THE MALONYL-COA-SENSITIVE FORM OF CARNITINE PALMITOYLTRANSFERASE IS NOT LOCALIZED EXCLUSIVELY IN THE OUTER-MEMBRANE OF RAT-LIVER MITOCHONDRIA

The data used to support the idea that malonyl-coenzyme A (CoA)-sensit ive carnitine palmitoyltransferase (CPT-I) is localized on the outer m itochondrial membrane are based on harsh techniques that disrupt mitoc hondrial physiology. We have turned to the use of the French press, wh ich produces a shearing force that denudes mitochondria of their outer membrane without the physiologically disruptive effects characteristi c of phosphate swelling. Our results indicate that the mitoplasts cont ain just 15-19% of the outer membrane marker enzyme activity while ret aining 85% of the total CPT activity and 50% of both CPT-I, as well as long-chain acyl-CoA synthase activity, the latter two supposed outer membrane enzymes. These mitoplasts were shown by electron microscopy t o have the configuration of mitochondria that merely have been diveste d of their outer membranes. Carnitine-dependent fatty acid oxidation w as retained in the mitoplasts, showing that they were physiologically intact. Moreover, protein immuno blotting analysis showed that CPT-I, as well as the inner CPT-II, was localized in the mitoplast fraction. The outer membrane fraction, which consisted of membrane ''ghosts,'' c ontained most (50-60%) of marker enzyme activity, monoamine oxidase-B and porin proteins, but only about 27-29% CPT-I activity. Because CPT- I and long-chain acyl-CoA synthetase appear to be associated with both inner and outer membranes, we postulate that these enzymes reside in contact sites, which represent a melding of both limiting membranes.