Acyl-CoA synthetase isoforms 1, 4, and 5 are present in different subcellular membranes in rat liver and can be inhibited independently

Inhibition studies have suggested that acyl-CoA synthetase (ACS, EC 6.2.1.3 ) isoforms might regulate the use of acyl-CoAs by different metabolic pathw ays. In order to determine whether the subcellular locations differed for e ach of the three ACSs present in liver and whether these isoforms were regu lated independently, non-cross-reacting peptide antibodies were raised agai nst ACS1, ACS4, and ACS5, ACS1 was identified in endoplasmic reticulum, mit ochondria-associated membrane (MAM), and cytosol, but not in mitochondria. ACS4 was present primarily in MAM, and the 76-kDa ACS5 protein was located in mitochondrial membrane, Consistent with these locations, N-ethylmaleimid e, an inhibitor of ACS4, inhibited ACS activity 47% in MAM and 28% in endop lasmic reticulum. Troglitazone, a second ACS4 inhibitor, inhibited ACS acti vity < 10% in microsomes and mitochondria and 45% in MAM. Triacsin C, a com petitive inhibitor of both ACS1 and ACS4, inhibited ACS activity similarly in endoplasmic reticulum, MAM, and mitochondria, suggesting that a hitherto unidentified triacsin-sensitive ACS is present in mitochondria. ACS1, ACS4 , and ACS5 were regulated independently by fasting and re-feeding. Fasting rats for 48 h resulted in a decrease in ACS4 protein, and an increase in AC S5, Refeeding normal chow or a high sucrose diet for 24 h after a 48-h fast increased both ACS1 and ACS4 protein expression 1.5-2.0-fold, consistent w ith inhibition studies, These results suggest that ACS1 and ACS4 may be lin ked to triacylglycerol synthesis. Taken together, the data suggest that acy l-CoAs may be functionally channeled to specific metabolic pathways through different ACS isoforms in unique subcellular locations.