Membrane trafficking and quality control of the plasma membrane ATPase in Saccharomyces cerevisiae
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To study membrane trafficking and quality control along the secretory pathway, I have characterized a targeting-defective pma1-7 mutant in which newly synthesized mutant Pma1 is mislocalized to the vacuole instead of being delivered to the plasma membrane. In the first chapter of this thesis, I describe genes encoding components of the membrane trafficking and quality control machinery by isolation of mutants that suppress the trafficking defect of pma1-7. A group of SOP (s&barbelow;uppressors o&barbelow;f p&barbelow;ma1-7) is identical with VPS (V&barbelow;acuolar P&barbelow;rotein S&barbelow;orting) genes required for delivery of newly synthesized proteins to the vacuole. A second group of SOP genes encode novel regulators of protein trafficking that are not required for delivery of the vacuolar proteins. One of these genes, SOP2, has sequence homology with mammalian nerve terminal protein synaptojanin. I have demonstrated that the synaptojanin-like protein in yeast plays a role in protein transport between the late Golgi compartment and the endosomes. In Chapter 2, I describe a pulse-chase immunofluorescence method to follow the movement of newly synthesized mutant Pma1 in different sop mutants. My analysis suggests that mutant Pma1 can move to the plasma membrane either directly from Golgi to surface or after its entry into the endosomal system. These observations provide evidence for an endosome-to-surface pathway that has not been previously established in yeast. In the last Chapter, I describe characterization of one of the SOP genes, SOP4. This work led to the discovery that export of newly synthesized Pma1 from the ER is regulated by transient association with Sop4. Therefore I have proposed that Sop4 may function as a specific facilitator for Pma1 at ER export. These data have important implications for understanding protein exit from the ER and quality control in the secretory pathway.