Analysis of the Role of the Hypervariable Region of Yeast Ras2p and Its Farnesylation in the Interaction with Exchange Factors and Adenylyl Cyclase
2000; Elsevier BV; Volume: 275; Issue: 23 Linguagem: Inglês
10.1074/jbc.m000729200
ISSN1083-351X
AutoresJean‐Bernard Créchet, Eric Jacquet, Alberto De Bernardi, Andrea Parmeggiani,
Tópico(s)Cellular transport and secretion
ResumoRas proteins from Saccharomyces cerevisiae differ from mammalian Ha-Ras in their extended C-terminal hypervariable region. We have analyzed the function of this region and the effect of its farnesylation with respect to the action of the GDP/GTP exchange factors (GEFs) Cdc25p and Sdc25p and the target adenylyl cyclase. Whereas Ras2p farnesylation had no effect on the interaction with purified GEFs from the Cdc25 family, this modification became a strict requirement for stimulation of the nucleotide exchange on Ras using reconstituted cell-free systems with GEFs bound to the cell membrane. Determination of GEF effects showed that in cell membrane the Cdc25p dependent activity on Ras2p was predominant over that of Sdc25p. In contrast to full-length GEFs, a membrane-bound C-terminal region containing the catalytic domain of Cdc25p was still able to react productively with unfarnesylated Ras2p. These results indicate that in membrane-bound full-length GEF the N-terminal moiety regulates the interaction between catalytic domain and farnesylated Ras2p·GDP. Differently from GEF, full activation of adenylyl cyclase did not require farnesylation of Ras2p·GTP, even if this step of maturation was found to facilitate the interaction. The use of Ha-Ras/Ras2p chimaeras of different length emphasized the key role of the hypervariable region of Ras2p in inducing maximum activation of adenylyl cyclase and for a productive interaction with membrane-bound GEF. Ras proteins from Saccharomyces cerevisiae differ from mammalian Ha-Ras in their extended C-terminal hypervariable region. We have analyzed the function of this region and the effect of its farnesylation with respect to the action of the GDP/GTP exchange factors (GEFs) Cdc25p and Sdc25p and the target adenylyl cyclase. Whereas Ras2p farnesylation had no effect on the interaction with purified GEFs from the Cdc25 family, this modification became a strict requirement for stimulation of the nucleotide exchange on Ras using reconstituted cell-free systems with GEFs bound to the cell membrane. Determination of GEF effects showed that in cell membrane the Cdc25p dependent activity on Ras2p was predominant over that of Sdc25p. In contrast to full-length GEFs, a membrane-bound C-terminal region containing the catalytic domain of Cdc25p was still able to react productively with unfarnesylated Ras2p. These results indicate that in membrane-bound full-length GEF the N-terminal moiety regulates the interaction between catalytic domain and farnesylated Ras2p·GDP. Differently from GEF, full activation of adenylyl cyclase did not require farnesylation of Ras2p·GTP, even if this step of maturation was found to facilitate the interaction. The use of Ha-Ras/Ras2p chimaeras of different length emphasized the key role of the hypervariable region of Ras2p in inducing maximum activation of adenylyl cyclase and for a productive interaction with membrane-bound GEF. guanine nucleotide exchange factor mouse RasGRF, a neuronal RasGEF polyacrylamide gel electrophoresis amino acid guanosine 5′-O-(thiotriphosphate) 4-morpholineethanesulfonic acid Ras proteins are GTPases cycling between the active GTP-bound state and the inactive GDP-bound state. They transmit extracellular signals that regulate cell growth and differentiation (1.Satoh T. Nakafuku M. Kaziro Y. J. Biol. Chem. 1992; 267: 24149-24152Abstract Full Text PDF PubMed Google Scholar). The level of activated Ras is controlled by the GTPase-activating protein and the GDP/GTP exchange factor (GEF)1 which in the case ofSaccharomyces cerevisiae are Ira1p/Ira2p (2.Buchberg A.M. Cleveland L.S. Jenkins N.A. Copeland N.G. Nature. 