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 transmembrane (TM) segments, specify both mitochondrial targeting and anchorage at the outer mitochondrial membrane (OMM). In the present study, we have identified the precise import sequence in this polytopic OMM protein. In vitro import studies with fusion and deletion CPT1 proteins demonstrated that none of its TM segments behave as a signal anchor sequence. Analysis of the regions flanking the TM segments revealed that residues 123-147, located immediately downstream of TM2, function as a noncleavable, matrix-targeting signal. They specify mitochondrial targeting, whereas the hydrophobic TM segment(s) acts as a stop-transfer sequence that stops and anchors the translocating CPT1 into the OMM. Heterologous expression in Saccharomyces cerevisiae of several deleted CPT1 proteins not only confirms the validity of the "stop-transfer" import model but also indicates that residues 1-82 of CPT1 contain a putative microsomal targeting signal whose cellular significance awaits further investigation. Finally, we identified a highly folded core within the C-terminal domain of CPT1 that is hidden in the entire protein by its cytosolic N-terminal residues. Functional analysis of the deleted CPT1 proteins 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.
Cohen
I
I
Endocrinologie, Métabolisme et Développement, CNRS-UPR 1524, 9 Rue J. Hetzel, 92190 Meudon, France.
Guillerault
F
F
Girard
J
J
Prip-Buus
C
C
D000818:Animals; D002334:Carnitine O-Palmitoyltransferase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D020134:Catalytic Domain; D008565:Membrane Proteins / Q000378:metabolism; D008930:Mitochondria, Liver / Q000201:enzymology*; D008954:Models, Biological; D017510:Protein Folding; D017434:Protein Structure, Tertiary; D021381:Protein Transport; D051381:Rats; D012441:Saccharomyces cerevisiae / Q000235:genetics; D017384:Sequence Deletion; D014162:Transfection
D008565:Membrane Proteins; D002334:Carnitine O-Palmitoyltransferase