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Packing of transmembrane domain 2 of carnitine palmitoyltransferase‐1A affects oligomerization and malonyl‐CoA sensitivity of the mitochondrial outer membrane protein
Author(s) -
Jenei Zsuzsanna A.,
Warren Gemma Z. L.,
Hasan Muhammad,
Zammit Victor A.,
Dixon Ann M.
Publication year - 2011
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fj.11-192005
Subject(s) - transmembrane protein , transmembrane domain , chemistry , carnitine , carnitine palmitoyltransferase i , carnitine o palmitoyltransferase , inner mitochondrial membrane , sensitivity (control systems) , mitochondrion , domain (mathematical analysis) , malonyl coa , microbiology and biotechnology , membrane , biophysics , biochemistry , biology , enzyme , beta oxidation , receptor , mathematical analysis , mathematics , electronic engineering , engineering
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 G XXX G(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 G XXX G(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 . Jenei, Z. A., Warren, G. Z. L., Hasan, M., Zammit, V. A., Dixon, A. M. Packing of transmembrane domain 2 of carnitine palmitoyltransferase‐1A affects oligomerization and malonyl‐CoA sensitivity of the mitochondrial outer membrane protein. FASEB J. 25, 4522–4530 (2011). www.fasebj.org