TOPOLOGY OF CARNITINE PALMITOYLTRANSFERASE-I IN THE MITOCHONDRIAL OUTER-MEMBRANE

The topology of carnitine palmitoyltransferase I (CPT I) in the outer membrane of rat liver mitochondria was studied using several approache s. 1. The accessibility of the active site and malonyl-CoA-binding sit e of the enzyme from the cytosolic aspect of the membrane was investig ated using preparations of octanoyl-CoA and malonyl-CoA immobilized on to agarose beads to render them impermeant through the outer membrane . Both immobilized ligands were fully able to interact effectively wit h CPT I. 2. The effects of proteinase K and trypsin on the activity an d malonyl-CoA sensitivity of CPT I were studied using preparations of mitochondria that were either intact or had their outer membranes rupt ured by hypo-osmotic swelling (OMRM). Proteinase K had a marked but si milar effect on CPT I activity irrespective of whether only the cytoso lic or both sides of the membrane were exposed to it. However, it affe cted sensitivity more rapidly in OMRM. By contrast, trypsin only reduc ed CPT I activity when incubated with OMRM. The sensitivity of the res idual CPT I activity was unaffected by trypsin. 3. The proteolytic fra gments generated by these treatments were studied by Western blotting using three anti-peptide antibodies raised against linear epitopes of CPT I. These showed that a proteinase K-sensitive site close to the N- terminus was accessible from the cytosolic side of the membrane. No tr ypsin-sensitive sites were accessible in intact mitochondria. In OMRM, both proteinase K and trypsin acted from the inter-membrane space sid e of the membrane. 4. The ability of intact mitochondria and OMRM to b ind to each of the three anti-peptide antibodies was used to study the accessibility of the respective epitopes on the cytosolic and inter-m embrane space sides of the membrane. 5. The results of all these appro aches indicate that CPT I adopts a bitopic topology within the mitocho ndrial outer membrane; it has two transmembrane domains, and both the N- and C-termini are exposed on the cytosolic side of the membrane, wh ereas the linker region between the transmembrane domains protrudes in to the intermembrane space.