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Coiled-coil Interaction of N-terminal 36 Residues of Cyclase-associated Protein with Adenylyl Cyclase Is Sufficient for Its Function in Saccharomyces cerevisiae Ras Pathway

1998; Elsevier BV; Volume: 273; Issue: 43 Linguagem: Inglês

10.1074/jbc.273.43.28019

ISSN

1083-351X

Autores

Yoshimitsu Nishida, Fumi Shima, Hiroyoshi Sen, Yasuhiro Tanaka, Chie Yanagihara, Yuriko Yamawaki‐Kataoka, Ken‐ichi Kariya, Tohru Kataoka,

Tópico(s)

Endoplasmic Reticulum Stress and Disease

Resumo

In the budding yeast Saccharomyces cerevisiae, association with the 70-kDa cyclase-associated protein (CAP) is required for proper response of adenylyl cyclase to Ras proteins. We show here that a small segment comprising the N-terminal 36 amino acid residues of CAP is sufficient for association with adenylyl cyclase as well as for its function in the Ras-adenylyl cyclase pathway as assayed by the ability to conferRAS2 Val-19-dependent heat shock sensitivity to yeast cells. The CAP-binding site of adenylyl cyclase was mapped to a segment of 119 amino acid residues near its C terminus. Both of these regions contained tandem repetitions of a heptad motif αXXαXXX (where α represents a hydrophobic amino acid and X represents any amino acid), suggesting a coiled-coil interaction. When mutants of CAP defective in associating with adenylyl cyclase were isolated by screening of a pool of randomly mutagenized CAP, they were found to carry substitution mutations in one of the key hydrophobic residues in the heptad repeats. Furthermore, mutations of the key hydrophobic residues in the heptad repeats of adenylyl cyclase also resulted in loss of association with CAP. These results indicate the coiled-coil mechanism as a basis of the CAP-adenylyl cyclase interaction. In the budding yeast Saccharomyces cerevisiae, association with the 70-kDa cyclase-associated protein (CAP) is required for proper response of adenylyl cyclase to Ras proteins. We show here that a small segment comprising the N-terminal 36 amino acid residues of CAP is sufficient for association with adenylyl cyclase as well as for its function in the Ras-adenylyl cyclase pathway as assayed by the ability to conferRAS2 Val-19-dependent heat shock sensitivity to yeast cells. The CAP-binding site of adenylyl cyclase was mapped to a segment of 119 amino acid residues near its C terminus. Both of these regions contained tandem repetitions of a heptad motif αXXαXXX (where α represents a hydrophobic amino acid and X represents any amino acid), suggesting a coiled-coil interaction. When mutants of CAP defective in associating with adenylyl cyclase were isolated by screening of a pool of randomly mutagenized CAP, they were found to carry substitution mutations in one of the key hydrophobic residues in the heptad repeats. Furthermore, mutations of the key hydrophobic residues in the heptad repeats of adenylyl cyclase also resulted in loss of association with CAP. These results indicate the coiled-coil mechanism as a basis of the CAP-adenylyl cyclase interaction. adenylyl cyclase-associated protein adenylyl cyclase glutathioneS-transferase polymerase chain reaction GAL4 DNA-binding domain GAL4 transactivation domain 2-(N-morpholino)ethanesulfonic acid. The budding yeast Saccharomyces cerevisiae has twoRAS genes, RAS1 and RAS2, whose protein products are structurally, functionally, and biochemically similar to mammalian Ras proto-oncoproteins (for reviews, see Refs. 1Broach J.R. Deschenes R.J. Adv. Cancer Res. 1990; 54: 79-139Crossref PubMed Scopus (161) Google Scholarand 2Gibbs J.B. Marshall M. Microbiol. Rev. 1989; 53: 171-185Crossref PubMed Google Scholar). The yeast Ras proteins are essential regulatory elements of adenylyl cyclase, which catalyzes the production of cAMP, a second messenger vital for cell growth (3Toda T. Uno I. Ishikawa T. Powers S. Kataoka T. Broek D. Cameron S. Broach J. Matsumoto K. Wigler M. Cell. 1985; 40: 27-36Abstract Full Text PDF PubMed Scopus (705) Google Scholar, 4Broek D. Samiy N. Fasano O. Fujiyama A. Tamanoi F. Northup J. Wigler M. Cell. 1985; 41: 763-769Abstract Full Text PDF PubMed Scopus (194) Google Scholar). The Ras-adenylyl cyclase pathway has been implicated in transduction of a signal triggered by glucose to an intracellular environment where a protein phosphorylation cascade is induced by cAMP. Yeast cells bearing the activatedRAS2 gene, RAS2 Val-19, exhibit an elevated level of intracellular cAMP and display abnormal phenotypes, including sensitivity to heat shock, sensitivity to nutritional starvation, and failure to sporulate (3Toda T. Uno I. Ishikawa T. Powers S. Kataoka T. Broek D. Cameron S. Broach J. Matsumoto K. Wigler M. Cell. 1985; 40: 27-36Abstract Full Text PDF PubMed Scopus (705) Google Scholar, 5Kataoka T. Powers S. McGill C. Fasano O. Strathern J. Broach J. Wigler M. Cell. 1984; 37: 437-445Abstract Full Text PDF PubMed Scopus (271) Google Scholar). Yeast adenylyl cyclase, encoded by the CYR1 gene, consists of 2026-amino acid residues that comprise at least four domains: the N-terminal, the middle leucine-rich repeat, the catalytic, and the C-terminal domains (6Kataoka T. Broek D. Wigler M. Cell. 1985; 43: 493-505Abstract Full Text PDF PubMed Scopus (265) Google Scholar, 7Yamawaki-Kataoka Y. Tamaoki T. Choe H.