Photolabeling identifies an interaction between phosphatidylcholine and glycerol-3-phosphate dehydrogenase (Gut2p) in yeast mitochondria

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  • Marjolein J F W Janssen
  • Frank van Voorst
  • Ginette E J Ploeger
  • Peter Mose Larsen
  • Martin Røssel Larsen
  • Anton I P M de Kroon
  • Ben de Kruijff

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In search of mitochondrial proteins interacting with phosphatidylcholine (PC), a photolabeling approach was applied, in which photoactivatable probes were incorporated into isolated yeast mitochondria. Only a limited number of proteins were labeled upon photoactivation, using either the PC analogue [125I]TID-PC or the small hydrophobic probe [125I]TID-BE. The most prominent difference was the very specific labeling of a 70 kDa protein by [125I]TID-PC. Mass spectrometric analysis of a tryptic digest of the corresponding 2D-gel spot identified the protein as the GUT2 gene product, the FAD-dependent mitochondrial glycerol-3-phosphate dehydrogenase. This was confirmed by the lack of specific labeling in mitochondria from a gut2 deletion strain. Only under conditions where the inner membrane was accessible to the probe, Gut2p was labeled by [125I]TID-PC, in parallel with increased labeling of the phosphate carrier (P(i)C) in the inner membrane. A hemagglutinin-tagged version of Gut2p was shown to be membrane-bound. Carbonate extraction released the protein from the membrane, whereas a high concentration of NaCl did not, demonstrating that Gut2p is a peripheral membrane protein bound to the inner membrane via hydrophobic interactions. The significance of the observed interactions between Gut2p and PC is discussed.
LanguageEnglish
JournalBiochemistry
Volume41
Issue number18
Pages (from-to)5702-11
Number of pages10
ISSN0006-2960
Publication statusPublished - 7 May 2002

    Research areas

  • Amino Acid Sequence, Blotting, Western, Electrophoresis, Gel, Two-Dimensional, Glycerolphosphate Dehydrogenase, Intracellular Membranes, Iodine Radioisotopes, Mass Spectrometry, Mitochondria, Molecular Probes, Molecular Sequence Data, Peptide Mapping, Phosphatidylcholines, Photochemistry, Protein Transport, Temperature, Yeasts