The GTPase-activating enzyme Gyp1p is required for recycling of internalized membrane material by inactivation of the Rab/Ypt GTPase Ypt1p.
Rab/Ypt GTPases are key regulators of membrane trafficking and together with SNARE proteins mediate selective fusion of vesicles with target compartments. A family of GTPase-activating enzymes (GAPs) specific for Rab/Ypt GTPases has been discovered, but little is known about their function and substrate specificity in vivo. Here we show that the GAP activity of Gyp1p, a yeast member of this family, is specifically required for recycling of the SNARE Snc1p and the membrane dye FM4-64, implying that inactivation of a Rab/Ypt GTPase may be necessary for recycling of membrane material. Interestingly, recycling of GFP-Snc1p in gyp1 Delta cells is partially restored by reducing the activity of Ypt1p. Moreover, GFP-Snc1p accumulated intracellularly in wild-type cells expressing a GTP-locked, mutant form of Ypt1p (Ypt1p-Q67L), suggesting that GTP hydrolysis of Ypt1p is essential for recycling. Ypt6p is known to be required for the fusion of recycling vesicles to the late Golgi compartment. Interestingly, the deletions of GYP1 and YPT6 were synthetic lethal, raising the possibility that at least two distinct pathways are involved in recycling of membrane material.
Lafourcade
Céline
C
Swiss Federal Institute of Technology Zurich (ETH), Institute of Biochemistry, ETH Hoenggerberg, 8093 Zurich, Switzerland.
Galan
Jean-Marc
JM
Gloor
Yvonne
Y
Haguenauer-Tsapis
Rosine
R
Peter
Matthias
M
D000595:Amino Acid Sequence; D000818:Animals; D001692:Biological Transport / Q000502:physiology; D002462:Cell Membrane / Q000378:metabolism*; D004705:Endocytosis / Q000502:physiology; D011992:Endosomes / Q000378:metabolism; D005089:Exocytosis / Q000502:physiology; D005456:Fluorescent Dyes / Q000378:metabolism; D020690:GTPase-Activating Proteins / Q000235:genetics / Q000378:metabolism*; D006056:Golgi Apparatus / Q000378:metabolism; D006801:Humans; D008565:Membrane Proteins / Q000378:metabolism*; D026901:Membrane Transport Proteins / Q000378:metabolism; D008969:Molecular Sequence Data; D011726:Pyridinium Compounds / Q000378:metabolism; D000644:Quaternary Ammonium Compounds / Q000378:metabolism; D050683:R-SNARE Proteins; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000166:cytology / Q000235:genetics / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics / Q000378:metabolism*; D016415:Sequence Alignment; D022161:Transport Vesicles / Q000378:metabolism*; D020691:rab GTP-Binding Proteins / Q000378:metabolism*
C092350:FM 4-64; D005456:Fluorescent Dyes; D020690:GTPase-Activating Proteins; C416529:GYP1 protein, S cerevisiae; D008565:Membrane Proteins; D026901:Membrane Transport Proteins; D011726:Pyridinium Compounds; D000644:Quaternary Ammonium Compounds; D050683:R-SNARE Proteins; D011993:Recombinant Fusion Proteins; C083241:SNC1 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins; C060755:YPT1 protein, S cerevisiae; D020691:rab GTP-Binding Proteins