Cholesterol loading in macrophages stimulates formation of ER-derived vesicles with elevated ACAT1 activity.
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.
Sakashita
Naomi
N
Department of Cell Pathology, Graduate School of Medical Sciences, Kumamoto University, Kumamoto 861-8556, Japan. nanaomi@gpo.kumamoto-u.ac.jp
Chang
Catherine C Y
CC
Lei
Xiaofeng
X
Fujiwara
Yukio
Y
Takeya
Motohiro
M
Chang
Ta-Yuan
TY
D000101:Acetyl-CoA C-Acetyltransferase / Q000378:metabolism*; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D002784:Cholesterol / Q000378:metabolism*; D002788:Cholesterol Esters / Q000378:metabolism; D022162:Cytoplasmic Vesicles / Q000378:metabolism*; D004721:Endoplasmic Reticulum / Q000378:metabolism*; D006801:Humans; D008264:Macrophages / Q000166:cytology* / Q000201:enzymology / Q000378:metabolism*; D018613:Microscopy, Confocal; D050765:Qa-SNARE Proteins / Q000378:metabolism; D014461:Ultracentrifugation
D002788:Cholesterol Esters; D050765:Qa-SNARE Proteins; D002784:Cholesterol; C506639:ACAT1 protein, human; D000101:Acetyl-CoA C-Acetyltransferase
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