In contrast, this study shows that the Faf pathway is a positive regulator of synaptic growth at the NMJ in which Lqf is an essential substrate. Hence, it is suggested that Hiw and Faf/Lqf are two distinct UPS pathways that regulate synapse development in Drosophila (Bao, 2008).

However, the relationship between the Faf and Hiw pathways in synapse development is rather complex. Intriguingly, the MAPKKK Wnd is required for synaptic overgrowth mediated by both Hiw and Faf pathways. One possibility is that Wnd acts downstream of Lqf to fulfill the function of both the Hiw and the Faf pathways. However, this idea is inconsistent with the observation that unlike lqf mutants, the wnd null mutant itself has no morphological or electrophysiological defect. More importantly, wnd mutations do not suppress the transmitter release defect seen in the hiw mutant, whereas the lqf mutant does. Alternatively, it is suggested that Hiw and Faf act through two parallel pathways and that the suppression of Faf-induced overgrowth by the wnd mutation may be mediated by Fos/Jun kinase signaling. Based on the observation that overexpression of Ubp2A increases neuronal Wnd levels, it is possible that Faf may also use Wnd as a substrate for synaptic overgrowth. However, this has yet to be tested experimentally (Bao, 2008).

Recent genetic studies have revealed an interesting feature of synapse growth and function that closely depends on protein turnover by specific UPS pathways. In Drosophila, faf or lqf mutations are capable of partially suppressing the defect in transmitter release in hiw mutants. This partial suppression is specific and should not be viewed simply as a reduction of transmitter release in faf or lqf mutant backgrounds by hiw mutations. If there were no partial suppression by faf or lqf mutations, the amplitude of EJPs would be similar to that in hiw single mutants. Because faf null mutations reduce Lqf levels, it is reasonable to suggest that Lqf acts downstream of Faf to inhibit synaptic transmission in hiw mutants. Unlike the functional interactions with hiw, however, faf or lqf mutations do not affect synaptic overgrowth in hiw mutants. Differing from lqf and faf mutations, wnd mutations fully suppress synaptic overgrowth but do not affect synaptic physiology in the hiw mutant. Hence, different ubiquitin pathways can specifically dissociate synapse growth from function (Bao, 2008).

The physiological stimuli involved in such selective modulation of synapse growth and function remain to be identified. Given the conserved role of the ubiquitin-proteasome system in synaptic plasticity across animal species, the findings reported in this study may have general neurobiological implications. In particular, it is noted that the Faf homolog in mouse, Usp9x or Fam is differentially expressed in different regions of the brain. Such spatial distribution patterns may provide a means for Usp9x to locally regulate synaptic function. Importantly, Usp9x is localized at synapses, where calcium influx rapidly regulates its enzymatic activity and deubiquitination of epsin 1. Hence, Faf and Lqf/epsin 1 are good candidate mediators of activity-dependent synaptic plasticity (Bao, 2008).


EVOLUTIONARY HOMOLOGS

A dominant insertional P-element mutation enhances position-effect variegation in Drosophila melanogaster. The mutation is homozygous, viable, and fertile and maps at 64E on the third chromosome. The corresponding gene was cloned by transposon tagging. Insertion of the transposon upstream of the open reading frame correlates with a strong reduction of transcript level. A transgene was constructed with the cDNA and found to have the opposite effect from that of the mutation, namely, the suppression of variegation. Sequencing of the cDNA reveals a large open reading frame encoding a putative ubiquitin-specific protease (Ubp). Ubiquitin marks various proteins, frequently for proteasome-dependent degradation. Ubps can cleave the ubiquitin part from these proteins. The deduced protein, termed D-Udp-64E, consists of 898 amino acids and shares a Cys and His domain structure with other ubiquitin C-terminal hydrolases. The transcript is expressed ubiquitously during embryonic development (Henchoz, 1996).

