Evidence that both the acyl-CoA- and malonyl-CoA binding sites of mitochondrial overt carnitine palmitoyltransferase (CPT I) are exposed on the cytosolic face of the outer membrane.

Abstract

Mitochondria1 overt carnitine palmitoyltransferase (CPT I) is an integral membrane protein which resides in the outer membrane of mitochondria [ 11. Its primary structure, deduced from its cDNA sequence [2], has two hydrophobic stretches which have been suggested [3] to represent transmembrane domains (TMDs). A bitopic topography of the protein within the membrane is also suggested by the hypothesis [ 11 that the active site and malonyl-CoA-binding site of the enzyme reside on opposite sides of the membrane. This has emerged from studies in which exposure of intact mitochondria to proteases results in the loss of sensitivity of CPT I to malonyl-CoA inhibition before the loss of catalytic activity [1,4,5]. However in these studies the possibility that the effect on sensitivity could be due to protease action on other membrane components was not considered. We performed limited proteulysis experiments on mitochondria in which the outer membrane was either left intact (incubations in iso-osmotic medium) or in which the membrane was ruptured (by swelling the mitochondna in hypo-osmotic medium). This approach allowed us to find out whether important determinants of catalytic activity andor malonyl-CoA sensitivity occurred on either side of the membrane. In addition, we have used agarose-immobilised malonyl-CoA and octanoyl-CoA (to make them impermeable through the outer membrane) to ascertain whether these ligands are able to interact with the outer (cytosolic) face of the membrane in intact mitochondna. The mitochondria1 preparations used were purified by Percollgradient centrifugation [6] to minimise contamination with microsomes and peroxisomes, which also contain malonyl-CoA-sensitive CPT activities [7,8]. The f d mitochondnal suspension was split into two and the ensuing pellets were resuspended (5mg protedml) in iso-osmotic (25OmM-sucrose, 5mM K phosphate, 0.lmM EGTA, pH 7.4) or hypoosmotic buffer (same but containing only 25mM-sucrose). Proteinase K (5p(g/ml) or trypsin (lOpcg/ml) were added and incubations were performed at 30°C for periods up to 30 min (proteinase K) or 60 min (trypsin). Protease action was stopped by addition of suitable inhibitors. The activity of CPT and of marker enzymes was measured. OctanoylCoA and malonyl-CoA were. unmobilised onto Sepharose 4 8 via 6or 14atom spacer arms via the amino N of the adenine moiety of CoA. hteinase K induced an identical rate of loss of CPT I activity in intact or swollen mitochondna, suggesting that cleavage at a site on the outer face of the membrane results in the disruption of a domain important for CPT I activity. By contrast, trypsin only had a significant effect on enzyme activity when the inner face of the membrane was also exposed. The response of the malonyl-CoA sensitivity of CPT I to protease action also depended on the identity of the protease. Proteinase K action resulted in the loss of sensitivity in intact and swollen mitochondna, but the effect was more rapid when both sides of the membrane were exposed to the protease. By contrast trqpsin did not affect malonyl-CoA sensitivity even when signhcant loss of activity occurred (in swollen mitochondria). The data suggest that the effects on CPT I characteristics were dependent not only on the sidedness of domains crucial for the expression of activity of CPT I or its sensitivity to malonyl-CoA, but also on the specificities of the proteases and the accessibility of the relevant amino acid residues to them. Consequently, limited proteolysis experiments cannot be used in isolation to determine the topography of CPT 1. Experiments using immobilised papain on intact mitochondria confirmed that site(s) crucial for both CPT I activity and malonyl-CoA-sensitivity are exposed on the cytosolic face of the outer membrane. In order to evaluate the possibility that the actual binding sites for both substrate (octanoyl-CoA) and malonyl-CoA could be located on the cytosol-facing aspect of the outer membrane, experiments were carried out in which the ability of agarose-immobilised octanoyl-CoA or malonylCoA to act as substrate and inhibitor, respectively, was assessed. The ligands were. linked to Sepharose 4B, through the amino N on the CoA Table 1. Agarose4mmobilised octanoyl-CoA or -malonyl-CoA can act as substrate or inhibitor for CPT I in intact mitochondria