A striking feature of eukaryotic cells, from yeast to human, is their compartmental organization into membrane-bound organelles, each with a unique composition and function. The long-term goal of this research project is to understand how the Goigi complex, a multi-compartment organelle, performs its essential role in the transport, modification and sorting of proteins in the secretory pathway. Towards this goal, the function of ADP-ribosylation factor (ARF) in vesicle-mediated transport from the Golgi complex is being examined. ARF appears to regulate the budding of both COPI- and clathrin-coated vesicles from the Golgi complex. The proposed studies stem from recent discoveries implicating a role for Drs2p, an integral membrane P-type ATPase and potential lipid translocase (or flippase), specifically in the ARF-dependent budding of clathrin coated vesicles. The yeast Saccharomyces cerevisiae will be used for this work because of the fundamental similarity of the secretory pathway in all eukaryotic cells coupled with the powerful genetic and molecular approaches available with this organism make it an ideal model system. The specific goals of this research project are to determine if Drs2p is required for recruiting ARF and clathrin to Golgi membranes, thus indicating a direct role for Drs2p in clathrin coated vesicle budding. The proposed lipid flippase activity of Drs2p will be tested as will the potential overlap in function between Drs2p and four Drs2p-related proteins encoded within the yeast genome. There are profound implications to human health that can be derived from this study. Both Drs2p and clathrin are required for the normal function of the Golgi complex in the transport and processing of many proteins. In addition, lipid flippases related to Drs2p are thought to restrict phosphatidylserine (PS) to the inner leaflet of 'the plasma membrane. Loss of this asymmetric distribution of PS in blood cells stimulates clot formation and in apoptotic cells allows for recognition and phagocytosis by macrophages.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM062367-04
Application #
6794057
Study Section
Cell Development and Function Integrated Review Group (CDF)
Program Officer
Shapiro, Bert I
Project Start
2001-09-15
Project End
2005-08-31
Budget Start
2004-09-01
Budget End
2005-08-31
Support Year
4
Fiscal Year
2004
Total Cost
$239,968
Indirect Cost
Name
Vanderbilt University Medical Center
Department
Biology
Type
Schools of Arts and Sciences
DUNS #
004413456
City
Nashville
State
TN
Country
United States
Zip Code
37212
Zhou, Xiaoming; Sebastian, Tessy T; Graham, Todd R (2013) Auto-inhibition of Drs2p, a yeast phospholipid flippase, by its carboxyl-terminal tail. J Biol Chem 288:31807-15
Xu, Peng; Baldridge, Ryan D; Chi, Richard J et al. (2013) Phosphatidylserine flipping enhances membrane curvature and negative charge required for vesicular transport. J Cell Biol 202:875-86
Graham, Todd R (2013) Arl1 gets into the membrane remodeling business with a flippase and ArfGEF. Proc Natl Acad Sci U S A 110:2691-2
Baldridge, Ryan D; Xu, Peng; Graham, Todd R (2013) Type IV P-type ATPases distinguish mono- versus diacyl phosphatidylserine using a cytofacial exit gate in the membrane domain. J Biol Chem 288:19516-27
Baldridge, Ryan D; Graham, Todd R (2013) Two-gate mechanism for phospholipid selection and transport by type IV P-type ATPases. Proc Natl Acad Sci U S A 110:E358-67
Sebastian, Tessy T; Baldridge, Ryan D; Xu, Peng et al. (2012) Phospholipid flippases: building asymmetric membranes and transport vesicles. Biochim Biophys Acta 1821:1068-77
Baldridge, Ryan D; Graham, Todd R (2012) Identification of residues defining phospholipid flippase substrate specificity of type IV P-type ATPases. Proc Natl Acad Sci U S A 109:E290-8
Graham, Todd R; Burd, Christopher G (2011) Coordination of Golgi functions by phosphatidylinositol 4-kinases. Trends Cell Biol 21:113-21
Brett, Christopher L; Kallay, Laura; Hua, Zhaolin et al. (2011) Genome-wide analysis reveals the vacuolar pH-stat of Saccharomyces cerevisiae. PLoS One 6:e17619
Graham, Todd R; Kozlov, Michael M (2010) Interplay of proteins and lipids in generating membrane curvature. Curr Opin Cell Biol 22:430-6

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