1990; 347: 291-294Crossref PubMed Scopus (178) Google Scholar, 3.Tanaka K. Nakafuku M. Tamanoi F. Kaziro Y. Matsumoto K. Toh-e A. Mol. Cell. Biol. 1990; 10: 4303-4313Crossref PubMed Scopus (241) Google Scholar) and Cdc25p, respectively (4.Parmeggiani A. Mistou M.Y. Jacquet E. Poullet P. Créchet J.-B. Lacal J.C. McCormick F. The Ras Superfamilies of GTPases. CRC Press, Inc., Boca Raton, FL1993: 467-488Google Scholar). This organism harbors a second RasGEF (Sdc25p, Ref.5.Créchet J.-B. Poullet P. Mistou M.Y. Parmeggiani A. Camonis J. Boy-Marcotte E. Damak F. Jacquet M. Science. 1990; 248: 866-868Crossref PubMed Scopus (91) Google Scholar) of unclear functions, that can complement Cdc25p (6.Broek D. Toda T. Michaeli T. Levin L. Birchmeier C. Zoller M. Powers S. Wigler M. Cell. 1987; 48: 789-799Abstract Full Text PDF PubMed Scopus (312) Google Scholar, 7.Damak F. Boy-Marcotte E. Le-Roscouet D. Guilbaud R. Jacquet M. Mol. Cell. Biol. 1991; 11: 202-212Crossref PubMed Scopus (58) Google Scholar, 8.Boy-Marcotte E. Ikonomi P. Jacquet M. Mol. Biol. Cell. 1996; 7: 529-539Crossref PubMed Scopus (38) Google Scholar). Ras1p and Ras2p regulate the activity of adenylyl cyclase and cAMP-dependent protein kinases (9.Matsumoto K. Uno I. Oshima Y. Ishikawa T. Proc. Natl. Acad. Sci. U. S. A. 1982; 79: 2355-2359Crossref PubMed Scopus (216) Google Scholar). One major difference between yeast and mammalian Ras proteins lies in their C-terminal hypervariable region which in the case of Ras from the former organism is much more extended (∼120 versus ∼20 aa residues). The function of this overextended C-terminal region is as yet unclear. Association with the cell membrane is an essential condition for the function of Ras proteins. Translocation of Ras to the inner surface of the membrane is promoted by sequential post-translational modifications of the C-terminal CAAX consensus box (10.Casey P.J. Curr. Opin. Cell Biol. 1994; 6: 219-225Crossref PubMed Scopus (169) Google Scholar). The first step, the farnesylation of cysteine, is followed by proteolytic cleavage of the AAX peptide, methyl-esterification of the exposed isoprenylated cysteine and in the case of human N-Ras, Ha-Ras, andS. cerevisiae Ras1p and Ras2p, palmitoylation of one or two cysteines located upstream to the CAAX motif (11.Hancock J.F. Magee A.I. Childs J.E. Marshall C.J. Cell. 1989; 57: 1167-1177Abstract Full Text PDF PubMed Scopus (1447) Google Scholar). After farnesylation, AAX proteolysis and methylation, Ras proteins are still mainly cytosolic; their tight association with the plasma membrane requires palmitoylation (12.Hancock J.F. Paterson H. Marshall C.J. Cell. 1990; 63: 133-139Abstract Full Text PDF PubMed Scopus (834) Google Scholar, 13.Cadwallader K.A. Paterson H. Macdonald S.G. Hancock J.F. Mol. Cell. Biol. 1994; 7: 4722-4730Crossref Scopus (152) Google Scholar, 14.Dudler T. Gelb H.H. J. Biol. Chem. 1996; 271: 11541-11547Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar) or for K-Ras a signal composed of a polybasic domain (14.Dudler T. Gelb H.H. J. Biol. Chem. 1996; 271: 11541-11547Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar). In mammalians, farnesylation was reported to be essential for the action of the ubiquitary exchange factor SOS (15.Porfiri E. Evans T. Chardin P. Hancock J.F. J. Biol. Chem. 1994; 269: 22672-22677Abstract Full Text PDF PubMed Google Scholar); it targets Raf to the cell membrane (16.Marais R. Light Y. Paterson H.F. Marshall C.J. EMBO J. 1995; 14: 3136-3145Crossref PubMed Scopus (520) Google Scholar, 17.Mc Geady P. Kuroda S. Shimizu K. Takai Y. Gelb M.H. J. Biol. Chem. 1995; 270: 26347-26351Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar, 18.Kikuchi A. Williams L.T. J. Biol. Chem. 1994; 269: 20054-20059Abstract Full Text PDF PubMed Google Scholar, 19.Okada T. Masuda T. Shinkai M. Kariya K. Kataoka T. J. Biol. Chem. 1996; 271: 4671-4678Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar) and is necessary for transformation (20.Kato K. Cox A.D. Hisaka M.M. Graham S.M. Buss J.E. Der C.D. