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 (am ino 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 mu M and the maximal stimulation of the Rasp GTPase a ctivity 6 000 times, The Ira2p activity was confirmed to be strictly s pecific for Ras2p, no stimulatory effect on human c-H-ras p21 GTPase b eing detectable, Comparison with the GAP-like domain of mammalian p120 -GAP and neurofibromin using yeast Ras2p as substrate showed that Ira2 p-383 has an affinity and turnover intermediary between GAP-334 and NF 1-414. The activity of lra2p-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 Rasp a multiple-round reaction of GTP hydrolysis and GDP/GTP exchange, sho wing that it is possible to reconstitute in vitro a S, cerevisiae syst em 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 1539-20 53), that showed the same K-m for Ras2p as Ira2p-383. was also inhibit ed by tubulin to the same extent but with a higher affinity than Ira2p -383. This indicates that the conserved catalytic domain contains a bi nding site for tubulin that is extended to its N-terminal flanking reg ion. These results show that the inhibition of neurofibromin by tubuli n [Bollag, G., McCormick. F. & Clark, R. (1993) EMBO J, 12, 1923-1927] is a property shared with Ira2p.