Enrichment of acyl coenzyme A : cholesterol O-acyltransferase near trans-Golgi network and endocytic recycling compartment

Acyl coenzyme A:cholesterol O-acyltransferase (ACAT) is the enzyme responsi ble for cholesterol esterification in macrophages leading to foam cell form ation. The determination of its localization is a critical step in understa nding its regulation by cholesterol, Using immunofluorescence and confocal microscopy, we previously showed that the enzyme colocalized with markers o f the endoplasmic reticulum, but in addition, ACAT was found in an unidenti fied paranuclear site. In the present study, we further define the localiza tion of paranuclear ACAT. First, we found that ACAT does not colocalize wit h sorting endosomes or late endosomes labeled with fluorescent alpha(2)-mac roglobulin. The paranuclear ACAT is close to the endocytic recycling compar tment labeled with fluorescent transferrin. We also show that the paranucle ar structure containing ACAT is very close to TGN38, a membrane protein of the trans-Golgi network (TGN), but farther from Gos28, a marker of cis, med ial, and trans Golgi. After treatment with nocodazole, the central localiza tion of ACAT did not colocalize with markers of the TGN. These data indicat e that a significant fraction of ACAT resides in membranes that may be a su bcompartment of the endoplasmic reticulum in proximity to the TGN and the e ndocytic recycling compartment. Because the TGN and the endocytic recycling compartment are engaged in extensive membrane traffic with the plasma memb rane, esterification of cholesterol in these membranes may play an importan t role in macrophage foam cell formation during atherogenesis.