Cytological evidence that the C-terminus of carnitine palmitoyltransferase I is on the cytosolic face of the mitochondrial outer membrane.
Carnitine palmitoyltransferase I (CPT I) is a key enzyme in the regulation of beta-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.
Department of Pediatrics, Research Laboratory CMCV 2nd floor, Groningen University Hospital, NL-9700 RB Groningen, The Netherlands. f.r.van.der.ley@med.rug.nl
F R
FR
van der Leij
A M
AM
Kram
B
B
Bartelds
H
H
Roelofsen
G B
GB
Smid
J
J
Takens
V A
VA
Zammit
J R
JR
Kuipers
D000595:Amino Acid Sequence; D002334:Carnitine O-Palmitoyltransferase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D003600:Cytosol / Q000201:enzymology* / Q000648:ultrastructure; D004705:Endocytosis; D049452:Green Fluorescent Proteins; D006367:HeLa Cells; D006801:Humans; D007425:Intracellular Membranes / Q000201:enzymology / Q000648:ultrastructure; D008164:Luminescent Proteins / Q000235:genetics; D008854:Microscopy, Electron; D008928:Mitochondria / Q000201:enzymology* / Q000648:ultrastructure; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism
D008164:Luminescent Proteins; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins; D002334:Carnitine O-Palmitoyltransferase