Expression of a cDNA for rat liver carnitine palmitoyltransferase I in yeast establishes that catalytic activity and malonyl-CoA sensitivity reside in a single polypeptide

N F Brown,V Esser,D W Foster,J D Mcgarry

doi:10.1016/s0021-9258(18)47213-4

N F Brown, V Esser + Show 2 more

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https://doi.org/10.1016/s0021-9258(18)47213-4

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Abstract

A cDNA encoding full-length carnitine palmitoyltransferase I (CPT I) from rat liver was expressed in Saccharomyces cerevisiae, a system devoid of endogenous CPT activity. The recombinant enzyme was of the expected size (as deduced from immunoblots), membrane-bound, and detergent-labile. It was also potently inhibited by malonyl-CoA, with an I50 value (concentration causing 50% inhibition) of approximately 5 microM, similar to that of the native enzyme in rat liver mitochondria. A truncated variant of the enzyme that lacked the amino-terminal 82 residues encompassing the first hydrophobic domain retained catalytic function but was much less sensitive to malonyl-CoA (I50 > 80 microM). Deletion of the cDNA segment encoding amino acids 31-148 (which includes both first and second hydrophobic stretches) resulted in no detectable product. The data establish unequivocally that a single polypeptide possesses both catalytic and malonyl-CoA binding domains, as well as the other properties previously attributed by us to native CPT I in mammalian mitochondria, and should thus put to rest the controversy surrounding this issue (Kerner, J., Zaluzec, E., Gage, D., and Bieber, L. L. (1994) J. Biol. Chem. 269, 8209-8219). In addition, the results strengthen the view that one site of interaction of malonyl-CoA with the rat liver enzyme involves the NH2-terminal region of the molecule.

Highlights

A truncated varianot f the enzyme that lacketdhe ami- is there anassociated malonyl-CoAbinding regulatory subunit no-terminal 82 residues encompassing the first hydro- that is presentonly on the outer mitochondrial membrane?
The data establish unequivocally that a single poly- that thisbody of work be summarized briefly. 1)Treatment of peptide possesses both catalytic and malonyl-CoA bind- rat liver mitochondrial membranes with the mild detergent, ing domains, as well as the other properties previously Tween 20, released a soluble carnitine palmitoyltransferase (CPT) activity that wasnot inhibattributed by us to native CPT I in mammalian mito- itable by malonyl-CoA but left a particulate fraction that was chondria, and should put to rest the controversy greatly enriched in malonyl-CoA-sensitive enzyme [3]
CoA was purified from rat liver mitochondria and its corresponding cDNAisolated [101, 1].Transfection of COS cells with this construct resulted in a 10-20-fold increase in malonylCoA-sensitive CPT activity [11].We believe that this cDNA encodes a CPT I polypeptide having both catalytic activity and malonyl-CoMetomoxir-CoAsensitivity

Summary

Introduction

A truncated varianot f the enzyme that lacketdhe ami- is there anassociated malonyl-CoAbinding regulatory subunit no-terminal 82 residues encompassing the first hydro- that is presentonly on the outer mitochondrial membrane?. Deletion strongly supportive of a model in which CPT I activity and its of the cDNA segmentencodingamino acids 31-148 sensitivity t o malonyl-CoA are properties of a single polypep-

Results

Conclusion