Regulation of mitochondrial outer-membrane carnitine palmitoyltransferase (CPT I): role of membrane-topology.
The topology of the outer membrane carnitine palmitoyltransferase (CPT I) of rat liver mitochondria was studied systematically using several experimental approaches. Studies with immobilized malonyl-CoA and octanoyl-CoA showed that functionally the active and regulatory sites of CPT I are exposed on the outer (cytosolic) surface of the mitochondrial outer membrane. Anti-peptide antibodies generated against three linear peptide sequences that occur in between and on either side of two hydrophobic, putative transmembrane domains were used to (a) ascertain which were bound by intact mitochondria and mitochondria in which the outer membrane was permeabilized to proteins; and (b) to determine the size of fragments generated by limited proteolysis (by trypsin or proteinase K) of CPT I in intact or outer membrane-ruptured mitochondria. The sizes and immunoreactivity of the proteolytic fragments generated were correlated with the effects of the proteases on CPT I activity and malonyl-CoA sensitivity. The results of all the different approaches suggested the following: (i) CPT I has two transmembrane domains; (ii) both the N- and C-termini are exposed on the cytosolic side of the membrane; (iii) the linker region between the two transmembrane domains protrudes into the intermembrane space; (iv) both the active site and the malonyl-CoA-binding site are exposed on the cytosolic side of the membrane; (v) the amino-terminus of the protein interacts with the C-terminal domain of the protein to maintain the optimal conformation required for activity of the enzyme and its sensitivity to malonyl-CoA.
Zammit
V A
VA
Hannah Research Institute, Ayr, U.K.
Fraser
F
F
Orstorphine
C G
CG
D000214:Acyl Coenzyme A / Q000096:biosynthesis* / Q000378:metabolism; D000818:Animals; D001665:Binding Sites; D015153:Blotting, Western; D002334:Carnitine O-Palmitoyltransferase / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000378:metabolism*; D010450:Endopeptidases / Q000378:metabolism; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004800:Enzymes, Immobilized / Q000378:metabolism; D007425:Intracellular Membranes / Q000201:enzymology*; D007700:Kinetics; D008316:Malonyl Coenzyme A / Q000378:metabolism; D008930:Mitochondria, Liver / Q000201:enzymology* / Q000378:metabolism; D010446:Peptide Fragments / Q000737:chemistry / Q000276:immunology; D051381:Rats; D017208:Rats, Wistar
D000214:Acyl Coenzyme A; D004791:Enzyme Inhibitors; D004800:Enzymes, Immobilized; D010446:Peptide Fragments; C034681:octanoyl-coenzyme A; D008316:Malonyl Coenzyme A; D002334:Carnitine O-Palmitoyltransferase; D010450:Endopeptidases