In eukaryotes, both natural and engineered ubiquitin (Ub) fusions, either to itself or to other proteins, are cleaved by processing proteases after the last amino acid residue of ubiquitin. YUH1 and UBP1, the genes for two ubiquitin-specific proteases of the yeast Saccharomyces cerevisiae, have been cloned previously and shown to encode nonhomologous proteins. Using an Escherichia coli-based genetic screen, two other yeast genes for ubiquitin-specific proteases have been isolated and named UBP2 and UBP3. Ubp2 (1,264 residues), Ubp3 (912 residues), and the previously cloned Ubp1 (809 residues) are largely dissimilar except for two short regions containing Cys and His which encompass their putative active sites. Neither of these proteases has sequence similarities to Yuh1. Both Ubp2 and the previously identified Ubp1 cleave in vitro at the C terminus of the ubiquitin moiety in natural and engineered fusions irrespective of their size, poly-Ub being the exception. However, both Ubp1 and Ubp2 are also capable of cleaving poly-Ub when coexpressed with it in E. coli, suggesting that such cleavage is largely cotranslational. Although inactive in E. coli extracts, Ubp3 is active with all of the tested ubiquitin fusions except poly-Ub when coexpressed with them in E. coli. Null yuh1 ubp1 ubp2 ubp3 quadruple mutants are viable and retain the ability to deubiquitinate ubiquitin fusions, indicating the presence of at least one more ubiquitin-specific processing protease in S. cerevisiae (Baker, 1992).

A necessary step in ubiquitin-dependent proteolysis is the addition of a polyubiquitin chain to the target protein. This ubiquitinated protein is degraded by a multisubunit complex known as the 26S proteasome. The polyubiquitin chain is probably not released until a late stage in the proteolysis by the proteasome. It is subsequently disassembled to yield functional ubiquitin monomers. A 93 kDa protein, isopeptidase T, has the properties expected for the enzymethatdisassembles these branched polyubiquitin chains. Protein and cDNA sequencing reveals that isopeptidase T is a member of the ubiquitin specific protease family (UBP). Isopeptidase T is a protein of 835 amino acids with conserved Cys and His boxes characteristic of UBPs. Isopeptidase T disassembles branched polyubiquitin chains (linked by the G76-K48 isopeptide bond) by a sequential exo mechanism, starting at the proximal end of the chain (the proximal ubiquitin contains a free carboxyl-terminus). Isopeptidase T prefers to disassemble chains in which there is an intact and unblocked RGG sequence at the C-terminus of the proximal subunit. Rates of disassembly are reduced when G76 of the proximal ubiquitin is modified, for example, by ligation to substrate protein, by esterification, by replacement of the proximal glycine with alanine (G76A), or by truncation. Linear proubiquitin provides only a poor substrate. Observed rates and specificity are consistent with isopeptidase T playing a major role in disassembly of polyubiquitin chains. The high discrimination against chains that are blocked or modified at the proximal end indicates that the enzyme acts after release of the chains from conjugated proteins or degradation intermediates. Thus, the proteolytic degradation signal is not disassembled by isopeptidase T before the ubiquitinated protein is degraded. These (and earlier) results suggest that UBP isozymes may exhibit significant substrate specificity, consistent with a role in the regulated catabolism of the polymeric ubiquitin, including the polyubiquitin protein degradation signal (Wilkinson, 1995).

Using a gene trap approach in embryonic stem cells, a murine gene has been isolated with extensive sequence similarity to the Drosophila faf gene, and been termed Fam (fat facets in mouse). The putative mouse protein shows colinearity and a high degree of sequence identity to the Drosophila protein over almost its entire length of 2554 amino acids. The two enzymatic sites characteristic of ubiquitin-specific proteases are very highly conserved between mice and Drosophila and this conservation extends to yeast. Fam is expressed in a complex pattern during postimplantation development. In situ hybridization detects Fam transcripts in the rapidly expanding cell populations of gastrulating and neurulating embryos, in post-mitotic cells of the CNS as well as in the apoptotic regions between the digits, indicating that it is not associated with a single developmental or cellular event. The strong sequence similarity to faf and the developmentally regulated expression pattern suggest that Fam and the ubiquitin pathway may play a role in determining cell fate in mammals, as has been established for Drosophila (Wood, 1997).

