Significance of GTP hydrolysis in Ypt1p-regulated endoplasmic reticulum to Golgi transport revealed by the analysis of two novel Ypt1-GAPs.
We here report on the identification and detailed biochemical characterization of two novel GTPase-activating proteins, Gyp5p and Gyp8p, whose efficient substrate is Ypt1p, a Ypt/Rab-GTPase essential for endoplasmic reticulum-to-Golgi trafficking in yeast. Gyp5p accelerated the intrinsic GTPase activity of Ypt1p 4.2 x 10(4)-fold and, surprisingly, the 40-fold reduced GTP hydrolysis rate of Ypt1(Q67L)p 1.5 x 10(4)-fold. At steady state, the two newly discovered GTPase-activating proteins (GAPs) as well as the previously described Gyp1p, which also uses Ypt1p as the preferred substrate, display different subcellular localization. To add to an understanding of the significance of Ypt1p-bound GTP hydrolysis in vivo, yeast strains expressing the GTPase-deficient Ypt1(Q67L)p and having different Ypt1-GAP genes deleted were created. Depending on the genetic background, different mutants exhibited growth defects at low temperature and, already at permissive temperature, various morphological alterations resembling autophagy. Transport of proteins was not significantly impaired. Growth defects of Ypt1(Q67L)-expressing cells could be suppressed on high expression of all three Ypt1-GAPs. We propose that permanently active Ypt1p leads to increased vesicle fusion, which might induce previously unnoticed autophagic degradation of exaggerated membrane-enclosed structures. The data indicate that hydrolysis of Ypt1p-bound GTP is a prerequisite for a balanced vesicle flow between endoplasmic reticulum and Golgi compartments.
De Antoni
Anna
A
Max-Planck-Institute for Biophysical Chemistry, Department of Molecular Genetics, D-37070 Göttingen, Germany.
Schmitzová
Jana
J
Trepte
Hans-Heinrich
HH
Gallwitz
Dieter
D
Albert
Stefan
S
D000595:Amino Acid Sequence; D001343:Autophagy; D001483:Base Sequence; D017931:DNA Primers; D004721:Endoplasmic Reticulum / Q000378:metabolism*; D020690:GTPase-Activating Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D006056:Golgi Apparatus / Q000378:metabolism*; D006160:Guanosine Triphosphate / Q000378:metabolism*; D006868:Hydrolysis; D008854:Microscopy, Electron; D008969:Molecular Sequence Data; D021381:Protein Transport; D012441:Saccharomyces cerevisiae / Q000254:growth & development / Q000378:metabolism / Q000648:ultrastructure; D029701:Saccharomyces cerevisiae Proteins; D017386:Sequence Homology, Amino Acid; D020691:rab GTP-Binding Proteins / Q000378:metabolism*
D017931:DNA Primers; D020690:GTPase-Activating Proteins; D029701:Saccharomyces cerevisiae Proteins; D006160:Guanosine Triphosphate; C060755:YPT1 protein, S cerevisiae; D020691:rab GTP-Binding Proteins