Identification of novel single amino acid changes that result in hyperactivation of the unique GTPase, Rheb, in fission yeast.
Rheb GTPase is a key player in the control of growth, cell cycle and nutrient uptake that is conserved from yeast to humans. To further our understanding of the Rheb pathway, we sought to identify hyperactivating mutations in the Schizosaccharomyces pombe Rheb, Rhb1. Hyperactive forms of Rhb1 were found to result from single amino acid changes at valine-17, serine-21, lysine-120 or asparagine-153. Expression of these mutants confers resistance to canavanine and thialysine, phenotypes which are similar to phenotypes exhibited by cells lacking the Tsc1/Tsc2 complex that negatively regulates Rhb1. The thialysine-resistant phenotype of the hyperactive Rhb1 mutants is suppressed by a second mutation in the effector domain. Purified mutant proteins exhibit dramatically decreased binding of GDP, while their GTP binding is not drastically affected. In addition, some of the mutant proteins show significantly decreased GTPase activities. Thus the hyperactivating mutations are expected to result in an increase in the GTP-bound/GDP-bound ratio of Rhb1. By using the hyperactive mutant, Rhb1(K120R), we have been able to demonstrate that Rhb1 interacts with Tor2, one of the two S. pombe TOR (Target of Rapamycin) proteins. These fission yeast results provide the first evidence for a GTP-dependent association of Rheb with Tor.
Urano
Jun
J
Department of Microbiology, Immunology and Molecular Genetics, Jonsson Comprehensive Cancer Center, Molecular Biology Institute, University of California, Los Angeles, CA 90095-1489, USA.
Comiso
Melissa J
MJ
Guo
Lea
L
Aspuria
Paul-Joseph
PJ
Deniskin
Roman
R
Tabancay
Angel P
AP
Kato-Stankiewicz
Juran
J
Tamanoi
Fuyuhiko
F
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000935:Antifungal Agents / Q000494:pharmacology; D002172:Canavanine / Q000494:pharmacology; D018797:Cell Cycle Proteins / Q000378:metabolism; D003545:Cysteine / Q000031:analogs & derivatives / Q000494:pharmacology; D025141:Drug Resistance, Fungal; D020558:GTP Phosphohydrolases / Q000032:analysis / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D006153:Guanosine Diphosphate / Q000378:metabolism; D006160:Guanosine Triphosphate / Q000378:metabolism; D008969:Molecular Sequence Data; D009154:Mutation; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism; D011485:Protein Binding; D011500:Protein Synthesis Inhibitors / Q000494:pharmacology; D012568:Schizosaccharomyces / Q000201:enzymology*; D029702:Schizosaccharomyces pombe Proteins / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D017386:Sequence Homology, Amino Acid
D000935:Antifungal Agents; D002172:Canavanine; D018797:Cell Cycle Proteins; D003545:Cysteine; D020558:GTP Phosphohydrolases; D006153:Guanosine Diphosphate; D006160:Guanosine Triphosphate; D019869:Phosphatidylinositol 3-Kinases; D011500:Protein Synthesis Inhibitors; D029702:Schizosaccharomyces pombe Proteins; C000204:S-2-aminoethyl cysteine; C513100:tor2 protein, S pombe; C412832:Rhb1 protein, S pombe