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Molecular cloning and characterization of Saccharomyces cerevisiae acyl-CoA:sterol acyltransferase and dissection of human ACAT oligomerization motif(s).

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Call No.	QP606.A3 Y8 1997
Author	Yu, Chunjiang
Title	Molecular cloning and characterization of Saccharomyces cerevisiae acyl-CoA:sterol acyltransferase and dissection of human ACAT oligomerization motif(s).
Date	1997
Abstract	Esterification of cholesterol by acyl-CoA:cholesterol acyltransferase (ACAT) is a key element in maintaining cholesterol homeostasis in cells of higher animals. In the budding yeast, Saccharomyces cerevisiae, accumulation of ergosteryl esters accompanies entry into stationary phase and sporulation. We have determined that two genes in yeast, SAT1 and SAT2, encode isozymes of acyl-CoA:sterol acyltransferase (ASAT) which are functionally related to ACAT. The SAT1 isozyme is the major catalytic isoform, accounting for at least 65-75% of total ASAT activity. Targeted deletions of one or both genes do not compromise mitotic cell growth or spore germination. However, diploids that are homozygous for a sat1 null mutation exhibit significantly reduced sporulation efficiency. Furthermore, a larger fraction of the sporulating sat1 null diploids arrest after the first meiotic division. Human ACAT expressed in sat1 sat2 mutant cells can catalyze esterification of cholesterol and, to a lesser extent, ergosterol in vitro, but restores ergosteryl oleate formation in vivo to only $\sim$8% of that catalyzed by yeast ASAT in wild-type cells. Similarly, yeast SAT1 and SAT2 only displayed very low ACAT activity in AC29 cells, a Chinese Hamster Ovary cell line that is deficient for ACAT. Neither gene was able to complement the ACAT deficiency in AC29 cells. Mammalian ACAT has been proposed to function as an allosteric enzyme in an oligomeric form. In order to identify the domain(s) mediating ACAT oligomerization, I used a Glutathione S-Transferase (GST) fusion protein strategy to dissect the ACAT protein sequences that may mediate ACAT oligomerization. With this expression system, I was able to analyze the oligomeric status for some of the expressed GST-ACAT fusion proteins, and found a potential oligomerization motif within the ACAT N-terminus (amino acids 1-131).
Description	1 online resource (xiii, 161 leaves) : illustrations
Format	application/pdf
Advisor	McClung, C. Robertson
Subject	Acyltransferases -- Genetic aspects. ; Esterification -- Genetic aspects. ; Saccharomyces cerevisiae -- Genetics. ; Cholesterol -- Genetic aspects.
College	Dartmouth College -- Department of Biological Sciences.
Issuing Body	Dartmouth Dissertations are made available through the Dartmouth Digital Library.
Degree	Thesis (Ph.D)--Dartmouth College.
Copyright	In Copyright. This material is protected by copyright and/or related rights. You are free to use this material in any way that is permitted by copyright law that applies to your use. For other uses, you need to obtain permission from the rights-holder(s). http://rightsstatements.org/page/InC/1.0/
DOI	https://doi.org/10.1349/ddlp.3510

Title	yu1997 1
Full Text	Molecular Cloning and Characterization of Saccharomyces cerevisiae Acyl-CoA:Sterol Acyltransferase and Dissection of Human ACAT oligomerization Motif(s) A Thesis Submitted to the Faculty in partial fulfillment of the requirements for the degree of Doctor of Philosophy in Biology by Chunjiang Yu DARTMOUTH COLLEGE Hanover, New Hampshire May, 1997

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Molecular cloning and characterization of Saccharomyces cerevisiae acyl-CoA:sterol acyltransferase and dissection of human ACAT oligomerization motif(s).

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