title: Packing of transmembrane domain 2 of carnitine palmitoyltransferase-1A affects oligomerization and malonyl-CoA sensitivity of the mitochondrial outer membrane protein.
abstract: The purpose of this study was to investigate the sequence-dependence of oligomerization of transmembrane domain 2 (TM2) of rat carnitine palmitoyltransferase 1A (rCPT1A), to elucidate the role of this domain in the function of the full-length enzyme. Oligomerization of TM2 was studied qualitatively using complementary genetic assays that facilitate measurement of helix-helix interactions in the Escherichia coli inner membrane, and multiple quantitative biophysical methods. The effects of TM2-mutations on oligomerization and malonyl-CoA inhibition of the full-length enzyme (expressed in the yeast Pichia pastoris) were quantified. Changes designed to disrupt close-packing of the GXXXG(A) motifs reduced the oligomeric state of the corresponding TM2 peptides from hexamer to trimer (or lower), a reduction also observed on mutation of the TM2 sequence in the full-length enzyme. Disruption of these GXXXG(A) motifs had a parallel effect on the malonyl-CoA sensitivity of rCPT1A, reducing the IC(50) from 30.3 ± 5.0 to 3.0 ± 0.6 μM. For all measurements, wild-type rCPT1A was used as a control alongside various appropriate (e.g., molecular mass) standards. Our results suggest that sequence-determined, TM2-mediated oligomerization is likely to be involved in the modulation of malonyl-CoA inhibition of CPT1A in response to short- and long-term changes in protein-protein and protein-lipid interactions that occur in vivo.
authors.affiliation: Department of Chemistry, University of Warwick, Coventry, CV4 7AL, UK.
authors.forename: Zsuzsanna A
authors.initials: ZA
authors.lastname: Jenei
authors.forename: Gemma Z L
authors.initials: GZ
authors.lastname: Warren
authors.forename: Muhammad
authors.initials: M
authors.lastname: Hasan
authors.forename: Victor A
authors.initials: VA
authors.lastname: Zammit
authors.forename: Ann M
authors.initials: AM
authors.lastname: Dixon
mesh_terms: D020816:Amino Acid Motifs; D000818:Animals; D001483:Base Sequence; D002334:Carnitine O-Palmitoyltransferase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D017931:DNA Primers / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D066298:In Vitro Techniques; D008316:Malonyl Coenzyme A / Q000378:metabolism; D051336:Mitochondrial Membranes / Q000378:metabolism; D024101:Mitochondrial Proteins / Q000378:metabolism; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D010843:Pichia / Q000235:genetics / Q000378:metabolism; D055503:Protein Multimerization; D017434:Protein Structure, Tertiary; D051381:Rats; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism
chemical_list: D017931:DNA Primers; D024101:Mitochondrial Proteins; D011994:Recombinant Proteins; D008316:Malonyl Coenzyme A; D002334:Carnitine O-Palmitoyltransferase