Mutational analysis of the putative effector domain of the GTP-binding Ypt1 protein in yeast suggests specific regulation by a novel GAP activity.
Ypt1p of Saccharomyces cerevisiae is a ras-related GTP-binding protein that fulfils an essential function in intracellular protein transport between the endoplasmic reticulum (ER) and the Golgi complex. Ypt proteins from yeasts and mammals that share an identical sequence in the region analogous to the ras effector domain are functionally interchangeable. We analyzed the function of the putative effector domain of yeast Ypt1p (amino acids 37-45) using site-directed mutagenesis and gene replacement. Four out of six point mutations leading to single amino acid substitutions (Y37F, S39A, T40S and V43E) did not cause any particular phenotype. ypt1(I41M) mutants were inviable whereas ypt1(D44N) mutant cells were temperature sensitive at 37 degrees C and accumulated core-glycosylated invertase at the nonpermissive temperature. This mutant also accumulated ER and small vesicles both at 25 degrees C and 37 degrees C. From porcine liver we identified and partially purified a GTPase-activating protein (yptGAP) that is similarly active with mouse ypt1p/rab1p and yeast Ypt1p but is inactive with H-ras protein as a substrate. Although none of the yeast ypt1 mutant proteins were significantly impaired in their ability to bind GTP, purified ypt1(D44N)p responded only partially and ypt1(I41M)p did not respond at all, to yptGAP. Thus we suggest that analogous to rasGAP/H-ras p21 interaction in mammalian cells, yptGAP is an intracellular target of Ypt1p, interacting with the effector domain and regulating its GTPase activity, and that this interaction is required for the functioning of yeast Ypt1p in intracellular protein transport.
Becker
J
J
Max-Planck-Institute for Biophysical Chemistry, Department of Molecular Genetics, Göttingen, Germany.
Tan
T J
TJ
Trepte
H H
HH
Gallwitz
D
D
D000595:Amino Acid Sequence; D001483:Base Sequence; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics; D005656:Fungal Proteins / Q000235:genetics; D020558:GTP Phosphohydrolases / Q000378:metabolism; D020690:GTPase-Activating Proteins; D005800:Genes, Fungal; D006026:Glycoside Hydrolases / Q000378:metabolism; D007700:Kinetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D015345:Oligonucleotide Probes; D010641:Phenotype; D011506:Proteins / Q000235:genetics* / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000235:genetics* / Q000254:growth & development / Q000378:metabolism / Q000648:ultrastructure; D029701:Saccharomyces cerevisiae Proteins; D013696:Temperature; D043324:beta-Fructofuranosidase; D020691:rab GTP-Binding Proteins; D020703:ras GTPase-Activating Proteins
D005656:Fungal Proteins; D020690:GTPase-Activating Proteins; D015345:Oligonucleotide Probes; D011506:Proteins; D029701:Saccharomyces cerevisiae Proteins; D020703:ras GTPase-Activating Proteins; D006026:Glycoside Hydrolases; D043324:beta-Fructofuranosidase; D020558:GTP Phosphohydrolases; C060755:YPT1 protein, S cerevisiae; D020691:rab GTP-Binding Proteins