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Topology of the microsomal glycerol-3-phosphate acyltransferase Gpt2p/Gat1p of Saccharomyces cerevisiae

  • Pagac, Martin Department of Biology, University of Fribourg, Switzerland - College of Pharmacy, University of Hawaii, Hilo, USA
  • Vazquez, Hector M. Department of Biology, University of Fribourg, Switzerland
  • Bochud, Arlette Department of Biology, University of Fribourg, Switzerland
  • Roubaty, Carole Department of Biology, University of Fribourg, Switzerland
  • Knöpfli, Cécile Department of Biology, University of Fribourg, Switzerland
  • Vionnet, Christine Department of Biology, University of Fribourg, Switzerland
  • Conzelmann, Andreas Department of Biology, University of Fribourg, Switzerland
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    18.09.2012
Published in:
  • Molecular Biology. - 2012, vol. 86, no. 5, p. 1156-1166
Dual topology reporter
cysteine accessibility
phosphatidic acid
lyso phosphatidic acid acyl Coenzyme A
microsomes
English All glycerophospholipids are made from phosphatidic acid, which, according to the traditional view, is generated at the cytosolic surface of the ER. In yeast, phosphatidic acid is synthesized de novo by two acyl-CoA dependent acylation reactions. The first is catalyzed by one of the two homologous glycerol-3-phosphate acyltransferases Gpt2p/Gat1p and Sct1p/Gat2p, the second by one of the two 1-acyl-sn-glycerol-3-phosphate acyltransferases Slc1p and Ale1p/Slc4p. To study the biogenesis and topology of Gpt2p we observed the location of dual topology reporters inserted after various transmembrane helices. Moreover, using microsomes, we probed the accessibility of natural and substituted cysteine residues to a membrane impermeant alkylating agent and tested the protease sensitivity of various epitope tags inserted into Gpt2p. Finally, we assayed the sensitivity of the acyltransferase activity to membrane impermeant agents targeting lysine residues. By all these criteria we find that the most conserved motifs of Gpt2p and its functionally relevant lysines are oriented towards the ER lumen. Thus, the first step in biosynthesis of phosphatidic acid in yeast seems to occur in the ER lumen and substrates may have to cross the ER membrane.
Faculty
Faculté des sciences et de médecine
Department
Département de Biologie
Language
  • English
Classification
Biological sciences
License
License undefined
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Persistent URL
https://folia.unifr.ch/unifr/documents/302541
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