Gene name - fat facets
Cytological map position - 100E1--100E3
Function - ubiquitin-specific protease
Symbol - faf
FlyBase ID: FBgn0005632
Genetic map position - 3-
Classification - conserved Cys and His domains ubiquitin-specific protease
Cellular location - cytoplasmic
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).
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).
cDNAs represent two transcripts of at least 8500 and 8900 nucleotides that differ in their 3' ends (Fischer-Vize, 1992).
Exons - 16
Apart from their large size, the Faf proteins have few remarkable features. At amino acids 262-290, there is a potential leucine zipper structural domain, shown to be a dimerization site within transcription factors. In addition there is a likely PEST sequence, often found in rapidly degraded proteins (Fischer-Vize, 1992). The isolation of several yeast ubiquitin-specific proteases (Ubps) on the basis of functional assays has revealed that these enzymes are similar to each other primarily in two small regions, the so-called Cys and His domains, centered on a single cysteine (Cys) and two histidine (His) residues thought to be the active site of the protease. Similar Cys and His domains identify the Faf protein as a potential Ubp (Huang, 1995).
date revised: 16 February 98
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