UCLA

The goal of this dissertation is to better understand the endosomal protein trafficking machinery; focusing on the role of the biogenesis of lysosome-related organelles complex-1 (BLOC-1), Adaptor Protein-3 (AP-3), and Rabaptin-5-associated exchange factor for Rab5 (Rabex-5). BLOC-1 is a stable protein complex implicated in protein trafficking between endosomes and lysosome-related organelles (LRO). Mutations in three subunits of BLOC-1 cause Hermansky-Pudlak syndrome (HPS) types 7, 8 and 9, and two of its subunits have been tentatively associated to schizophrenia. A data-mining approach was developed to prioritize over 100 candidate-binding partners for fly and human BLOC-1. The top candidate in the ranking was the Rab GTPase Rab11. Experiments done in Drosophila melanogaster revealed a synthetic lethal genetic interaction between Rab11 and Rab32/38; the later encoded by the gene lightoid. AP-3 is a stable heterotretameric complex also involved in trafficking between endosomes and LROs. Mutations in one subunit of AP-3 results in HPS type 2. Homologues of AP-3 genes in Drosophila melanogaster are involved in pigment granule biogenesis. A large-scale screening was conducted to identify genetic modifier of AP-3 function in the fly eye. Deletions in two regions in chromosome 2 and two regions in chromosome 3 modified the AP-3 mutant g2 eye pigment color in heterozygous form. Further experiments demonstrated that Gap69C and Atg2 are genetic modifiers of AP-3. Rabex-5 is a guanine nucleotide exchange factor of Rab5, a Rab GTPase important in the early endosome trafficking. To understand Rabex-5 physiological function a reverse genetic approach was undertaken to generate a mutant form of the Rabex-5 encoding gene, Rbx5. Homozygous loss-of-function (Rbx5^ex1) mutant flies displayed a "giant larvae" phenotype and did not survive to adulthood. Mutant larval tissues including the brain and wing imaginal discs displayed growth abnormalities. Rescue experiments suggested that Rbx5^ex1 adult lethality was due to affecting Rab5 function.