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 6403-6407Crossref PubMed Scopus (552) Google Scholar). In yeast farnesylation of Ras2p was found to be important for the interaction with the adenylyl cyclase-CAP complex (21.Kuroda Y. Suzuki N. Kataoka T. Science. 1993; 259: 683-686Crossref PubMed Scopus (119) Google Scholar, 22.Shima F. Yamawaki-Kataoka Y. Yanagihara C. Tamada M. Okada T. Kariya K.I. Kataoka T. Mol. Cell. Biol. 1997; 17: 1057-1064Crossref PubMed Scopus (47) Google Scholar). Information on the role of farnesylation in the activity of yeast Cdc25p and Sdc25p is so far limited to the observation that the isolated catalytic domain of Cdc25p promoted the nucleotide exchange on prenylated Ras and even more strongly on unprocessed Ras (15.Porfiri E. Evans T. Chardin P. Hancock J.F. J. Biol. Chem. 1994; 269: 22672-22677Abstract Full Text PDF PubMed Google Scholar).In this work we have analyzed the role of the C-terminal hypervariable region of Ras proteins and its farnesylation in the interaction with both full-length exchange factors Cdc25p and Sdc25p, and in the activation of adenylyl cyclase. As methodological approach, a well defined reconstituted in vitro system using membrane preparations from isogenic yeast strains was utilized in order to mimicin vivo conditions and compensate for the fact that the isolated full-length Cdc25p and Sdc25p are not yet available despite considerable efforts. In this context the activities of these two GEFs as components bound to the cell membrane were characterized and compared with the activity of membrane-bound GEF C-terminal region. The obtained results have further enlighted the regulatory role of the N-terminal region of GEF on the C-terminal catalytic domain and demonstrated the absolute requirement of Ras2p farnesylation for a productive interaction. The construction of Ha-Ras/Ras2p chimaeras has selectively defined the importance of the hypervariable region of yeast Ras for the activation of adenylyl cyclase.DISCUSSIONThe reconstituted cell-free system used in this study reproducesin vitro the physiological conditions of the interaction between Ras and GEFs or adenylyl cyclase as cell membrane-bound components. Our observation that farnesylation of Ras2p did not affect the GDP dissociation mediated by purified catalytic domains of yeast GEFs or full-length CDC25 Mm emphasizes the need for a system in which these Ras ligands are associated with the cell membrane to study physiologically relevant interactions. The use of CRI4 adenylyl cyclase strains allowed the isolation of Ras- and GEF-free membranes, and enabled the selective study of the effect of Ras2p farnesylation on Cdc25p or Sdc25p interaction by bypassing the lethality of the deletions. In this system the activity of membrane-associated full-length GEFs was strictly dependent on farnesylation of Ras2p. A relevant residual exchange activity was observed in membranes from strains with disrupted CDC25 gene, that was also strictly dependent on farnesylation of Ras2p and could be attributed to Sdc25p. For the first time, it was so possible to define the extent of cell-membrane exchange activity dependent on Sdc25p acting as a second yeast GEF. Genetic analysis has shown that the SDC25 gene can functionally complement a cdc25 mutation and that theSDC25 and CDC25 genes are differently transcribed, the former being expressed late during growth (8.Boy-Marcotte E. Ikonomi P. Jacquet M. Mol. Biol. Cell. 1996; 7: 529-539Crossref PubMed Scopus (38) Google Scholar). The same K m and V max of membranes with overproduced Cdc25p or Sdc25p strongly suggests that the weaker activity of Sdc25p versus Cdc25p in native yeast