Integration of transport pathways in Saccharomyces cerevisiae

Author: 
Date created: 
2017-02-28
Identifier: 
etd10018
Keywords: 
Yeast compensatory endocytosis
Non-vesicular transport
Transport pathway integration
SEC4
OSH4
ARV1
Abstract: 

Plasma membrane (PM) homeostasis is essential for viability and depends on maintaining a constant balance between the amount of membrane material arriving at the cell cortex and the amount recycled. This membrane transport is mediated by two distinct mechanisms: (i) vesicular transport, utilizing membrane-enclosed vesicles for bulk transport of membrane proteins and lipids; and (ii) non-vesicular lipid transport. While the molecular mechanisms of these processes are well defined, they are generally considered independent events but how they are integrated is poorly understood. To gain insight into how these transport pathways are coordinated at the PM, three avenues of research were conducted using a combination of genetic, biochemical and live-cell microscopy assays. First, I showed that the Oxysterol-binding protein-related protein (ORP) Osh4p, implicated in non-vesicular sterol transfer, was found to associate with exocytic vesicles and formed complexes with regulators of polarized exocytosis, including the small GTPases Sec4p, Cdc42p, and Rho1p. Second, I tested the function of the evolutionarily conserved endoplasmic reticulum (ER)-associated protein Arv1p, also suggested to be involved in non-vesicular sterol transfer. Ultimately Arv1p was found to be dispensable for sterol exchange between the ER and PM but instead it was shown to play an important role in maintaining ER ultrastructure; Arv1p might be involved in regulating insertion of tail-anchored proteins into membranes. Finally, the essential yeast Rab GTPase Sec4p, principally known to be a key regulator of exocytosis, was shown to mechanistically couple polarized exocytosis with cortical actin polymerization, which activates Las17p (the yeast Wiskott–Aldrich syndrome [WASp])-dependent endocytosis. Las17p activation results in actin filament nucleation, which pulls the PM inward for endocytic vesicle biogenesis. Sec4p thereby represents the first direct regulatory link that couples exocytosis and endocytosis, which we termed \"yeast compensatory endocytosis.\" By identifying novel mechanisms that coordinate intracellular transport pathways, these studies provided important new insights into how PM homeostasis is regulated and maintained.

Document type: 
Thesis
Rights: 
This thesis may be printed or downloaded for non-commercial research and scholarly purposes. Copyright remains with the author.
File(s): 
S1 Video. Photobleaching enables single particle tracking of GFP-Sec4p at the cell cortex.
S2 Video. Las17p-RFP and GFP-Sec4p particles co-localize at cortical actin patches.
S3 Video. Sla1p-RFP and GFP-Sec4p co-localize at cortical actin patches.
S4 Video. GFP-Sec4p particles co-localize with Abp1p-RFP at cortical actin patches.
S5 Video. SEC4 and SEC2 function are required for normal actin patch polarization and dynamics.
Senior supervisor: 
Christopher Beh
Department: 
Science: Department of Molecular Biology and Biochemistry
Thesis type: 
(Thesis) Ph.D.
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