Self-association of transmembrane domain 2 (TM2), but not TM1, in carnitine palmitoyltransferase 1A: role of GXXXG(A) motifs.
Carnitine palmitoyltransferase 1 (CPT1) controls the rate of entry of long-chain fatty acids into the mitochondrial matrix for beta-oxidation and has been reported to exist as an oligomer. We have investigated the in vivo oligomerization of full-length rat CPT1A (rCPT1A) along with those of the N-terminal truncation/deletion mutants Delta(1-82), Delta(1-18), and Delta(19-30) expressed in yeast mitochondria. The data indicate that in liver mitochondria in vivo CPT1A exists as a hexamer but that during preparation and storage of mitochondria the order of oligomerization is rapidly reduced to the trimer, such that a mixture of hexamer and trimer is observed in isolated mitochondria in vitro. Mutants bearing deletions of different segments of the N terminus (including the more N-terminal of the two transmembrane domains) have the same pattern of oligomerization when expressed in yeast mitochondria. The self-association of the individual rCPT1A transmembrane (TM) domains (TM1, TM2) was also studied using the TOXCAT assay (which measures TM self-association in the Escherichia coli inner membrane). There was minimal self-association of the sequence corresponding to TM1 but significant self-association of TM2 in TOXCAT. Chemical cross-linking and analytical ultracentrifugation of a TM2-derived synthetic peptide showed oligomerization with a similar trimer/hexamer equilibrium to that observed for native rCPT1A in isolated mitochondria. Therefore, there was a correlation between the oligomerization behavior of TM2 peptide and that of the full-length protein. In silico molecular modeling of rCPT1A TM2 highlighted the favorable orientation of GXXXG and GXXXA motifs in the formation of the TM2 hexamer.
Jenei
Zsuzsanna A
ZA
Department of Chemistry and Clinical Sciences Research Institute, Warwick Medical School, University of Warwick, Coventry CV4 7AL, United Kingdom.
Borthwick
Karen
K
Zammit
Victor A
VA
Dixon
Ann M
AM
D020816:Amino Acid Motifs / Q000502:physiology; D000595:Amino Acid Sequence; D000818:Animals; D002334:Carnitine O-Palmitoyltransferase / Q000235:genetics / Q000378:metabolism*; D002462:Cell Membrane / Q000201:enzymology / Q000235:genetics; D003198:Computer Simulation; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D008297:Male; D008930:Mitochondria, Liver / Q000201:enzymology* / Q000235:genetics; D024101:Mitochondrial Proteins / Q000235:genetics / Q000378:metabolism*; D008958:Models, Molecular; D010084:Oxidation-Reduction; D020836:Protein Structure, Quaternary / Q000502:physiology; D017434:Protein Structure, Tertiary / Q000502:physiology; D051381:Rats; D017208:Rats, Wistar; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D017384:Sequence Deletion
D024101:Mitochondrial Proteins; D011994:Recombinant Proteins; D002334:Carnitine O-Palmitoyltransferase