FUNCTIONAL-CHARACTERIZATION OF MITOCHONDRIAL CARNITINE PALMITOYLTRANSFERASE-I AND PALMITOYLTRANSFERASE-II EXPRESSED IN THE YEAST PICHIA-PASTORIS

The rate-limiting step in beta oxidation is the conversion of long-cha in acyl-CoA to acylcarnitine, a reaction catalyzed by the outer mitoch ondrial membrane enzyme carnitine palmitoyltransferase I (CPTI) and in hibited by malonyl-CoA. The acylcarnitine is then translocated across the inner mitochondrial membrane by the carnitine/acylcarnitine transl ocase and converted back to acyl-CoA by CPTIl. Although CPTII has been examined in detail, studies on CPTI have been hampered by an inabilit y to purify CPTI in an active form from CPTII. In particular, it has n ot been conclusively demonstrated that CPTI is even catalytically acti ve, or whether sensitivity of CPTI to malonyl-CoA is an intrinsic prop erty of the enzyme or is contained in a separate regulatory subunit th at interacts with CPTI. To address these questions, the genes for CPTI and CPTII were separately expressed in Pichia pastoris, a yeast with no endogenous CPT activity. High levels of CPT activity were present i n purified mitochondrial preparations from both CPTI- and CPTII-expres sing strains. Furthermore, CPTI activity was highly sensitive to inhib ition by malonyl-CoA while CPTII was not. Thus, CPT catalytic activity and malonyl-CoA sensitivity are contained within a single CPTI polype ptide in mammalian mitochondrial membranes. We describe the kinetic ch aracteristics for the yeast-expressed CPTs, the first such report for a CPTI enzyme in the absence of CPTII. Yeast-expressed CPTI is inactiv ated by detergent solubilization. However, removal of the detergent in the presence of phospholipids resulted in the recovery of malonyl-CoA -sensitive CPTI activity, suggesting that CPTI requires a membranous e nvironment. CPTI is thus reversibly inactivated by detergents.