Cytological evidence that the C-terminus of carnitine palmitoyltransferase I is on the cytosolic face of the mitochondrial outer membrane

Feike R. Van Der Leij*, Anita M. Kram, Beatrijs Bartelds, Han Roelofsen, Gioia B. Smid, Janny Takens, Victor A. Zammit, Jaap R.G. Kuipers

*Corresponding author for this work

Research output: Contribution to journalArticleAcademicpeer-review

31 Citations (Scopus)

Abstract

Carnitine palmitoyltransferase I (CPT I) is a key enzyme in the regulation of β-oxidation. The topology of this enzyme has been difficult to elucidate by biochemical methods. We studied the topology of a fusion protein of muscle-type CPT I (M-CPT I) and green fluorescent protein (GFP) by microscopical means. To validate the use of the fusion protein, designated CPT I-GFP, we checked whether the main characteristics of native CPT I were retained. CPT I-GFP was expressed in HeLa cells after stable transfection. Confocal laser scanning microscopy in living cells revealed an extranuclear punctate distribution of CPT I-GFP, which coincided with a mitochondrial fluorescent marker. Immunogold electron microscopy localized CPT I-GFP almost exclusively to the mitochondrial periphery and showed that the C-terminus of CPT I must be on the cytosolic face of the mitochondrial outer membrane. Western analysis showed a protein that was 6 kDa smaller than predicted, which is consistent with previous results for the native M-CPT I. Mitochondria from CPT I-GFP-expressing cells showed an increased CPT activity that was inhibited by malonyl-CoA and was lost on solubilization with Triton X-100. We conclude that CPT I-GFP adopts the same topology as native CPT I and that its C-terminus is located on the cytosolic face of the mitochondrial outer membrane. The evidence supports a recently proposed model for the domain structure of CPT I based on biochemical evidence.

Original languageEnglish
Pages (from-to)777-784
Number of pages8
JournalBiochemical Journal
Volume341
Issue number3
DOIs
Publication statusPublished - 1 Aug 1999
Externally publishedYes

Bibliographical note

The Biochemical Society, London © 1999

Fingerprint

Dive into the research topics of 'Cytological evidence that the C-terminus of carnitine palmitoyltransferase I is on the cytosolic face of the mitochondrial outer membrane'. Together they form a unique fingerprint.

Cite this