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van der Leij, F.R. et al.

van der Leij, Kram, Bartelds, Roelofsen, Smid, Takens, Zammit, Kuipers,

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.[1]

References

Cytological evidence that the C-terminus of carnitine palmitoyltransferase I is on the cytosolic face of the mitochondrial outer membrane. van der Leij, F.R., Kram, A.M., Bartelds, B., Roelofsen, H., Smid, G.B., Takens, J., Zammit, V.A., Kuipers, J.R. Biochem. J. (1999) [Pubmed]

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