EST 221 derived from human adult testis detects homology to the Drosophila fat facets gene (fat) and has related sequences on both the X and Y chromosomes mapping to Xp11.4 and Yq11.2, respectively. These two loci have been termed DFFRX and DFFRY for Drosophila fat facets related X and Y. The major transcript detected by EST 221 is about 8 kb in size and is expressed widely in a range of 16 human adult tissues. RT-PCR analysis of 13 different human embryonic tissues, with primers specific for the X and Y sequences demonstrates that both loci are expressed in developing tissues. Quantitative RT-PCR analysis of lymphoblastoid cell lines carrying different numbers of X chromosomes reveals that the X-linked gene escapes X-inactivation. The amino acid sequence (2547 residues) for the complete open reading frame of the X gene has 44% identity and 88% similarity to the Drosophila sequence and contains the conserved Cys and His domains characteristic of deubiquitinating enzymes, suggesting its biochemical function may be the hydrolysis of ubiquitin from protein-ubiquitin conjugates. The requirement of faf for normal oocyte development in Drosophila combined with the map location and escape from X-inactivation of DFFRX raises the possibility that the human homolog plays a role in the defects of oocyte proliferation and subsequent gonadal degeneration found in Turner's syndrome (Jones, 1996).

Human DFFRY (the Y-linked homologue of the DFFRX Drosophila fat-facets related X gene) maps to proximal Yq11.2 within the interval defining the AZFa spermatogenic phenotype. The complete coding region of DFFRY has been sequenced and shows 89% identity to the X-linked gene at the nucleotide level. In common with DFFRX , the potential amino acid sequence contains the conserved Cys and His domains characteristic of ubiquitin C-terminal hydrolases. The human DFFRY mRNA is expressed in a wide range of adult and embryonic tissues, including testis, whereas the homologous mouse Dffry gene is expressed specifically in the testis. Analysis of three azoospermic male patients has shown that DFFRY is deleted from the Y chromosome in these individuals. Two patients have a testicular phenotype, which resembles Sertoli cell-only syndrome, and the third shows diminished spermatogenesis. In all three patients, the deletions extend from close to the 3' end into the gene, removing the entire coding sequence of DFFRY . The mouse Dffry gene maps to the Sxrb deletion interval on the short arm of the mouse Y chromosome; its expression in mouse testis can first be detected between 7.5 and 10. 5 days after birth when type A and B spermatogonia and pre-leptotene and leptotene spermatocytes are present (Brown, 1998).

A cDNA encoding a new ubiquitin-specific protease, UBP41, in chick skeletal muscle was cloned using an Escherichia coli-based in vivo screening method. Nucleotide sequence analysis of the cDNA containing an open reading frame of 1,071 base pairs reveals that the protease consists of 357 residues with a calculated molecular mass of 40,847 Da, and that it is related to members of the UBP family containing highly conserved Cys and His domains. Chick UBP41 was expressed in E. coli and purified from the cells to apparent homogeneity. The purified enzyme behaves as an approximately 43-kDa protein under both denaturing and nondenaturing conditions, suggesting that it consists of a single polypeptide chain. Like other deubiquitinating enzymes, it was sensitive to inhibition by ubiquitin-aldehyde and sulfhydryl blocking agents, such as N-ethylmaleimide. The UBP41 protease cleaves at the C terminus of the ubiquitin moiety; thus, the protease is active against ubiquitin-beta-galactosidase as well as ubiquitin C-terminal extension protein of 80 amino acids. UBP41 also releases free ubiquitin from poly-His-tagged di-ubiquitin. It converts poly-ubiquitinated lysozyme conjugates to mono-ubiquitinated forms of about 24 kDa, although the latter molecules are not further degraded to free ubiquitin and lysozyme. These results suggest that UBP41 may play an important role in the recycling of ubiquitin by hydrolysis of branched poly-ubiquitin chains generated by the action of 26 S proteasome on poly-ubiquitinated protein substrates, as well as in the production of free ubiquitin from linear poly-ubiquitin chains and of certain ribosomal proteins from ubiquitin fusion proteins (Baek, 1997).

