Jenei, Zsuzsanna A., Borthwick, Karen, Zammit, Victor A. and Dixon, Ann M.. (2009) Self-association of transmembrane domain 2 (TM2), but Not TM1, incCarnitine palmitoyltransferase 1A role of GXXXG(A) motifs. Journal of Biological Chemistry, Vol.284 (No.11). pp. 6988-6997. ISSN 0021-9258

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Abstract

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.

Item Type:	Journal Article
Subjects:	Q Science > QD Chemistry Q Science > QH Natural history > QH301 Biology
Divisions:	Faculty of Science > Chemistry Faculty of Medicine > Warwick Medical School > Clinical Sciences Research Institute (CSRI) Faculty of Medicine > Warwick Medical School > Metabolic and Vascular Health Faculty of Medicine > Warwick Medical School
Library of Congress Subject Headings (LCSH):	Mitochondria, Oligomers, Biochemistry
Journal or Publication Title:	Journal of Biological Chemistry
Publisher:	American Society for Biochemistry and Molecular Biology, Inc.
ISSN:	0021-9258
Date:	13 March 2009
Volume:	Vol.284
Number:	No.11
Number of Pages:	10
Page Range:	pp. 6988-6997
Identification Number:	10.1074/jbc.M808487200
Status:	Peer Reviewed
Publication Status:	Published
Access rights to Published version:	Open Access
Funder:	Diabetes UK, University of Warwick Postgraduate Research Fellowship
URI:	http://wrap.warwick.ac.uk/id/eprint/28381

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