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Title:Mutational analysis of the Vam3 transmembrane domain and its effects on vacuole fusion in Saccharomyces cerevisiae
Author(s):Wu, Ming
Advisor(s):Fratti, Rutilio A.
Department / Program:Biochemistry
Discipline:Biochemistry
Degree Granting Institution:University of Illinois at Urbana-Champaign
Degree:M.S.
Genre:Thesis
Subject(s):soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE)
Membrane fusion
Vam3
transmembrane domain
Saccharomyces cerevisiae
Abstract:Vam3 is a SNARE protein essential in yeast vacuole homotypic fusion. The specific role of the Vam3 trans-membrane domain (TMD) during vacuole fusion remains unclear. Previous work had shown that replacing Vam3 TMD with a prenyl lipid anchor (CCIIM) results in attenuated fusion and accumulation of trans-SNARE complexes, indicating CCIIM can not transit enough energy to stimulate membrane fusion as Vam3 TMD does. Hypothesis were proposed that the either the length or the specific Vam3 TMD interactions with its cognate SNAREs, or a combination of both, result in the less efficient fusion with Vam3-CCIIM. In this study, I further studied the Vam3-CCIIM vacuole fusion and vacuole morphology. To test the above hypotheses, I constructed truncations at the C-terminus of the Vam3 TMD to study the effect of varying length of Vam3 TMD. To study the specific TMD interactions with other SNAREs, I mutated three non-aliphatic amino acids in the Vam3 TMD that may contribute to inter-helical interactions with cognate SNAREs. I also constructed the yeast mutant strains and found those mutations cause vacuole fragmentation. These mutants could be used for further studying their effect on fusion and protein complex formation, and deciphering Vam3 TMD's role during fusion.
Issue Date:2013-05-24
URI:http://hdl.handle.net/2142/44229
Rights Information:Copyright 2013 Ming Wu
Date Available in IDEALS:2013-05-24
Date Deposited:2013-05


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