Localization of Human Acyl-Coenzyme A:Cholesterol Acyltransferase-1 (ACAT-1) in Macrophages and in Various Tissues

Abstract

To investigate the distribution of acyl-coenzyme A:cholesterol acyltransferase-1 (ACAT-1) in various human tissues , we examined tissues of autopsy cases immunohistochemically. ACAT-1 was demonstrated in macrophages , antigen-presenting cells , steroid hormone-producing cells , neurons , cardiomyocytes, smooth muscle cells , mesothelial cells , epithelial cells of the urinary tracts , thyroid follicles , renal tubules , pituitary , prostatic , and bronchial glands, alveolar and intestinal epithelial cells , pancreatic acinar cells , and hepatocytes. These findings showed that ACAT-1 is present in a variety of human tissues examined. The immunoreactivities are particularly prominent in the macrophages , steroid hormoneproducing cells , followed by hepatocytes , and intestinal epithelia. In cultured human macrophages, immunoelectron microscopy revealed that ACAT-1 was located mainly in the tubular rough endoplasmic reticulum; immunoblot analysis showed that the ACAT-1 protein content did not change with or without cholesterol loading; however , on cholesterol loading , about 30 to 40% of the total immunoreactivity appeared in small-sized vesicles. These vesicles were also enriched in 78-kd glucose-regulated protein (GRP 78), a specific marker for the endoplasmic reticulum. Immunofluorescent microscopy demon- Acyl-coenzyme A:cholesterol acyltransferase (ACAT) is a key enzyme involved in cellular cholesterol metabolism. It catalyzes the formation of cholesteryl esters from cholesterol and long-chain fatty acyl-coenzyme A. 1 ACAT activities are present in various tissues such as liver, intestines, adrenal glands, and aorta and are involved in intracellular cholesterol storage, lipoprotein assembly, steroid hormone production, and dietary cholesterol absorption. 2 Previous studies showed that ACAT is a membrane-bound enzyme; its activity is found only in the membrane fractions of intracellular organelles, especially in the rough endoplasmic reticulum. 3,4 More recently, molecular probes of ACAT have become available. In 1993, the first cDNA of ACAT, designated as ACAT-1, was cloned from a human THP-1 cell cDNA library by using a somatic cell and molecular genetic approach. 5 The sequence of human ACAT-1 cDNA led to the clonings of its homologues from various other species, including mice. 6 ACAT-1 gene knockout mice were produced. 7,8 Analyses of these mice showed that ACAT activities were significantly decreased in selected tissues examined but not in the livers, strongly suggesting that one or more additional ACAT genes, distinct from the ACAT-1 gene, probably exist in mice. More recently, a different ACAT cDNA, designated ACAT-2, was cloned. 9 -11 The sequence of ACAT-2 is homologous but different from that of ACAT-1. Earlier, two different ACAT-like genes were found in the simple eukaryote Sachromycetes saravesea. 12,13 The exact roles of ACAT-1 and ACAT-2 in different species remain unknown. Their physiological functions are currently under intensive investigation in several laboratories.Recently, specific polycl...

Transmembrane Lipid Transfer Is Crucial for Providing Neutral Lipids during Very Low Density Lipoprotein Assembly in Endoplasmic Reticulum

“…Incorporation of [ 35 TM 35 S cell labeling mix, 1.85 MBq/ml/dish; Amersham Biosciences, Tokyo, Japan) containing 40 M verapamil. After washing three times with PBS, the cells were homogenized using a probe-type sonicator in cell-lysis buffer (20 mM Tris-HCl, pH 7.4, containing 0.2% Triton X-100, 0.2% SDS, 4 mM EDTA, and the following protease inhibitors: 20 g/ml each of leupeptin, antipain, chymostatin, and pepstatin, 200 g/ml phenylmethylsulfonyl fluoride and 80 g/ml of N-acetyl-leucyl-leucyl-norleucinal (ALLN).…”

“…A recent report pointed out that ACAT is present in small cytosolic vesicles in addition to ER membrane (35). Although some DGAT activity was found in the microsomal lumen, there was, however, no direct evidence for the synthesis of lipids on the luminal side of ER.…”

Human Acyl-Coenzyme A:Cholesterol Acyltransferase 1 (acat1) Sequences Located in Two Different Chromosomes (7 and 1) Are Required to Produce a Novel ACAT1 Isoenzyme with Additional Sequence at the N Terminus

“…Thus, similar to the 50-kDa ACAT1, the 56-kDa ACAT1 protein is also mainly localized in the ER. We had previously showed by immunoelectron microscopy that, in human macrophages, the 50-kDa ACAT1 is mainly located in the rough ER (12). In our current work, we used the same antibody DM10 and performed immunoelectron microscopy to study the subcellular localization of ACAT1 in transfected AC29 cells.…”

“…Immunoelectron Microscopy-Immunoelectron microscopy was performed according to a method described previously (12). Briefly, various transfected AC29 cells were fixed with 4% paraformaldehyde in PBS, pH 7.2, for 30 min at 4°C.…”

“…The 56-kDa hACAT1 Is Also Localized in the ER-We had previously shown that the 50-kDa ACAT1 protein is mainly localized in the endoplasmic reticulum (10,12). To compare the subcellular localization of the 56-and the 50-kDa ACAT1s, we performed transient transfections in AC29 cells, using either plasmid K1D2m, which only expresses the 56-kDa ACAT1, or plasmid K1D4, which only expresses the 50-kDa ACAT1.…”