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Permanent link (DOI): https://doi.org/10.7939/R3734K

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Functional and Topological Analysis of Acyl-CoA:Diacylglycerol Acyltransferase 2 From Saccharomyces cerevisiae Open Access

Descriptions

Other title
Subject/Keyword
Mutagenesis
TAG
Yeast
DGAT2
Structure/Function
Topology
Type of item
Thesis
Degree grantor
University of Alberta
Author or creator
Liu, Qin
Supervisor and department
Wesealake, Randall (Agricultural, Food and Nutritional Science)
Examining committee member and department
Kav, Nat (Agricultural, Food and Nutritional Science, Univeristy of Alberta)
Gaenzle, Michael (Agricultural, Food and Nutritional Science, Univeristy of Alberta)
McMaster, Christopher (Biochemistry and Molecular Biology, Dalhousie University)
Sykes, Brian (Biochemistry, University of Alberta)
Department
Department of Agricultural, Food, and Nutritional Science
Specialization

Date accepted
2011-01-31T23:29:24Z
Graduation date
2011-06
Degree
Doctor of Philosophy
Degree level
Doctoral
Abstract
Acyl-CoA:diacylglycerol acyltransferase (EC 2.3.1.20, DGAT or DAGAT) is a membrane protein found mainly in the endoplasmic reticulum (ER). It catalyzes the final step in the biosynthesis of triacylglyerol (TAG or TG), which is the principal repository of fatty acids for energy utilization and membrane formation. Several lines of evidence have indicated that DGAT has a substantial effect on carbon flux into TAG. DGAT has at least two discrete family members (DGAT1 and DGAT2) with different physiological roles. High-resolution structures of both DGATs, however, are absent due to difficulties in purification. In order to gain insight into structural and functional relationships of DGATs, a functional DGAT2 protein from the yeast Saccharomyces cerevisiae (ScDGAT2, also known as Dgalp) was selected. The structural and functional role of cysteine residues in ScDGAT2 was studied using site-directed mutagenesis (SDM) in combination with chemical modification. Although ScDGAT2 is susceptible to thiol-modifying reagents, none of the cysteines are essential for the catalytic activity or involved in structure support though disulfide linkages. Inhibition of DGAT activity by thiol-specific modification was localized to cysteine314, which is in the proximity of a highly conserved motif in DGAT2s. Thus, cysteine314 may reside in a crucial position near a possible active site or related to proper protein folding. The functional importance and topological orientation of signature motifs in ScDGAT2 were also studied using the same methods. Both the N- and C-termini of ScDGAT2 are oriented toward the cytosol. A highly conserved motif, 129YFP131, and a hydrophilic segment exclusive to ScDGAT2, reside in the ER and play essential roles in enzyme catalysis. In addition, the strongly conserved H195, which may be part of the active site of DGAT2, is likely embedded in the membrane. Although ScDGAT2 has a topology similar to that of murine DGAT2, there are striking differences which suggest that the topological organization of DGAT2 is not ubiquitously conserved.
Language
English
DOI
doi:10.7939/R3734K
Rights
Permission is hereby granted to the University of Alberta Libraries to reproduce single copies of this thesis and to lend or sell such copies for private, scholarly or scientific research purposes only. Where the thesis is converted to, or otherwise made available in digital form, the University of Alberta will advise potential users of the thesis of these terms. The author reserves all other publication and other rights in association with the copyright in the thesis and, except as herein before provided, neither the thesis nor any substantial portion thereof may be printed or otherwise reproduced in any material form whatsoever without the author's prior written permission.
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