Abstract
ACAT1 is normally a resident enzyme in the endoplasmic reticulum (ER). We previously showed that treating macrophages with denatured LDL causes a large increase in ER-derived, ACAT1-positive vesicles. Here, we isolated ER membranes and ER-derived vesicles to examine their ACAT enzyme activity in vitro. The results showed that when macrophages are grown under normal conditions, ACAT1 is located in high density ER membrane; its enzymatic activity is relatively low. Loading macrophages with cholesterol did not increase the total cellular ACAT1 protein content significantly but caused more ACAT1 to appear in ER-derived vesicles. These vesicles exhibit lower density and are associated with markers of both ER and the trans-Golgi network. When normalized with equal ACAT1 protein mass, the enzymatic activities of ACAT1 in ER-derived vesicles were 3-fold higher than those present in ER membrane. Results using reconstituted ACAT enzyme assay showed that the increase in enzyme activity in ER-derived vesicles is not due to an increase in the cholesterol content associated with these vesicles. Overall, our results show that macrophages cope with cholesterol loading by using a novel mechanism: they produce more ER-derived vesicles with elevated ACAT1 enzyme activity without having to produce more ACAT1 protein.
Highlights
ACAT1 is normally a resident enzyme in the endoplasmic reticulum (ER)
Likewise, when primary human macrophages grown in medium A were treated with aggregated LDL, cellular cholesterol content increased from 10.4 to 34.1 μg/mg protein; cholesteryl ester content increased from 5.1 to 148 μg/mg protein (Fig. 1A, right panels)
The results showed that in basal “medium A” conditions, most of the ACAT1 signal appeared in the medium-density fractions where the ER marker protein calnexin and the trans-Golgi network (TGN) marker protein syntaxin 6 were found (Fig. 2A)
Summary
ACAT1 is normally a resident enzyme in the endoplasmic reticulum (ER). We previously showed that treating macrophages with denatured LDL causes a large increase in ER-derived, ACAT1-positive vesicles. In human macrophages, when maintained in normal medium, the ACAT1 signal is mainly with the tubular ER; adding modified LDL to the growth medium of these cells caused up to 30–40% of the total ACAT1 immunoreactive signals to become associated with small, ER-derived vesicles 80–100 nm in diameter [13]. These studies suggest that in macrophages, ACAT1 can be associated with ER-derived vesicles, especially when cells are grown in cholesterol-rich conditions. Journal of Lipid Research Volume 51, 2010 1263 positive membrane fractions and evaluated the ACAT enzyme activities in these fractions
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