Please use this identifier to cite or link to this item: http://scholarbank.nus.edu.sg/handle/10635/13582
Title: Cloning, expression and characterization of oxysterol- binding protein homologue 7 (OSH7) in yeast Saccharomyces cerevisiae
Authors: LI HONGZHE
Keywords: OSBP; OSH; Vps4p; yeast two-hybrid system; subcellular fractionation; endocytosis; vacular protein sorting
Issue Date: 5-Nov-2003
Source: LI HONGZHE (2003-11-05). Cloning, expression and characterization of oxysterol- binding protein homologue 7 (OSH7) in yeast Saccharomyces cerevisiae. ScholarBank@NUS Repository.
Abstract: Oxysterols are potent regulators of cellular sterol homeostasis. The mammalian oxysterol binding protein (OSBP) was able to bind oxysterols directly; therefore, OSBP and its related proteins (ORPs) are believed to mediate some of the effects by oxysterols. However, recent data suggested that OSBP and ORPs might interact with other lipids, such as phosphatidylinositides, and might have functions other than controlling cellular sterol metabolism. The molecular mechanisms underlying the function of the entire OSBP family of proteins remain to be elucidated. The yeast OSH genes (OSH1-OSH7), which encode a family of homologues of OSBP, are believed to play important roles in the maintenance of intracellular lipid distribution, endocytosis and the integrity of vacuole morphology. In our study, we demonstrated using yeast-two-hybrid system that the coiled-coil domain of Osh7p could interact with Vps4p, which belongs to the protein family of AAA-type ATPases. The interaction was further confirmed by a GST-pull down assay. Subcellular fractionation was performed to localize Osh7p mainly to the cytosolic fraction in wild-type cells, however, in vps4D yeast cells, a significant portion of the Osh7p redistributed to a membranous fraction. Sucrose density gradient analysis further confirmed the redistribution of Osh7p in vps4D strain. Meanwhile, we demonstrated that endocytosis and vacuolar protein sorting were not affected by OSH7 deletion. Concomitantly, the interaction between Osh7p and phospholipids was investigated using protein-lipid overlay assay. In this study, Osh7p showed the ability to bind to PI(4)P and PI(5)P. Finally, we presented evidence to suggest that the loss of Osh7p function influence sterol esterification.
URI: http://scholarbank.nus.edu.sg/handle/10635/13582
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