Premium
Topology of the microsomal glycerol‐3‐phosphate acyltransferase Gpt2p / Gat1p of S accharomyces cerevisiae
Author(s) -
Pagac Martin,
Vazquez Hector M.,
Bochud Arlette,
Roubaty Carole,
Knöpfli Cécile,
Vionnet Christine,
Conzelmann Andreas
Publication year - 2012
Publication title -
molecular microbiology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.857
H-Index - 247
eISSN - 1365-2958
pISSN - 0950-382X
DOI - 10.1111/mmi.12047
Subject(s) - phosphatidic acid , biology , acyltransferases , acyltransferase , biochemistry , membrane topology , biosynthesis , saccharomyces cerevisiae , transmembrane domain , amino acid , yeast , enzyme , phospholipid , membrane
Summary 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 catalysed 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.