Photolabeling identifies an interaction between phosphatidylcholine and glycerol-3-phosphate dehydrogenase (Gut2p) in yeast mitochondria.
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.
Janssen
Marjolein J F W
MJ
Department Biochemistry of Membranes, Centre for Biomembranes and Lipid Enzymology, Institute of Biomembranes, Utrecht University, Padualaan 8, NL-3584 CH Utrecht, The Netherlands.
van Voorst
Frank
F
Ploeger
Ginette E J
GE
Larsen
Peter Mose
PM
Larsen
Martin R
MR
de Kroon
Anton I P M
AI
de Kruijff
Ben
B
D000595:Amino Acid Sequence; D015153:Blotting, Western; D015180:Electrophoresis, Gel, Two-Dimensional; D005993:Glycerolphosphate Dehydrogenase / Q000235:genetics / Q000378:metabolism*; D007425:Intracellular Membranes / Q000201:enzymology / Q000378:metabolism; D007457:Iodine Radioisotopes; D013058:Mass Spectrometry; D008928:Mitochondria / Q000201:enzymology* / Q000378:metabolism; D015335:Molecular Probes / Q000737:chemistry; D008969:Molecular Sequence Data; D010449:Peptide Mapping; D010713:Phosphatidylcholines / Q000378:metabolism*; D010777:Photochemistry; D021381:Protein Transport; D013696:Temperature; D015003:Yeasts / Q000166:cytology / Q000201:enzymology* / Q000235:genetics / Q000378:metabolism
D007457:Iodine Radioisotopes; D015335:Molecular Probes; D010713:Phosphatidylcholines; D005993:Glycerolphosphate Dehydrogenase