The Extreme C Terminus of Rat Liver Carnitine Palmitoyltransferase I Is Not Involved in Malonyl-CoA Sensitivity but in Initial Protein Folding

Yong Pan,Isabelle Cohen,Fanny Guillerault,Bruno Fève,Jean Girard,Carina Prip-Buus

doi:10.1074/jbc.m208055200

Abstract

We previously reported that the N-terminal domain (1-147 residues) of rat liver carnitine palmitoyltransferase I (L-CPTI) was essential for import into the outer mitochondrial membrane and for maintenance of a malonyl-CoA-sensitive conformation. Malonyl-CoA binding experiments using mitochondria of Saccharomyces cerevisiae strains expressing wild-type L-CPTI or previously constructed chimeric CPTs (Cohen, I., Kohl, C., McGarry, J.D., Girard, J., and Prip-Buus, C. (1998) J. Biol. Chem. 273, 29896-29904) indicated that the N-terminal domain was unable, independently of the C-terminal domain, to bind malonyl-CoA with a high affinity, suggesting that the modulation of malonyl-CoA sensitivity occurred through N/C intramolecular interactions. To assess the role of the C terminus in malonyl-CoA sensitivity, a series of C-terminal deletion mutants was generated. The kinetic properties of Delta772-773 and Delta767-773 deletion mutants were similar to those of L-CPTI, indicating that the last two highly conserved Lys residues in all known L-CPTI species were not functionally essential. By contrast, Delta743-773 deletion mutant was totally inactive and unfolded, as shown by its sensitivity to trypsin proteolysis. Because the C terminus of the native folded L-CPTI could be cleaved by trypsin without inducing protein unfolding, we concluded that the last 31 C-terminal residues constitute a secondary structural determinant essential for the initial protein folding of L-CPTI.

Highlights

We previously reported that the N-terminal domain (1–147 residues) of rat liver carnitine palmitoyltransferase I (L-CPTI) was essential for import into the outer mitochondrial membrane and for maintenance of a malonyl-CoA-sensitive conformation
We examined whether the N-terminal domain of L-CPTI exhibits a high affinity for malonyl-CoA binding independently of the presence of the C-terminal domain of L-CPTI, and we investigated for the first time whether the extreme C terminus of L-CPTI, which exhibits notable differences with its M-CPTI counterpart, could be involved in the N/C intramolecular interactions that determine the degree of malonyl-CoA sensitivity
N-terminal Domain of L-CPTI and Malonyl-CoA Sensitivity—The N-terminal domain of rat L-CPTI is responsible for protein targeting to the outer mitochondrial membrane but is essential for malonyl-CoA sensitivity [13,14,15, 17, 20]

Summary

Introduction

We previously reported that the N-terminal domain (1–147 residues) of rat liver carnitine palmitoyltransferase I (L-CPTI) was essential for import into the outer mitochondrial membrane and for maintenance of a malonyl-CoA-sensitive conformation. A unique feature of CPTI is its potent inhibition by malonyl-CoA, the first committed intermediate of fatty acid biosynthesis [2] This provides a mechanism for physiological regulation of ␤-oxidation in liver. The N-terminal domain (1–147 residues) of L-CPTI is responsible for mitochondrial targeting and import into the OMM but is involved in maintenance of a folded active and malonyl-CoA-sensitive conformation of the enzyme [13]. TM2 was shown to be essential for the correct folding of the catalytic domain of L-CPTI, suggesting that some N/C intramolecular interactions may be directly involved in the establishment and/or maintenance of the native functional conformation of L-CPTI [14].

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