membranes is related to a low level of expression.Sdc25p and Cdc25p are associated with the membrane via hydrophobic sequences located in the C-terminal region (7.Damak F. Boy-Marcotte E. Le-Roscouet D. Guilbaud R. Jacquet M. Mol. Cell. Biol. 1991; 11: 202-212Crossref PubMed Scopus (58) Google Scholar, 48.Garreau H. Geymonat M. Renault G. Jacquet M. Mol. Biol. Cell. 1996; 86: 93-102Google Scholar). Membrane-associated overexpressed C-Cdc25p 877–1589 was found in this work to convert farnesylated Ras2p·GDP to the active state, as do membranes with intact Cdc25p or Sdc25p, but differently from these, it did not require farnesylation of Ras2p to stimulate the exchange activity. This shows that farnesylation is not involved in increasing the association of Ras2p with the cell membrane but favors specific protein-protein interactions. In fact, it is known that farnesylation is not sufficient for a stable anchoring of Ras to the plasma membrane, palmitoylation of the upstream cysteine being required (12.Hancock J.F. Paterson H. Marshall C.J. Cell. 1990; 63: 133-139Abstract Full Text PDF PubMed Scopus (834) Google Scholar, 13.Cadwallader K.A. Paterson H. Macdonald S.G. Hancock J.F. Mol. Cell. Biol. 1994; 7: 4722-4730Crossref Scopus (152) Google Scholar, 14.Dudler T. Gelb H.H. J. Biol. Chem. 1996; 271: 11541-11547Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar). Our observations are suggestive for a function of the N-terminal domain regulating the activity of the catalytic domain as proposed in Ref. 49.Munder T. Küntzel H. FEBS Lett. 1989; 242: 341-345Crossref PubMed Scopus (54) Google Scholar. Farnesylation of Ras could induce a topological orientation allowing the accessibility to the catalytic domain of Cdc25p, where the region 1374–1444 is essential for this interaction (50.Park W. Mosteller R.D. Broek D. Mol. Cell. Biol. 1994; 14: 8117-8122Crossref PubMed Scopus (23) Google Scholar). Evidence in vitro and in vivo has indicated that the N-terminal moiety of mammalian RasGEFs CDC25 Mm (38.Baouz S. Jacquet E. Bernardi A. Parmeggiani A. J. Biol. Chem. 1997; 272: 6671-6676Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar) and SOS (51.Corbalan-Garcia S. Margarit S.M. Galron D. Yang S-S. Bar-Sagi D. Mol. Cell. Biol. 1998; 18: 880-886Crossref PubMed Scopus (83) Google Scholar) down-regulates the activity of the catalytic region, despite a modular organization fully different from the N-terminal region of Cdc25p or Sdc25p.Another relevant aspect of this work is the effect of Ha-Ras farnesylation on the response to GEF and adenylyl cyclase. As for Ras2p, farnesylation of Ha-Ras is required for a response to membrane-associated Cdc25p, but differently from Ras2p, unprenylated Ha-Ras is insensitive to membrane-associated C-Cdc25p 877–1589 and becomes as responsive as Ras2p only after fusion with the hypervariable region of Ras2p (residues 182–322). This demonstrates the essential role of this region for a productive interaction with the C-terminal domain of Cdc25p and disagrees with a suggested negative regulatory role of the C-terminal portion of Ras2p, which was proposed from the observation that Ras proteins lacking the C-terminal domain can bypasscdc25 mutations (52.Marshall M.S. Gibbs J.B. Scolnick E.M. Sigal I.S. Mol. Cell. Biol. 1987; 7: 2309-2315Crossref PubMed Scopus (39) Google Scholar). We observed that purified C-Cdc25p can exert a GDP dissociating activity on Ha-Ras or Ha-Ras 1–173/Ras2p 307–322 even better than on Ras2p. These results show that with membrane-associated components protein interaction becomes subject to more selective constraints than with soluble components. Differently from region 8–181 (homologous to region 1–173 in Ha-ras) which is acidic, the hypervariable region 182–322 displays a highly positive net charge, that together with the presence of extensive hydrophobic stretches is likely essential for binding to membrane-associated