Cloning, sequencing, and expression of a cDNA encoding rat liver carnitine palmitoyltransferase I

Direct evidence that a single polypeptide is involved in inhibitor interaction and catalytic function

Victoria Esser, Charles H. Britton, Brian C. Weis, Daniel W. Foster, J. Denis McGarry

Research output: Contribution to journalArticle

177 Citations (Scopus)

Abstract

We report the isolation and characterization of a full-length cDNA encoding rat liver carnitine palmitoyltransferase I (CPT I). Oligonucleotides corresponding to two tryptic peptides derived from the malonyl-CoA/ etomoxir-CoA-binding protein of rat liver mitochondria (Esser, V., Kuwajima, M., Britton, C. H., Krishnan, K., Foster, D. W., and McGarry, J. D. (1993) J. Biol. Chem. 268, 5810-5816) were used to screen a rat liver cDNA library constructed in the plasmid cloning vector, pcDV. The clone obtained consisted of a 102-nucleotide 5′-untranslated region, a single open reading frame of 2,319 bases predicting a protein of 773 amino acids (Mr = 88,150), and a 3′-untranslated segment of 1,957 nucleotides followed by the poly(A)+ tail. A 0.9-kilobase fragment of the cDNA recognized a single species of mRNA (∼4.7 kilobases in size) in rat liver. The identity of the cDNA was confirmed by the findings that (i) the open reading frame encoded all four peptides found in the original protein; (ii) transfection of COS cells with the cDNA subcloned into the expression vector, pCMV6, resulted in a selective and 10-20-fold induction of a malonyl-CoA- and etomoxir-CoA-sensitive CPT activity; and (iii) the overexpressed product was readily detected on Western blots by an antibody raised against the starting material. It seems likely that the de novo synthesized enzyme is targeted to the mitochondrial outer membrane via a leader peptide and that the mature protein achieves membrane anchoring through a stretch of 20 amino acids present near its amino terminus. The predicted amino acid sequence of the protein shows regions of strong identity with those of three other rat acyltransferases, namely, liver CPT II, liver carnitine octanoyltransferase, and brain choline acetyltransferase. The findings provide the first insight into the structure of a CPT I isoform. They also establish unequivocally that CPT I and CPT II are distinct proteins and that inhibitors of CPT I interact within its catalytic domain, not with an associated regulatory component.

Original languageEnglish (US)
Pages (from-to)5817-5822
Number of pages6
JournalJournal of Biological Chemistry
Volume268
Issue number8
StatePublished - Mar 15 1993

Fingerprint

Carnitine O-Palmitoyltransferase
Cloning
Liver
Rats
Organism Cloning
Complementary DNA
Peptides
Malonyl Coenzyme A
Proteins
Amino Acids
Open Reading Frames
Nucleotides
Membranes
Acyltransferases
Genetic Vectors
Messenger RNA
Mitochondria
Choline O-Acetyltransferase
5' Untranslated Regions
Protein Sorting Signals

ASJC Scopus subject areas

  • Biochemistry

Cite this

Cloning, sequencing, and expression of a cDNA encoding rat liver carnitine palmitoyltransferase I : Direct evidence that a single polypeptide is involved in inhibitor interaction and catalytic function. / Esser, Victoria; Britton, Charles H.; Weis, Brian C.; Foster, Daniel W.; McGarry, J. Denis.

In: Journal of Biological Chemistry, Vol. 268, No. 8, 15.03.1993, p. 5817-5822.

Research output: Contribution to journalArticle

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title = "Cloning, sequencing, and expression of a cDNA encoding rat liver carnitine palmitoyltransferase I: Direct evidence that a single polypeptide is involved in inhibitor interaction and catalytic function",
abstract = "We report the isolation and characterization of a full-length cDNA encoding rat liver carnitine palmitoyltransferase I (CPT I). Oligonucleotides corresponding to two tryptic peptides derived from the malonyl-CoA/ etomoxir-CoA-binding protein of rat liver mitochondria (Esser, V., Kuwajima, M., Britton, C. H., Krishnan, K., Foster, D. W., and McGarry, J. D. (1993) J. Biol. Chem. 268, 5810-5816) were used to screen a rat liver cDNA library constructed in the plasmid cloning vector, pcDV. The clone obtained consisted of a 102-nucleotide 5′-untranslated region, a single open reading frame of 2,319 bases predicting a protein of 773 amino acids (Mr = 88,150), and a 3′-untranslated segment of 1,957 nucleotides followed by the poly(A)+ tail. A 0.9-kilobase fragment of the cDNA recognized a single species of mRNA (∼4.7 kilobases in size) in rat liver. The identity of the cDNA was confirmed by the findings that (i) the open reading frame encoded all four peptides found in the original protein; (ii) transfection of COS cells with the cDNA subcloned into the expression vector, pCMV6, resulted in a selective and 10-20-fold induction of a malonyl-CoA- and etomoxir-CoA-sensitive CPT activity; and (iii) the overexpressed product was readily detected on Western blots by an antibody raised against the starting material. It seems likely that the de novo synthesized enzyme is targeted to the mitochondrial outer membrane via a leader peptide and that the mature protein achieves membrane anchoring through a stretch of 20 amino acids present near its amino terminus. The predicted amino acid sequence of the protein shows regions of strong identity with those of three other rat acyltransferases, namely, liver CPT II, liver carnitine octanoyltransferase, and brain choline acetyltransferase. The findings provide the first insight into the structure of a CPT I isoform. They also establish unequivocally that CPT I and CPT II are distinct proteins and that inhibitors of CPT I interact within its catalytic domain, not with an associated regulatory component.",
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T1 - Cloning, sequencing, and expression of a cDNA encoding rat liver carnitine palmitoyltransferase I

