Identification of yeast component A: reconstitution of the geranylgeranyltransferase that modifies Ypt1p and Sec4p.
Members of a large family of small GTP-binding proteins, termed Rabs in mammalian cells or Ypt and Sec4 in yeast, regulate vesicular traffic in all eukaryotic cells. These proteins are able to bind to membranes because they are modified by the type II geranylgeranyltransferase (GGTase-II), a multisubunit complex. Component A, encoded by the choroideremia gene in humans, is an escort protein that brings Rabs to component B, the catalytic alpha/beta heterodimer. Mutations in the catalytic subunits of the yeast GGTase-II (Bet2p/Mad2p) disrupt the membrane attachment of Ypt1p and Sec4p and this in turn blocks membrane traffic. In mammalian cells, deletions in choroideremia lead only to retinal degeneration, even though GGTase-II activity is defective. The yeast MRS6 gene encodes a protein that is approximately 30% identical to the choroideremia gene product. Here we show that the addition of recombinant Mrs6p to bacterially expressed Bet2p (beta subunit) and Mad2p (alpha subunit) reconstitutes GGTase-II activity in vitro, demonstrating that Mrs6p is yeast component A. Like Bet2p and Mad2p, Mrs6p is required for the membrane attachment of Ypt1p and Sec4p in vivo. In contrast to what has been observed before for the loss of function of the choroideremia gene, the depletion of Mrs6p from yeast cells blocks vesicular transport. Thus, these findings suggest that there is one essential escort protein in yeast, while more than one may exist in mammalian cells.
Jiang
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Y
Department of Cell Biology, Howard Hughes Medical Institute, Yale University School of Medicine, New Haven, CT 06510.
Ferro-Novick
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S
D019883:Alkyl and Aryl Transferases; D000818:Animals; D002462:Cell Membrane / Q000378:metabolism; D015794:Choroideremia / Q000235:genetics; D005656:Fungal Proteins / Q000378:metabolism; D019204:GTP-Binding Proteins / Q000302:isolation & purification / Q000378:metabolism*; D017353:Gene Deletion; D005800:Genes, Fungal; D006801:Humans; D007700:Kinetics; D008322:Mammals; D011401:Promoter Regions, Genetic; D012162:Retinal Degeneration / Q000235:genetics; D012441:Saccharomyces cerevisiae / Q000201:enzymology / Q000235:genetics / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins; D014166:Transferases / Q000378:metabolism*; D020691:rab GTP-Binding Proteins
D005656:Fungal Proteins; D029701:Saccharomyces cerevisiae Proteins; D014166:Transferases; D019883:Alkyl and Aryl Transferases; C070117:Geranylgeranyltransferase Type II protein, S cerevisiae; D019204:GTP-Binding Proteins; C060755:YPT1 protein, S cerevisiae; C064320:SEC4 protein, S cerevisiae; D020691:rab GTP-Binding Proteins