Cytokines regulate cell growth by inducing the expression of specific target genes. Using the differential display method, cytokine-inducible immediate early gene, DUB-1 (for deubiquitinating enzyme), has been cloned. DUB-1 is related to members of the UBP superfamily of deubiquitinating enzymes that includes the oncoprotein Tre-2. A glutathione S-transferase-DUB-1 fusion protein cleaves ubiquitin from a ubiquitin-beta-galactosidase protein. When a conserved cysteine residue of DUB-1, required for ubiquitin-specific thiol protease activity, is mutated to serine (C60S), deubiquitinating activity is abolished. Continuous expression of DUB-1 from a steroid-inducible promoter induces growth arrest in the G1 phase of the cell cycle. Cells arrested by DUB-1 expression remain viable and resume proliferation upon steroid withdrawal. These results suggest that DUB-1 regulates cellular growth by modulating either the ubiquitin-dependent proteolysis or the ubiquitination state of an unknown growth regulatory factor(s) (Zhu, 1996).

The Ras target AF-6 has been shown to serve as one of the peripheral components of cell-cell adhesions, and is thought to participate in cell-cell adhesion regulation downstream of Ras. An AF-6-interacting protein with a molecular mass of approximately 220 kD (p220) waa purified to investigate the function of AF-6 at cell-cell adhesions. The peptide sequences of p220 are identical to the amino acid sequences of mouse Fam. Fam is homologous to a deubiquitinating enzyme in Drosophila, the product of the fat facets gene. Recent genetic analyses indicate that the deubiquitinating activity of the fat facets product plays a critical role in controlling the cell fate. Fam accumulates at the cell-cell contact sites of MDCKII cells, but not at free ends of plasma membranes. Fam is partially colocalized with AF-6 and interacts with AF-6 in vivo and in vitro. AF-6 is ubiquitinated in intact cells, and Fam prevents the ubiquitination of AF-6 (Taya, 1998).

In the ubiquitin-proteasome pathway, the ubiquitinated substrates either undergo degradation by the proteasome or stabilization through the action of the deubiquitinating enzyme. The deubiquitinating enzyme Fam is colocalizes with AF-6, one of the effectors of the Ras small GTPase, at cell-cell contact sites in epithelial cells and interacts with AF-6 in vivo and in vitro. Fam has deubiquitinating activity in vitro and prevents the ubiquitination of AF-6 in intact cells. The degradation of beta-catenin, which accumulates at the cell-cell contact sites as a cadherin/catenin complex, is thought to be regulated by the ubiquitin-proteasome pathway. These observations prompted an examination of the possible Fam regulation of the stabilization of beta-catenin. It was found that Fam interacts with beta-catenin both in vivo and in vitro. The Fam-binding site of beta-catenin maps to the region close to the APC or Axin-binding site of beta-catenin. Over-expression of Fam in mouse L cells results in an elevation of beta-catenin levels and in an elongation of the half-life of beta-catenin. In these L cells, Fam is colocalized with beta-catenin at the dot-like structures in the cytoplasm. These results indicate that Fam interacts with and stabilizes beta-catenin in vivo, presumably through the deubiquitination of beta-catenin (Taya, 1999).

The Drosophila fat facets and canoe genes regulate non-neural cell fate decisions during ommatidium formation. The FAM (Fat facets in mouse) de-ubiquitinating enzyme regulates the function of AF-6 (mammalian Canoe homolog) in the MDCK epithelial cell line. The expression of the FAM and AF-6 proteins overlaps extensively in the mouse eye from embryogenesis to maturity, especially in the non-neural epithelia including the retinal pigment epithelium, subcapsular epithelium of the lens and corneal epithelium. Expression is not limited to the epithelia however, because FAM and AF-6 also co-localize during lens fiber development as well as in sub-populations of the neural retina (Kanai-Azuma, 2000).

Fat facets is a Drosophila deubiquitinating enzyme required for eye development and early embryogenesis. Genetic evidence suggests that Fat facets deubiquitinates and thereby prevents the proteasomal degradation of specific substrates. The Drosophila Liquid facets protein is implicated as the critical substrate of Fat facets in the eye. A mouse homolog of Fat facets, called Fam, has been identified. The results of biochemical experiments implicate two different proteins, Af-6 and beta-catenin, as substrates for Fam. The functional relationship between Fat facets and Fam has been explored. Fam can substitute for Fat facets in all of its essential functions in Drosophila. In addition, the hypothesis was tested that Canoe and Armadillo, the Drosophila homologs of Af-6 and beta-catenin, respectively, are important substrates for Fat facets in the Drosophila eye. No genetic evidence has been found to support a role for either Canoe or Armadillo in the essential Fat facets pathways in Drosophila eye development. The significance of these results is discussed in light of the biochemical experiments that suggest that Af-6 and beta-catenin are substrates of Fam (Chen, 2000a).