GEF.Ha-Ras p21 was reported to be able to substitute for yeast Ras proteins in sustaining growth and adenylyl cyclase activation, but complementation of the defect of yeast Ras genes was not efficient (53.Broek D. Samiy N. Fasano O. Fujiyama A. Tamanoi F. Northup J. Wigler M. Cell. 1985; 41: 763-769Abstract Full Text PDF PubMed Scopus (195) Google Scholar, 54.Kataoka T. Powers S. Cameron S. Fasano O. Golfarb M. Broach J. Wigler M. Cell. 1985; 40: 19-26Abstract Full Text PDF PubMed Scopus (169) Google Scholar). In our hands unprenylated recombinant Ha-Ras was practically unable to stimulate adenylyl cyclase and only its farnesylation enabled some stimulation. Even with farnesylated Ha-Ras maximum activation of adenylyl cyclase was much lower than with Ras2p, an effect that was more pronounced with wild-type adenylyl cyclase than with its CRI4 mutant. However, farnesylated Ha-Ras showed a strong affinity for both CRI4 and CYR1 gene products, almost as high as that of farnesylated Ras2p. In order to clarify the reasons for the functional differences, we extended the observations that an activated Ha-Ras Val-12/Ras2p chimaera containing the first 73 amino acids of Ha-Ras Val-12 stimulates adenylyl cyclase more efficiently than Ha-Ras (53.Broek D. Samiy N. Fasano O. Fujiyama A. Tamanoi F. Northup J. Wigler M. Cell. 1985; 41: 763-769Abstract Full Text PDF PubMed Scopus (195) Google Scholar). For this purpose, we constructed two N-terminal Ha-Ras/C-ter Ras2p chimaeras including C-terminal regions of Ras2p of different length (Ha-Ras 1–81/Ras2p 89–322 and Ha-Ras 1–173/Ras2p 182–322) comprising the extended hypervariable region of Ras2p. Both constructs revealed the same profile of adenylyl cyclase activation as Ras2p, thereby defining the hypervariable domain as an additional important element for full reconstitution of the activity. Other major determinants for the interaction with adenylyl cyclase are the effector region (residues 32–40 in Ha-Ras and 39–47 in Ras2p) (31.Fasano O. Créchet J.-B. De Vendittis E. Zahn R. Feger G. Vitelli A. Parmeggiani A. EMBO J. 1988; 7: 3375-3383Crossref PubMed Scopus (25) Google Scholar, 55.Marshall M.S. Gibbs J.B. Scolnick E.M. Sigal I.S. Mol. Cell. Biol. 1988; 8: 52-61Crossref PubMed Scopus (29) Google Scholar, 56.Schaber M.D. Garsky V.M. Boylan D. Hill W.S. Scolnick E.M. Marshall M.S. Sigal I.S. Gibbs J.B. 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Chem. 1996; 271: 5353-5360Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar). By means of the CRI4-encoded product, which is highly sensitive to Ras2p even if unfarnesylated, we showed that maximum stimulation of adenylyl cyclase by unprenylated Ras2p or Ha-Ras/Ras2p chimaeras is identical to that obtained with prenylated Ras2p. This emphasizes the crucial role of the hypervariable region for maximum activation and shows that the major effect of farnesylation is to increase the affinity between Ras2p and adenylyl cyclase rather than to stimulate adenylyl cyclase activity.Even if there is still some uncertainty about a direct interaction between Ras proteins and adenylyl cyclase, due to the need of the adenylyl cyclase tightly associated protein CAP (60.Fedor-Chaiken M.R. Deschenes J. Broach J.R. Cell. 1990; 61: 329-340Abstract Full Text PDF PubMed Scopus (190) Google Scholar, 61.Field J. Vojtek A. Ballester R. Bolger G. Colicelli J. Ferguson K. Gerst J. Kataoka T. Michaeli T. Powers S. Riggs M. Rodgers L. Wieland I. Wheland B. Wigler M. Cell. 1990; 61: 319-327Abstract Full Text PDF PubMed Scopus (181) Google Scholar) for a proper response to post-translationally modified Ha-Ras (22.Shima F. Yamawaki-Kataoka Y. Yanagihara C. Tamada M. Okada T. Kariya K.I. Kataoka T. Mol. Cell. Biol. 1997; 17: 1057-1064Crossref PubMed Scopus (47) Google Scholar), it is possible that the basic nature of the C-terminal region of Ras2p could favor an efficient interaction with the