The N-terminal domain of rat liver carnitine palmitoyltransferase 1 contains an internal mitochondrial import signal and residues essential for folding of its C-terminal catalytic domain

We have previously shown that the first 147 N-terminal residues of the rat liver carnitine palmitoyltransferase 1 (CPT1), encompassing its two transme mbrane (TM) segments, specify both mitochondrial targeting and anchorage at the outer mitochondrial membrane (OMM), In the present study, we have iden tified the precise import sequence in this polytopic OMM protein. In vitro import studies with fusion and deletion CPT1 proteins demonstrated that non e of its TRI segments behave as a signal anchor sequence, Analysis of the r egions flanking the TM segments revealed that residues 123-147, located imm ediately downstream of TM2, function as a noncleavable, matrix-targeting si gnal, They specify mitochondrial targeting, whereas the hydrophobic TM segm ent(s) acts as a stop-transfer sequence that stops and anchors the transloc ating CPT1 into the OMM, Heterologous expression in Saccharomyces cerevisia e of several deleted CPT1 proteins not only confirms the validity of the "s top-transfer" import model but also indicates that residues 1-82 of CPT1 co ntain a putative microsomal targeting signal whose cellular significance aw aits further investigation. Finally, we identified a highly folded core wit hin the C-terminal domain of CPT1 that is hidden in the entire protein by i ts cytosolic N-terminal residues. Functional analysis of the deleted CPT1 p roteins indicates that this folded C-terminal core, which may belong to the catalytic domain of CPT1, requires TM2 for its correct folding achievement and is in close proximity to residues 1-47.