While Faf is similar to other Ubps mainly in the small Cys and His domains that define the catalytic region, Faf and Fam share approximately 50% identity and 70% similarity in amino acid sequence along the entire length of the two proteins. The high degree of primary sequence conservation in the two proteins suggests that Faf and Fam functions might be conserved. Since Fam-expressing transgenes can substitute for the endogenous faf gene in the eye and the ovary, the only two tissues in which faf function is critical in Drosophila, it is concluded that the Faf and Fam protein functions have indeed been conserved. Although yeast Ubp2 and Ubp3 bear minimal primary amino acid sequence similarity with Faf outside the catalytic domain, the Ubp2 and Ubp3 genes also can substitute to some extent for faf in the Drosophila eye. These observations do not weaken the present argument that Faf and Fam are functionally homologous for two reasons: (1) neither Ubp2 nor Ubp3 transgenes complement the faf mutant eye phenotype nearly as well as do faf or Fam transgenes; (2) Ubp2 expression in the Drosophila eye results in a mutant phenotype; this was never observed with any faf or Fam transgenes. Thus, faf and Fam behave identically when expressed in the Drosophila eye, while Ubp2 and Ubp3 behave differently from each other and from faf and Fam (Chen, 2000a).

The genetic interaction experiments presented in this study suggest that Cno and Arm, two cell-cell junction proteins thought to be important for integrating signaling with cell adhesion, and whose vertebrate homologs (Af-6 and beta-Catenin) are implicated as substrates of Fam, are not likely to be important substrates of Faf in the eye. In addition, the results of previous experiments implicate the Lqf protein, a Drosophila homolog of the vertebrate endocytosis complex component epsin, as the one critical substrate of Faf in the eye (Cadavid, 2000). If beta-Catenin and Af-6 are in vivo substrates of Fam, how can the failure to detect genetic interactions between arm or cno and faf be explained? One possibility is that Faf could regulate Arm and/or Cno in the eye, but that this role for Faf is non-essential in Drosophila. In fact, faf has a normally redundant role in Ras1 signal transduction in the eye. This idea was tested genetically for cno in the eye and no evidence of a role for faf in a cno pathway was revealed (Chen, 2000a).

A second possibility is that Arm and/or Cno is the essential substrate of Faf in the ovary. Embryos from females homozygous for faf mutations die very early during development; many rounds of nuclear division take place and some abnormally clustered pole cells (primordial germ cells) form but somatic cellularization never occurs. It is unknown whether cno or lqf are expressed or required in the ovary. The arm gene, however, is normally expressed in the female germline where it is required for a multitude of cell adhesion functions. The failure of cellularization in faf mutant embryos and the improper clustering of the pole cells suggest the possibility of a role for Arm in this pathway. Further experiments are required to determine if any of the three proteins are critical substrates for Faf in the Drosophila ovary (Chen, 2000a).

If Arm and Cno are true substrates for Fam in the mouse, it is unknown whether their interaction is essential for mouse development. The Fam gene is expressed widely in the mouse but Fam function may not be essential in all of these tissues. Indeed, Drosophila faf is expressed in many fly tissues (male sex organs, gut, fat body) where it is non-essential. A third possibility is that Arm/beta-catenin and Cno/Af-6 are not substrates of Faf/Fam in vivo. The catalytic domain of Fam alone was shown to bind to Af-6. Since the catalytic domains of Ubps are conserved, it is possible that, in vivo, another Ubp normally binds to Cno and/or Af-6. It has not been reported whether other Ubp catalytic domains can bind Cno and/or Af-6. Recently, it has been shown that the ENTH (epsin N-terminal homology) domain of epsin/Lqf bears structural similarity with the Armadillo repeats present in Arm/beta-catenin. Since beta-catenin binds Fam through these repeats (Taya, 1999), it is possible that Fam/beta-catenin binding is an artifact of the similarity between Lqf and beta-catenin. Alternatively, the structural similarity between the ENTH domain and the Armadillo repeats may indicate that the Faf/Fam substrates constitute a family of proteins containing this structural domain. The region of vertebrate Af-6 that binds Fam is not conserved at the primary amino acid level with its Drosophila homolog, Cno. Perhaps there is some underlying structural similarity between Af-6 and Cno in this region, which could also be shared by the ENTH domain and Arm repeats, and which is not apparent at the primary amino acid sequence level. These speculations highlight appealing avenues for further experiments (Chen, 2000a).