leucine repeat-rich region following the N-terminal region of adenylyl cyclase. This interaction could induce a suitable conformation for maximum activation of the catalytic domain. Leucine-rich repeat regions (61.Field J. Vojtek A. Ballester R. Bolger G. Colicelli J. Ferguson K. Gerst J. Kataoka T. Michaeli T. Powers S. Riggs M. Rodgers L. Wieland I. Wheland B. Wigler M. Cell. 1990; 61: 319-327Abstract Full Text PDF PubMed Scopus (181) Google Scholar) are critical in mediating protein-protein interaction (62.Kobe B. Deisenhofer J. Trends Biochem. Sci. 1994; 19: 415-421Abstract Full Text PDF PubMed Scopus (1035) Google Scholar), as has been recently shown in SUR-8, a conserved Ras-binding protein that contains this core consensus and positively regulates Ras-mediated signaling in Caenorhabditis elegans (63.Sieburth D.S. Sun Q. Han M. Cell. 1998; 94: 119-130Abstract Full Text Full Text PDF PubMed Scopus (166) Google Scholar).Farnesylation of Ras2p is not required for adenylyl cyclase activation, as has been tested with CRI4-adenylyl cyclase. However, as already reported for the wild-type CYR1 product (21.Kuroda Y. Suzuki N. Kataoka T. Science. 1993; 259: 683-686Crossref PubMed Scopus (119) Google Scholar), farnesylation of Ras2·GTP increases the affinity for adenylyl cyclase. Compared with the wild-type one, CRI4-adenylyl cyclase shows not only an increased sensitivity to Ras2p (26.Feger G. De Vendittis E. Vitelli A. Masturzo P. Zahn R. Verrotti A.C. Kavounis C. Pal G.P. Fasano O. EMBO J. 1991; 10: 349-359Crossref PubMed Scopus (14) Google Scholar, 46.De Vendittis E. Vitelli A. Zahn R. Fasano O. EMBO J. 1986; 5: 3657-3663Crossref PubMed Scopus (50) Google Scholar) but also a higher affinity for farnesylated Ras. One should stress that the differences between these two adenylyl cyclases are only quantitative.Adenylyl cyclase is not as strongly associated with the plasma membrane as Cdc25p and Sdc25p, does not contain a hydrophobic region resembling a membrane-spanning domain (64.Kataoka T. Broek D. Wigler M. Cell. 1985; 43: 493-505Abstract Full Text PDF PubMed Scopus (265) Google Scholar) and in ras1ras2bcy1 cells it is located in the soluble fraction (65.Engelberg D. Simchen G. Levitzki A. EMBO J. 1990; 9: 641-651Crossref PubMed Scopus (35) Google Scholar). Overexpression of Cdc25p has been reported to translocate adenylyl cyclase to the membrane fraction (65.Engelberg D. Simchen G. Levitzki A. EMBO J. 1990; 9: 641-651Crossref PubMed Scopus (35) Google Scholar), while disruption of IRA1 gene dislocates it from the membrane (66.Mitts M. Bradshaw-Rouse J.R. Heideman W. Mol. Cell. Biol. 1991; 11: 4591-4598Crossref PubMed Scopus (31) Google Scholar), indicating that both Cdc25p and Ira1p are involved in anchoring adenylyl cyclase to the membrane. A complex between the Cdc25p SH3 domain and adenylyl cyclase not mediated by CAP has been demonstrated (67.Freeman N.L. Lila T. Mintzer K.A. Chen Z. Pahk A.J. Ren R. Drubin D.G. Field J. Mol. Cell. Biol. 1996; 16: 548-556Crossref PubMed Scopus (112) Google Scholar). Taken together these data suggest the existence in the cell of a large oligomeric complex including Cdc25p, Ira1p, and adenylyl cyclase. The association of Ras2p, Cdc25p, and adenylyl cyclase (68.Gross E. Marbach I. Engelberg D. Segal M. Simchen G. Levitzki A. Mol. Cell. Biol. 1992; 12: 2653-2661Crossref PubMed Scopus (23) Google Scholar) is further supported by this work highlighting the common function of the C-terminal hypervariable region of Ras2p and its farnesylation in promoting the interaction with and/or activation of membrane-bound Cdc25p and adenylyl cyclase. However, neither Cdc25p nor Sdc25p, even when overexpressed, are able to increase the affinity of farnesylated Ras2p for adenylyl cyclase.In conclusion, this work shows that the cellular localization of Ras2p, its regulators Cdc25p and Sdc25p and target adenylyl cyclase requires structural