The overall goal of this research is to develop a mechanistic understanding of the vacuolar biogenesis pathways and endosome function in the yeast Saccharomycescerevisiae. Genetic analysis has led to the identification of a very large number of components required along the vacuolar assembly pathway in yeast, and two of these proteins, Vps55 and Vps68 are particularly of interest because these are integral membrane proteins that function at the endosome. Vps55 and Vps68 form a complex together with UipSp, a 25 kDa member of the large DUP240 gene family. The role of this integral membrane protein complex is being investigated as a docking/tethering site for the large collection of soluble Vps proteins that are recruited to the Golgi and endosome membrane during protein sorting and vacuole membrane traffic. "Class E" proteins proteins function at the endosome in the formation of the multi-vesicular body (MVB) and the sorting of cargo proteins into these intralumenal vesicles. Ten of these Vps proteins form three separate ESCRT protein complexes (Endosome Sorting Complex Required for Transport), and the AAA- ATPase Vps4 is required to disassemble the ESCRT complexes. We are investigating the role of three other Class E Vps proteins that are required to recruit the Vps4 complex to the endosome and to stimulate ESCRT disassembly by activating the Vps4 ATPase activity. We will also exploit our new genetic screen for mutants defective for membrane invagination at the endosome;analysis of these genes should reveal important insight into the mysterious process of MVB membrane invagination. We are investigating the connection between endosome function and the ability of yeast to sense glucose in the extracellular environment. Some of the yeast Class E mutants are Sucrose NonFermenting (Snf-), and only a subset of each of the ESCRT complex polypeptides are required for yeast to be Snf+. The high-affinity glucose sensor in yeast, Snf3, localizes to MVBs in yeast mutants defective for MVB function. Studies of membrane traffic in yeast have resulted in a deeper understanding of membrane transport in all eukaryotic cells, and these studies in yeast are also providing important insights into our understanding of lysosomal storage diseases and the cellular requirements for HIV viral particle formation.

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
Method to Extend Research in Time (MERIT) Award (R37)
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Special Emphasis Panel (NSS)
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Ainsztein, Alexandra M
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University of Oregon
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Coonrod, Emily M; Stevens, Tom H (2010) The yeast vps class E mutants: the beginning of the molecular genetic analysis of multivesicular body biogenesis. Mol Biol Cell 21:4057-60
Schluter, Cayetana; Lam, Karen K Y; Brumm, Jochen et al. (2008) Global analysis of yeast endosomal transport identifies the vps55/68 sorting complex. Mol Biol Cell 19:1282-94
Lottridge, Jillian M; Flannery, Andrew R; Vincelli, Jennifer L et al. (2006) Vta1p and Vps46p regulate the membrane association and ATPase activity of Vps4p at the yeast multivesicular body. Proc Natl Acad Sci U S A 103:6202-7
Bowers, Katherine; Stevens, Tom H (2005) Protein transport from the late Golgi to the vacuole in the yeast Saccharomyces cerevisiae. Biochim Biophys Acta 1744:438-54
Bowers, Katherine; Lottridge, Jillian; Helliwell, Stephen B et al. (2004) Protein-protein interactions of ESCRT complexes in the yeast Saccharomyces cerevisiae. Traffic 5:194-210
Kweon, Youngseok; Rothe, Anca; Conibear, Elizabeth et al. (2003) Ykt6p is a multifunctional yeast R-SNARE that is required for multiple membrane transport pathways to the vacuole. Mol Biol Cell 14:1868-81
Conibear, Elizabeth; Cleck, Jessica N; Stevens, Tom H (2003) Vps51p mediates the association of the GARP (Vps52/53/54) complex with the late Golgi t-SNARE Tlg1p. Mol Biol Cell 14:1610-23
Gerrard, S R; Bryant, N J; Stevens, T H (2000) VPS21 controls entry of endocytosed and biosynthetic proteins into the yeast prevacuolar compartment. Mol Biol Cell 11:613-26
Conibear, E; Stevens, T H (2000) Vps52p, Vps53p, and Vps54p form a novel multisubunit complex required for protein sorting at the yeast late Golgi. Mol Biol Cell 11:305-23
Gerrard, S R; Mecklem, A B; Stevens, T H (2000) The yeast endosomal t-SNARE, Pep12p, functions in the absence of its transmembrane domain. Traffic 1:45-55

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