T2 - Direct evidence that a single polypeptide is involved in inhibitor interaction and catalytic function

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AU - Foster, Daniel W.

AU - McGarry, J. Denis

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N2 - We report the isolation and characterization of a full-length cDNA encoding rat liver carnitine palmitoyltransferase I (CPT I). Oligonucleotides corresponding to two tryptic peptides derived from the malonyl-CoA/ etomoxir-CoA-binding protein of rat liver mitochondria (Esser, V., Kuwajima, M., Britton, C. H., Krishnan, K., Foster, D. W., and McGarry, J. D. (1993) J. Biol. Chem. 268, 5810-5816) were used to screen a rat liver cDNA library constructed in the plasmid cloning vector, pcDV. The clone obtained consisted of a 102-nucleotide 5′-untranslated region, a single open reading frame of 2,319 bases predicting a protein of 773 amino acids (Mr = 88,150), and a 3′-untranslated segment of 1,957 nucleotides followed by the poly(A)+ tail. A 0.9-kilobase fragment of the cDNA recognized a single species of mRNA (∼4.7 kilobases in size) in rat liver. The identity of the cDNA was confirmed by the findings that (i) the open reading frame encoded all four peptides found in the original protein; (ii) transfection of COS cells with the cDNA subcloned into the expression vector, pCMV6, resulted in a selective and 10-20-fold induction of a malonyl-CoA- and etomoxir-CoA-sensitive CPT activity; and (iii) the overexpressed product was readily detected on Western blots by an antibody raised against the starting material. It seems likely that the de novo synthesized enzyme is targeted to the mitochondrial outer membrane via a leader peptide and that the mature protein achieves membrane anchoring through a stretch of 20 amino acids present near its amino terminus. The predicted amino acid sequence of the protein shows regions of strong identity with those of three other rat acyltransferases, namely, liver CPT II, liver carnitine octanoyltransferase, and brain choline acetyltransferase. The findings provide the first insight into the structure of a CPT I isoform. They also establish unequivocally that CPT I and CPT II are distinct proteins and that inhibitors of CPT I interact within its catalytic domain, not with an associated regulatory component.

AB - We report the isolation and characterization of a full-length cDNA encoding rat liver carnitine palmitoyltransferase I (CPT I). Oligonucleotides corresponding to two tryptic peptides derived from the malonyl-CoA/ etomoxir-CoA-binding protein of rat liver mitochondria (Esser, V., Kuwajima, M., Britton, C. H., Krishnan, K., Foster, D. W., and McGarry, J. D. (1993) J. Biol. Chem. 268, 5810-5816) were used to screen a rat liver cDNA library constructed in the plasmid cloning vector, pcDV. The clone obtained consisted of a 102-nucleotide 5′-untranslated region, a single open reading frame of 2,319 bases predicting a protein of 773 amino acids (Mr = 88,150), and a 3′-untranslated segment of 1,957 nucleotides followed by the poly(A)+ tail. A 0.9-kilobase fragment of the cDNA recognized a single species of mRNA (∼4.7 kilobases in size) in rat liver. The identity of the cDNA was confirmed by the findings that (i) the open reading frame encoded all four peptides found in the original protein; (ii) transfection of COS cells with the cDNA subcloned into the expression vector, pCMV6, resulted in a selective and 10-20-fold induction of a malonyl-CoA- and etomoxir-CoA-sensitive CPT activity; and (iii) the overexpressed product was readily detected on Western blots by an antibody raised against the starting material. It seems likely that the de novo synthesized enzyme is targeted to the mitochondrial outer membrane via a leader peptide and that the mature protein achieves membrane anchoring through a stretch of 20 amino acids present near its amino terminus. The predicted amino acid sequence of the protein shows regions of strong identity with those of three other rat acyltransferases, namely, liver CPT II, liver carnitine octanoyltransferase, and brain choline acetyltransferase. The findings provide the first insight into the structure of a CPT I isoform. They also establish unequivocally that CPT I and CPT II are distinct proteins and that inhibitors of CPT I interact within its catalytic domain, not with an associated regulatory component.

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