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.
Article navigation
Research Article|
July 26 1999
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
;
Feike R. VAN DER LEIJ
1
*
Department of Pediatrics, Research Laboratory CMCV 2nd floor, Groningen University Hospital, Groningen University Institute for Drug Exploration, University of Groningen, P.O. Box 30001, NL-9700 RB Groningen, The Netherlands1
To whom correspondence should be addressed (e-mail f.r.van.der.ley@med.rug.nl).
Search for other works by this author on:
Anita M. KRAM
;
Anita M. KRAM
†
Department of Microbiology, Groningen Biomolecular Sciences and Biotechnology Institute, University of Groningen, Kerklaan 30, NL-9751 NN Haren, The Netherlands
Search for other works by this author on:
Beatrijs BARTELDS
;
Beatrijs BARTELDS
*
Department of Pediatrics, Research Laboratory CMCV 2nd floor, Groningen University Hospital, Groningen University Institute for Drug Exploration, University of Groningen, P.O. Box 30001, NL-9700 RB Groningen, The Netherlands
Search for other works by this author on:
Han ROELOFSEN
;
Han ROELOFSEN
*
Department of Pediatrics, Research Laboratory CMCV 2nd floor, Groningen University Hospital, Groningen University Institute for Drug Exploration, University of Groningen, P.O. Box 30001, NL-9700 RB Groningen, The Netherlands‡
Department of Internal Medicine, Groningen University Institute for Drug Exploration, University of Groningen, P.O. Box 30001 NL-9700 RB Groningen, The Netherlands
Search for other works by this author on:
Gioia B. SMID
;
Gioia B. SMID
*
Department of Pediatrics, Research Laboratory CMCV 2nd floor, Groningen University Hospital, Groningen University Institute for Drug Exploration, University of Groningen, P.O. Box 30001, NL-9700 RB Groningen, The Netherlands
Search for other works by this author on:
Janny TAKENS
;
Janny TAKENS
*
Department of Pediatrics, Research Laboratory CMCV 2nd floor, Groningen University Hospital, Groningen University Institute for Drug Exploration, University of Groningen, P.O. Box 30001, NL-9700 RB Groningen, The Netherlands
Search for other works by this author on:
Victor A. ZAMMIT
;
Victor A. ZAMMIT
§
Department of Cellular Biochemistry, Hannah Research Institute, Mauchline Road, Ayr, KA6 5HL Scotland, U.K.
Search for other works by this author on:
Jaap R. G. KUIPERS
Jaap R. G. KUIPERS
*
Department of Pediatrics, Research Laboratory CMCV 2nd floor, Groningen University Hospital, Groningen University Institute for Drug Exploration, University of Groningen, P.O. Box 30001, NL-9700 RB Groningen, The Netherlands
Search for other works by this author on:
Biochem J (1999) 341 (3): 777-784.
Article history
Received:
March 03 1999
Revision Received:
April 22 1999
Accepted:
May 24 1999
Citation
Feike R. VAN DER LEIJ, Anita M. KRAM, Beatrijs BARTELDS, Han ROELOFSEN, Gioia B. SMID, Janny TAKENS, Victor A. ZAMMIT, Jaap R. G. KUIPERS; Cytological evidence that the C-terminus of carnitine palmitoyltransferase I is on the cytosolic face of the mitochondrial outer membrane. Biochem J 1 August 1999; 341 (3): 777–784. doi: https://doi.org/10.1042/bj3410777
Download citation file:
Close
Sign in
Don't already have an account? Register
Sign in to your personal account
You could not be signed in. Please check your email address / username and password and try again.
Biochemical Society Member Sign in
Sign InSign in via your Institution
Sign in via your Institution
2
Views
0
Citations
Cited By
Related Articles
Amino acid sequence and structural repeats in schistosome paramyosin match those of myosin
Biosci Rep (January, 1987)
Partial sequence of hemoglobin from cobra ( Naja naja naja )
Biosci Rep (October, 1987)
Characterization and cloning of avian-hepatic glutathione S-transferases
Biochem J (September, 1999)
Import of a major mitochondrial enzyme depends on synergy between two distinct helices of its presequence
Biochem J (September, 2016)