modifications that are dispensable under conditions of soluble purified components. We have highlighted the essential role of Ras2p farnesylation for GEF responsiveness and the involvement of its C-terminal hypervariable region in the interaction with Cdc25p. Compared with Ha-Ras, this region has been found to contain structural elements essential for the activation of adenylyl cyclase, the farnesylation facilitating this interaction. Ras proteins are GTPases cycling between the active GTP-bound state and the inactive GDP-bound state. They transmit extracellular signals that regulate cell growth and differentiation (1.Satoh T. Nakafuku M. Kaziro Y. J. Biol. Chem. 1992; 267: 24149-24152Abstract Full Text PDF PubMed Google Scholar). The level of activated Ras is controlled by the GTPase-activating protein and the GDP/GTP exchange factor (GEF)1 which in the case ofSaccharomyces cerevisiae are Ira1p/Ira2p (2.Buchberg A.M. Cleveland L.S. Jenkins N.A. Copeland N.G. Nature. 1990; 347: 291-294Crossref PubMed Scopus (178) Google Scholar, 3.Tanaka K. Nakafuku M. Tamanoi F. Kaziro Y. Matsumoto K. Toh-e A. Mol. Cell. Biol. 1990; 10: 4303-4313Crossref PubMed Scopus (241) Google Scholar) and Cdc25p, respectively (4.Parmeggiani A. Mistou M.Y. Jacquet E. Poullet P. Créchet J.-B. Lacal J.C. McCormick F. The Ras Superfamilies of GTPases. CRC Press, Inc., Boca Raton, FL1993: 467-488Google Scholar). This organism harbors a second RasGEF (Sdc25p, Ref.5.Créchet J.-B. Poullet P. Mistou M.Y. Parmeggiani A. Camonis J. Boy-Marcotte E. Damak F. Jacquet M. Science. 1990; 248: 866-868Crossref PubMed Scopus (91) Google Scholar) of unclear functions, that can complement Cdc25p (6.Broek D. Toda T. Michaeli T. Levin L. Birchmeier C. Zoller M. Powers S. Wigler M. Cell. 1987; 48: 789-799Abstract Full Text PDF PubMed Scopus (312) Google Scholar, 7.Damak F. Boy-Marcotte E. Le-Roscouet D. Guilbaud R. Jacquet M. Mol. Cell. Biol. 1991; 11: 202-212Crossref PubMed Scopus (58) Google Scholar, 8.Boy-Marcotte E. Ikonomi P. Jacquet M. Mol. Biol. Cell. 1996; 7: 529-539Crossref PubMed Scopus (38) Google Scholar). Ras1p and Ras2p regulate the activity of adenylyl cyclase and cAMP-dependent protein kinases (9.Matsumoto K. Uno I. Oshima Y. Ishikawa T. Proc. Natl. Acad. Sci. U. S. A. 1982; 79: 2355-2359Crossref PubMed Scopus (216) Google Scholar). One major difference between yeast and mammalian Ras proteins lies in their C-terminal hypervariable region which in the case of Ras from the former organism is much more extended (∼120 versus ∼20 aa residues). The function of this overextended C-terminal region is as yet unclear. Association with the cell membrane is an essential condition for the function of Ras proteins. Translocation of Ras to the inner surface of the membrane is promoted by sequential post-translational modifications of the C-terminal CAAX consensus box (10.Casey P.J. Curr. Opin. Cell Biol. 1994; 6: 219-225Crossref PubMed Scopus (169) Google Scholar). The first step, the farnesylation of cysteine, is followed by proteolytic cleavage of the AAX peptide, methyl-esterification of the exposed isoprenylated cysteine and in the case of human N-Ras, Ha-Ras, andS. cerevisiae Ras1p and Ras2p, palmitoylation of one or two cysteines located upstream to the CAAX motif (11.Hancock J.F. Magee A.I. Childs J.E. Marshall C.J. Cell. 1989; 57: 1167-1177Abstract Full Text PDF PubMed Scopus (1447) Google Scholar). After farnesylation, AAX proteolysis and methylation, Ras proteins are still mainly cytosolic; their tight association with the plasma membrane requires palmitoylation (12.Hancock J.F. Paterson H. Marshall C.J. Cell. 1990; 63: 133-139Abstract Full Text PDF PubMed Scopus (834) Google Scholar, 13.Cadwallader K.A. Paterson H. Macdonald S.G. Hancock J.F. Mol. Cell. Biol. 1994; 7: 4722-4730Crossref Scopus (152) Google Scholar, 14.Dudler T. Gelb H.H. J. Biol. 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