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).

GENE STRUCTURE

cDNAs represent two transcripts of at least 8500 and 8900 nucleotides that differ in their 3' ends (Fischer-Vize, 1992).

Exons - 16

PROTEIN STRUCTURE

Amino Acids - 2691

Structural Domains

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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