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Functional characterization of stero...

Guo, Zhongmin.

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Functional characterization of sterol esterification enzymes in yeast.

紀錄類型:	書目-電子資源 : Monograph/item
正題名/作者:	Functional characterization of sterol esterification enzymes in yeast./
作者:	Guo, Zhongmin.
出版者:	Ann Arbor : ProQuest Dissertations & Theses, : 2000,
面頁冊數:	142 p.
附註:	Source: Dissertations Abstracts International, Volume: 62-03, Section: B.
Contained By:	Dissertations Abstracts International62-03B.
標題:	Nutrition. -
電子資源:	http://pqdd.sinica.edu.tw/twdaoapp/servlet/advanced?query=9970202
ISBN:	9780599751682

Functional characterization of sterol esterification enzymes in yeast.
Guo, Zhongmin.

Functional characterization of sterol esterification enzymes in yeast. - Ann Arbor : ProQuest Dissertations & Theses, 2000 - 142 p.

Source: Dissertations Abstracts International, Volume: 62-03, Section: B.

Thesis (Ph.D.)--Columbia University, 2000.

This item must not be sold to any third party vendors.

In all eukaryotes, free sterol is an essential component of membranes. However, high levels of free sterol can be toxic. Cells maintain sterol homeostasis, in part, by converting excess free sterol to sterol ester. In mammals this reaction is mediated by the enzyme acyl-coenzyme A: cholesterol acyltransferase (ACAT). ACAT activity has been implicated in multiple physiologic processes, and in the pathological development of atherosclerosis. Two yeast ACAT homologs, ARE1 and ARE2 (for ACAT related enzymes 1 and 2, respectively), were identified, disruption of both genes produced a viable yeast cell with no detectable sterol ester. The goal of this study was to investigate the mechanism and function of sterol esterification. enzymes using yeast as a model system. We show that the functional regions of yeast Are2p (the major isoform) reside in the carboxyl terminus that is most conserved in ACAT family members. We identified two conserved motifs in yeast Are2p that are essential for its enzyme activity, H/YSF and FYxDWVVN. The same two sequence motifs in human ACAT1 were also critical for activity when assayed in the are1Δ are2Δ yeast cell. The FYxDWWN motif in Are2p is likely involved in binding of fatty acyl CoA substrate since a single amino acid change in this motif caused an ∼6-fold increase in the apparent Km of the enzyme. In contrast, deletion of 30 residues from the amino terminus led to increased activity, indicating that this region is not required for activity and may play a negative, regulatory role. Antibodies against Are2p were generated. The wildtype Are2p was found to be membrane-bound, with an apparent molecular weight of about 79 kDa. No evidence of phosphorylation or N-glycosylation were found in Are2p. The HA epitope-tagged Are1 protein was also membrane-associated, and migrated as a heterogeneous protein of 55-60 kDa. The expression level of HA-Are1p was much lower than that of HA-Are2p, which correlates with its low enzyme activity. Furthermore, we demonstrate by yeast two-hybrid and co-immunoprecipitation experiments that Are1p or Are2p homo-multimerizes, but do not hetero-multimerize. We identified five candidate genes whose products may interact with Are2p in the cell.

ISBN: 9780599751682Subjects--Topical Terms:

517777
Nutrition.
Subjects--Index Terms:

ACAT

Functional characterization of sterol esterification enzymes in yeast.
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520 $a In all eukaryotes, free sterol is an essential component of membranes. However, high levels of free sterol can be toxic. Cells maintain sterol homeostasis, in part, by converting excess free sterol to sterol ester. In mammals this reaction is mediated by the enzyme acyl-coenzyme A: cholesterol acyltransferase (ACAT). ACAT activity has been implicated in multiple physiologic processes, and in the pathological development of atherosclerosis. Two yeast ACAT homologs, ARE1 and ARE2 (for ACAT related enzymes 1 and 2, respectively), were identified, disruption of both genes produced a viable yeast cell with no detectable sterol ester. The goal of this study was to investigate the mechanism and function of sterol esterification. enzymes using yeast as a model system. We show that the functional regions of yeast Are2p (the major isoform) reside in the carboxyl terminus that is most conserved in ACAT family members. We identified two conserved motifs in yeast Are2p that are essential for its enzyme activity, H/YSF and FYxDWVVN. The same two sequence motifs in human ACAT1 were also critical for activity when assayed in the are1Δ are2Δ yeast cell. The FYxDWWN motif in Are2p is likely involved in binding of fatty acyl CoA substrate since a single amino acid change in this motif caused an ∼6-fold increase in the apparent Km of the enzyme. In contrast, deletion of 30 residues from the amino terminus led to increased activity, indicating that this region is not required for activity and may play a negative, regulatory role. Antibodies against Are2p were generated. The wildtype Are2p was found to be membrane-bound, with an apparent molecular weight of about 79 kDa. No evidence of phosphorylation or N-glycosylation were found in Are2p. The HA epitope-tagged Are1 protein was also membrane-associated, and migrated as a heterogeneous protein of 55-60 kDa. The expression level of HA-Are1p was much lower than that of HA-Are2p, which correlates with its low enzyme activity. Furthermore, we demonstrate by yeast two-hybrid and co-immunoprecipitation experiments that Are1p or Are2p homo-multimerizes, but do not hetero-multimerize. We identified five candidate genes whose products may interact with Are2p in the cell.
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