Cell polarity is maintained via a balance of exocytosis and endocytosis; the protein machinery that mediates these transport processes must be co-ordinated with membrane lipid signals. This lipid signalling is, in part, dependent on the establishment of membrane domains through lipid transport. Cholesterol is transported via a poorly defined route that is independent of vesicle-mediated secretory protein transport. This “non-vesicular” sterol transport is postulated to involve the conserved family of Oxysterol binding protein (OSBP) Related Proteins (ORPs), which are proposed to be sterol lipid transport proteins (LTPs). To test if ORPs primarily act as sterol LTPs or alternatively as sterol-responsive signalling proteins, the function of Saccharomyces cerevisiae OSBP Homologues (OSH1-OSH7) were analyzed. Depletion of all Osh proteins in yeast cells inhibited growth, and defects in endocytosis, polarized exocytosis, and sterol homeostasis, were observed. Consistent with a direct role in exocytosis, Oshdepletion disrupted the polarized localization of vesicle transport regulators (Rho- and Rab-GTPases, and exocyst complex subunits) and the Osh protein Osh4p was observed to travel on exocytic vesicles to sites of polarized growth. Osh4p also formed complexes in vivo with specific Rho- and Rab-GTPases, and exocyst complex subunits. Contrary to the postulated role of ORPs as LTPs, a designed mutation in Osh4p that disrupts its ability to bind and thereby transport sterols, did not inactivate the protein but caused a gain-of-function phenotype affecting exocytosis. Our experiments suggested that ORPs are not sterol LTPs and implied that sterols act as signalling ligands that repress Osh4p, and potentially other ORPs. To understand how Osh proteins might simultaneously affect both exocytosis and endocytosis, I tested whether the regulation of the exocytic and endocytic machinery are directly coupled. I found that the Rab GTPase Sec4p, which is an integral component of exocytosis, directly interacted with specific endocytic proteins at actin patches. SEC4 was required for proper endocytic site assembly and actin patch polarization, indicating that Sec4p links exocytosis and endocytosis to maintain cell polarization. Because these novel mechanisms involving sterol signalling and cell polarization are likely to be conserved, I propose these studies have broader medical implications applicable to cancer cell growth and metastasis.
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Thesis advisor: Beh, Christopher
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