During the Drosophila oogenic processes, Fat facets (Faf), an ubiquitin-specific protease essential for normal development of oocyte and eye, becomes localized at the posterior pole and is incorporated into the pole cells. This is dependent on Oskar, a key factor for pole cell determination, and suggests a role for Faf in germ cell differentiation and development. This study shows that Usp9x, an X-linked ortholog of Faf, is predominantly expressed in both germ cell and supporting cell lineages during mouse gonadal development in stage- and sex-dependent manners. Usp9x was first detected in PGCs at 10.5 days post coitum (dpc), and thereafter its expression both at mRNA and protein levels is enhanced in PGCs of both sexes at 11.5-13.5 dpc. In testis, Usp9x expression rapidly decreases to an undetectable level by 15.5 dpc and after birth to adult, no expression is found in any spermatogenic cells, except for weak expression in Sertoli cells. In the ovary, Usp9x expression in embryonic oocytes is also reduced at the newborn stage, its expression reappears in oocytes at the secondary follicle stage, and its products are highly accumulated in the cytoplasm of Graaffian follicles in adults. Although follicular epithelial cells also express Usp9x at a moderate level during postnatal development, its expression is downregulated from early secondary follicle stage. Thus, the present study is not only the first to demonstrate a conserved expression of fat facets in PGCs between mouse and fly, but also sex- and stage-dependent changes of a specific component of the deubiquitylation system during mammalian gonadal development (Noma, 2002).


REFERENCES

Search PubMed for articles about Drosophila fat facets

Baek, S. H., et al. (1997). Molecular cloning of a novel ubiquitin-specific protease, UBP41, with isopeptidase activity in chick skeletal muscle. J. Biol. Chem. 272(41): 25560-25565. PubMed Citation: 9325273

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Bao, H., Reist, N. E. and Zhang, B. (2008). The Drosophila epsin 1 is required for ubiquitin-dependent synaptic growth and function but not for synaptic vesicle recycling. Traffic 9(12): 2190-205. PubMed Citation: 18796008

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Brown, G. M., et al. (1998). Characterisation of the coding sequence and fine mapping of the human DFFRY gene and comparative expression analysis and mapping to the sxrb interval of the mouse Y chromosome of the dffry gene. Hum. Mol. Genet. 7(1): 97-107. PubMed Citation: 9384609

Cadavid, A. L. M., Ginzel, A. and Fischer, J. A. (2000). The function of the Drosophila Fat facets deubiquitinating enzyme in limiting photoreceptor cell number is intimately associated with endocytosis. Development 127: 1727-1736. 10725248

Chen, H., et al. (1998). Epsin is an EH-domain-binding protein implicated in clathrin-mediated endocytosis. Nature 394: 793-797. 9723620

Chen, X., et al. (2000a). On the conservation of function of the Drosophila Fat facets deubiquitinating enzyme and Fam, its mouse homolog. Dev. Genes Evol. 210: 603-610. 21025767

Chen, X., Li, Q. and Fischer, J. A. (2000b). Genetic analysis of the Drosophila DNAprim gene. The function of the 60-kd primase subunit of Dna polymerase opposes the fat facets signaling pathway in the developing eye. Genetics 156(4): 1787-95. 11102374

Chen, X., Zhang, B. and Fischer, J. A. (2002). A specific protein substrate for a deubiquitinating enzyme: Liquid facets is the substrate of Fat facets. Genes Dev. 16: 289-294. 11825870

Diantonio, A., et al. (2001). Ubiquitination-dependent mechanisms regulate synaptic growth and function. Nature 412: 449-452. 11473321

