THESIS
2013
xv, 115 p. : ill. (some col.) ; 30 cm
Abstract
The transport protein particle (TRAPP) is a multi-subunit tethering complex that provides specificity to the membrane fusion process by bridging vesicles to target membranes. TRAPP complexes exist in three forms and each of which is responsible for different trafficking events. Trs20p is a subunit common to all TRAPP complexes and is encoded by an essential gene. Trs20p’s evolutionary conserved aspartate at position 46 (D46) has been predicted to be critical for TRAPP function although the role of this residue has remained elusive. Surprisingly, my study revealed that amino acid substitutions at D46 resulted in modest defects in endosome to Golgi traffic, rather that deficiencies in endoplasmic reticulum (ER) to Golgi transport. The rather modest phenotype attributable to the trs20 muta...[
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The transport protein particle (TRAPP) is a multi-subunit tethering complex that provides specificity to the membrane fusion process by bridging vesicles to target membranes. TRAPP complexes exist in three forms and each of which is responsible for different trafficking events. Trs20p is a subunit common to all TRAPP complexes and is encoded by an essential gene. Trs20p’s evolutionary conserved aspartate at position 46 (D46) has been predicted to be critical for TRAPP function although the role of this residue has remained elusive. Surprisingly, my study revealed that amino acid substitutions at D46 resulted in modest defects in endosome to Golgi traffic, rather that deficiencies in endoplasmic reticulum (ER) to Golgi transport. The rather modest phenotype attributable to the trs20 mutation prompted me to search for homologues of Trs20p in the yeast proteome.
These studies resulted in the identification of Tca17p. By generating double mutants of tca17 and trs20, I showed that the phenotypes brought by trs20 mutants were indeed masked by TCA17. My findings suggested that Tca17p worked together with Trs20p in TRAPP complexes, and consistent with this reasoning I showed that trs20 mutants disrupted Tca17p’s associating with TRAPP complexes. In addition, my study also uncovered the existence of parallel pathways in endosome to Golgi transport mediated by the TRAPP II, COG and GARP complexes.
My yeast two-hybrid experiments revealed that both Tca17p and Trs20p bound two TRAPP II-specific subunits – Trs130p and Trs120p, respectively. Substitutions to Tca17p’s and Trs20p’s aspartate residue disrupted binding to these TRAPP II subunits thereby revealing the role of these residues in TRAPP complexes. In addition to its crucial role in endosome to Golgi transport, my study also revealed that substitutions to aspartate 46 of Trs20p also resulted in deficiencies in autophagy – as judged the processing defects in the cytoplasm-to-vacuole substrate amino peptidase one (API).
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