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Vol. 11, Issue 11, 3675-3687, November 2000
and
§
Arteriosclerosis Research Program, Departments of *Pathology,
A second form of the enzyme acyl-CoA:cholesterol acyltransferase,
ACAT2, has been identified. To explore the hypothesis that the two ACAT
enzymes have separate functions, the membrane topologies of ACAT1 and
ACAT2 were examined. A glycosylation reporter and FLAG epitope tag
sequence was appended to a series of ACAT cDNAs truncated after each
predicted transmembrane domain. Fusion constructs were assembled into
microsomal membranes, in vitro, and topologies were determined based on
glycosylation site use and accessibility to exogenous protease. The
accessibility of the C-terminal FLAG epitope in constructs was
determined by immunofluorescence microscopy of permeabilized
transfected cells. Both ACAT1 and ACAT2 span the membrane five times
with their N termini in the cytosol and C termini in the ER lumen. The
fourth transmembrane domain is located in a different region for each
protein, placing the putative active site ACAT1 serine
(Ser269) in the cytosol and the analogous residue in ACAT2
(Ser249) in the ER lumen. Mutation of these serines inactivated the ACAT enzymes. The outcome is consistent with the hypothesis that cholesterol ester formation by ACAT2 may be coupled to
lipoprotein particle assembly and secretion, whereas ACAT1 may function
primarily to maintain the balance of free and esterified cholesterol intracellularly.
Biochemistry, and Internal Medicine, Wake
Forest University School of Medicine, Winston-Salem, North Carolina
27157
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