Properties and regulation of the catalytic domain of Ira2p, a Saccharomyces cerevisiae GTPase-activating protein of Ras2p.
This work describes the biochemical characterization of the catalytic domain of Ira2p, a Saccharomyces cerevisiae GTPase-activating protein (GAP) regulating the RAS gene products. A fragment of 383 residues (amino acids 1644-2026) was produced in Escherichia coli as glutathione S-transferase fusion protein (GST-Ira2p-383) and highly purified (> 90%) by affinity chromatography. The affinity of Ras2p for the GST-fused Ira2p-383 was 18 microM and the maximal stimulation of the Ras2p GTPase activity 6,000 times. The Ira2p activity was confirmed to be strictly specific for Ras2p, no stimulatory effect on human c-H-ras p21 GTPase being detectable. Comparison with the GAP-like domain of mammalian p120-GAP and neurofibromin using yeast Ras2p as substrate showed that Ira2p-383 has an affinity and turnover intermediary between GAP-334 and NF1-414. The activity of Ira2p-383 was strongly inhibited by monovalent and divalent salts. The simultaneous presence of the catalytic domains of Ira2p and the yeast GDP/GTP exchange factor Cdc25p induced on Ras2p a multiple-round reaction of GTP hydrolysis and GDP/GTP exchange, showing that it is possible to reconstitute in vitro a S. cerevisiae system suitable for the study of the regulation of the Ras2p GDP/GTP cycle. The tubulin partially inhibited (25%) the GAP activity of the Ira2p-383. A larger Ira2p catalytic fragment, Ira2p-505 (amino acids 1549-2053), that showed the same Km for Ras2p as Ira2p-383, was also inhibited by tubulin to the same extent but with a higher affinity than Ira2p-383.(ABSTRACT TRUNCATED AT 250 WORDS)
Parrini
M C
MC
Groupe de Biophysique, Ecole Polytechnique, Palaiseau, France.
Jacquet
E
E
Bernardi
A
A
Jacquet
M
M
Parmeggiani
A
A
D001665:Binding Sites; D004591:Electrophoresis, Polyacrylamide Gel; D005656:Fungal Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D020558:GTP Phosphohydrolases / Q000378:metabolism; D020690:GTPase-Activating Proteins; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D025542:Neurofibromin 1; D010446:Peptide Fragments / Q000378:metabolism; D011506:Proteins / Q000037:antagonists & inhibitors / Q000378:metabolism*; D016283:Proto-Oncogene Proteins p21(ras) / Q000037:antagonists & inhibitors; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics* / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins; D013696:Temperature; D014404:Tubulin / Q000494:pharmacology; D020703:ras GTPase-Activating Proteins; D018631:ras Proteins / Q000378:metabolism*
D005656:Fungal Proteins; D020690:GTPase-Activating Proteins; C064960:IRA2 protein, S cerevisiae; D025542:Neurofibromin 1; D010446:Peptide Fragments; D011506:Proteins; D011993:Recombinant Fusion Proteins; D029701:Saccharomyces cerevisiae Proteins; D014404:Tubulin; D020703:ras GTPase-Activating Proteins; D020558:GTP Phosphohydrolases; C501009:HRAS protein, human; D016283:Proto-Oncogene Proteins p21(ras); D018631:ras Proteins