Effects of cardiolipin on carnitine palmitoyltransferase kinetics

Abstract

We previously reported [Biochem. J. (1994) 299, 761-767] that certain phospholipids added to intact rat liver mitochondria increased sensitivity of carnitine palmitoyltransferase-I (CPT1A) to inhibition by malonyl-CoA. The most effective phospholipid was cardiolipin, which enhanced inhibition so that it mimicked the normal change in malonyl-CoA sensitivity occurring in the fasting to fed transition. Since cardiolipin is a normal component of eukaryotic mitochondrial membranes, we used mitochondria from Saccharomyces cerevisiae to test the hypothesis that the absence of cardiolipin in mitochondria in vivo would change the enzyme kinetics of CPT1. Yeast do not oxidize fatty acids in mitochondria. They do not express any of the components of the CPT system for facilitating transport of fatty acids into mitochondria. From the Greenberg lab we obtained a cardiolipin synthase knock-out strain of S. cerevisiae lacking cardiolipin and having mitochondrial function deficiencies [J. Biol. Chem. (2000) 275, 22387-94], and we obtained its parent strain for use as controls. We transformed both strains with an expression vector for CPT1A, and harvested mitochondria from the cells after breaking cells with glass beads. CPT1A in mitochondria from the parent wild-type yeast had a Km for carnitine of 0.65 ± 0.05 mM, while mitochondria from the cardiolipin synthase knockout yeast had a Km of 0.31 ± 0.03 mM. We also tested malonyl-CoA sensitivity and found mitochondria from wild-type yeast to have an I-50 (concentration of malonyl-CoA producing 50% inhibition) = 0.50 ± 0.07 microM, while mitochondria from cardiolipin synthase knockout yeast had an I-50 = 2.0 ± 0.3 microM. These results suggest that membrane cardiolipin in vivo alters CPT1A to increase its sensitivity to inhibition by malonyl-CoA, and this may be a physiologically significant mechanism for regulating fatty acid oxidation. (Supported by NIH Grant HL-66924)