Structure/function analysis of the Drosophila fat facets deubiquitinating enzyme and analysis of the fat-dependent signaling pathway

I am interested in a cell communication pathway that determines cell fate in Drosophila eye. This pathway, in particular, is mediated by a deubiquitinating enzyme (DUB) called Fat facets (Faf). Further studies revealed another key component, Liquid facets (Lqf), which is a Drosophila homolog of epsin. Epsin in vertebrates is known to function in clathrin-mediated endocytosis. Thus, Ub pathway and endocytosis are both essential for cell patterning in Drosophila eye development. My goal is to understand the role of Faf in cell fate determination in the eye. First, I performed a structure/function analysis of Faf protein in three different ways: (1) Deleted forms of the faf gene were generated in vitro and introduced into the fly genome to express partial Faf proteins. Each of the 6 deleted Faf proteins was tested for its ability to complement the faf mutant eye phenotype in vivo; (2) The DNA sequence aberrations in 14 mutant faf alleles were determined and these data defined specific domains and amino acids required for the normal function of Faf; (3) The homolog of faf in mouse, Fam, was tested for its activities in Drosophila. I found that Fam is able to substitute faf in all of its essential functions in vivo, indicating that they are indeed functional homologs. In addition, all Drosophila UBPs were retrieved from Genome Project and aligned for any conserved functional motifs (this part of work is done by J. Fischer). DUBs are a large family of proteins about which little is known. This part of my work contributes to a better understanding of DUB function. The second part of my thesis is to analyze the faf-dependent signaling pathway. (1) The lqf mutation was identified in an enhancer screen for faf mutations. To identify more components of this pathway, a former graduate student, Qinghong Li, performed a screen for suppressors of the faf mutant eye phenotype. Two suppressors were found to have essential functions by themselves. One mutation is in UbcD1 which encodes an E2. I analyzed the second suppressor and discovered that it is allelic to DNAprim, which encodes the 60-kD subunit of DNA polymerase. I think that the interaction between DNAprim and faf is likely to be indirect. Overall, this part of my work contributes to better understanding the faf-dependent pathway in eye development; (2) Genetic experiments performed by a former graduate student, Angelica Cadavid, indicated Lqf is a candidate substrate of Faf in the eye. But there is no biochemical evidence so the conclusion is highly speculative. In collaboration with Dr. Bing Zhang, we generated antibodies to Lqf, and I used it in a variety of biochemistry experiments. The data are consistent with our genetic results. For the first time, a DUB is demonstrated to regulate the ubiquitination state of a specific substrate and this event is critical to a cell communication pathway in development.