Fischer-Vize, J. A., Rubin, G. M. and Lehmann, R. (1992). The fat facets gene is required for Drosophila eye and embryo development. Development 116: 985-1000

Fischer, J. A.,Leavell, S. K. and Li, Q. (1997). Mutagenesis screens for interacting genes reveal three roles for fat facets during Drosophila eye development. Dev. Genet. 21(2): 167-174

Henchoz, S., et al. (1996). The dose of a putative ubiquitin-specific protease affects position-effect variegation in Drosophila melanogaster. Mol. Cell. Biol. 16(10): 5717-5725

Hochstrasser, M. (1995). Ubiquitin, proteasomes, and the regulation of intracellular protein degradation. Curr. Opin. Cell Biol. 7: 215-223.

Horiuchi, D., Collins, C. A., Bhat, P., Barkus, R. V., Diantonio, A. and Saxton, W. M. (2007). Control of a kinesin-cargo linkage mechanism by JNK pathway kinases. Curr. Biol. 17(15): 1313-7. Medline abstract: 17658258

Huang, Y., Baker, R. T. and Fischer-Vize, J. A. (1995). Control of cell fate by a deubiquitinating enzyme encoded by the fat facets gene. Science 270: 1828-1831

Huang, Y. and Fischer-Vize, J. A. (1996). Undifferentiated cells in the developing Drosophila eye influence facet assembly and require the Fat facets ubiquitin-specific protease. Development 122: 3207-3216

Isaksson, A., et al. (1997). The deubiquitination enzyme fat facets negatively regulates RTK/Ras/MAPK signalling during Drosophila eye development. Mech. Dev. 68(1-2): 59-67

Itoh, T., et al. (2001). Role of the ENTH domain in phosphatidylinositol-4,5-bisphosphate binding and endocytosis. Science 291: 1047-1051. 11161217

Jones, M. H., et al. (1996). The Drosophila developmental gene fat facets has a human homologue in Xp11.4 which escapes X-inactivation and has related sequences on Yq11.2. Hum. Mol. Genet. 5(11): 1695-1701

Kanai-Azuma, M., et al. (2000). Co-localization of FAM and AF-6, the mammalian homologues of Drosophila faf and canoe, in mouse eye development. Mech. Dev. 91(1-2): 383-6.

Li, Q., et al. (1997). Genetic interactions with Rap1 and Ras1 reveal a second function for the fat facets deubiquitinating enzyme in Drosophila eye development. Proc. Natl. Acad. Sci. 94(23): 12515-12520

Liu, N., Dansereau, D. A, and Lasko, P. (2003). Fat facets interacts with Vasa in the Drosophila pole plasm and protects it from degradation. Curr. Biol. 13: 1905-1909. 14588248

Noma, T., et al. (2002). Stage- and sex-dependent expressions of Usp9x, an X-linked mouse ortholog of Drosophila Fat facets, during gonadal development and oogenesis in mice. Gene Expr. Patterns 2: 87-91. 12617843

Overstreet, E., Fitch, E. and Fischer, J. A. (2004). Fat facets and Liquid facets promote Delta endocytosis and Delta signaling in the signaling cells. Development. 131(21): 5355-66. 15469967

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Taya, S., et al. (1999) The deubiquitinating enzyme Fam interacts with and stabilizes beta-catenin. Genes Cells 4: 757-767.

Treier, M., Bohmann, D. and Mlodzik, M. (1995). JUN cooperates with the ETS domain protein pointed to induce photoreceptor R7 fate in the Drosophila eye. Cell 83: 753-760

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Wood, S. A., et al. (1997). Cloning and expression analysis of a novel mouse gene with sequence similarity to the Drosophila fat facets gene. Mech. Dev. 63 (1): 29-38.

Wu, Z., et al. (1999). Genetic analysis of the role of the Drosophila fat facets gene in the ubiquitin pathway. Dev. Genet. 25(4): 312-20.

Zhu, Y., et al. (1996). DUB-1, a deubiquitinating enzyme with growth-suppressing activity. Proc. Natl. Acad. Sci. 93(8): 3275-3279


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date revised: 15 October 2011

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