-R. Tanaka H. Kataoka T. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 5693-5697Crossref PubMed Scopus (82) Google Scholar). The leucine-rich repeat domain contains a binding site for Ras proteins (8Suzuki N. Choe H.-R. Nishida Y. Yamawaki-Kataoka Y. Ohnishi S. Tamaoki T. Kataoka T. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 8711-8715Crossref PubMed Scopus (100) Google Scholar, 9Minato T. Wang J. Akasaka K. Okada T. Suzuki N. Kataoka T. J. Biol. Chem. 1994; 269: 20845-20851Abstract Full Text PDF PubMed Google Scholar). Adenylyl cyclase forms a complex with 70-kDa CAP.1 CAP was identified biochemically as the only protein associated tightly with adenylyl cyclase and also by genetic screening of a gene whose mutation abolished the RAS2 Val-19-dependent heat shock sensitivity (10Field 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, 11Fedor-Chaiken M. Deschenes R.J. Broach J.R. Cell. 1990; 61: 329-340Abstract Full Text PDF PubMed Scopus (190) Google Scholar). Studies on the function of CAP revealed that CAP is a multifunctional protein. It was shown that the N-terminal region, mapped to residues 1–168, is required for acquisition of heat shock sensitivity in theRAS2 Val-19 background while the C-terminal region, mapped to residues 369–526, is required for normal cell morphology and responsiveness to nutrient deprivation and excess (12Gerst J.E. Ferguson K. Vojtek A. Wigler M. Field J. Mol. Cell. Biol. 1991; 11: 1248-1257Crossref PubMed Scopus (116) Google Scholar). The C-terminal function appears to be related to regulation of the actin cytoskeleton as evidenced by complementation of its defect by overexpression of profilin or SNC1 (13Vojtek A.B. Harrer B. Field J. Gerst J. Pollard T.D. Brown S. Wigler M. Cell. 1991; 66: 497-505Abstract Full Text PDF PubMed Scopus (148) Google Scholar, 14Gerst J.E. Rodgers L. Riggs M. Wigler M. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 4338-4342Crossref PubMed Scopus (104) Google Scholar) and by demonstration of its direct association with actin monomer and of its actin-sequestering activity (15Freeman L.N. Chen Z. Horenstein J. Weber A. Field J. J. Biol. Chem. 1995; 270: 5680-5685Abstract Full Text Full Text PDF PubMed Scopus (114) Google Scholar, 16Zelicof A. Protopopov V. David D. Lin X.-Y. Lustgarten V. Gerst J.E. J. Biol. Chem. 1996; 271: 18243-18252Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar). In addition, CAP possesses two proline-rich sequences in its middle region intervening between the two regions, with which associations of actin-binding protein 1, elongation factor 1α, and ribosomal protein L3 were recently shown (17Freeman L.N. Lila T. Mintzer A.K. Chen A.K. Pahk J.A. Ren R. Drubin G.D. Field J. Mol. Cell. Biol. 1996; 16: 548-556Crossref PubMed Scopus (112) Google Scholar, 18Yanagihara C. Shinkai M. Kariya K. Yamawaki-Kataoka Y. Hu C.-D. Masuda T. Kataoka T. Biochem. Biophys. Res. Commun. 1997; 232: 503-507Crossref PubMed Scopus (12) Google Scholar). The N-terminal region of CAP binds to the C-terminal region of adenylyl cyclase (19Wang J. Suzuki N. Nishida Y. Kataoka T. Mol. Cell. Biol. 1993; 13: 4087-4097Crossref PubMed Scopus (26) Google Scholar), and this association appears to be required for the proper in vivo response of adenylyl cyclase to Ras, because its loss by mutation of either CAP or adenylyl cyclase resulted in disappearance of theRAS2 Val-19-dependent heat shock sensitivity and in a reduced cAMP response to glucose stimulation (19Wang J. Suzuki N. Nishida Y. Kataoka T. Mol. Cell. Biol. 1993; 13: 4087-4097Crossref PubMed Scopus (26) Google Scholar). This function resides solely in the CAP N-terminal region and is separable from the functions of the other regions as reported (12Gerst J.E. Ferguson K. Vojtek A. Wigler M. Field J. Mol. Cell. Biol. 1991; 11: 1248-1257Crossref PubMed Scopus (116) Google Scholar). We have recently shown biochemically that the association of adenylyl cyclase with the CAP N-terminal region is responsible for efficient stimulation of adenylyl cyclase activity by the posttranslationally modified form of Ras, although the molecular mechanism underlying this process remains to be clarified (20Shima F. Yamawaki-Kataoka Y. Yanagihara C. Tamada M. Okada T. Kariya K. Kataoka T. Mol. Cell. Biol. 1997; 17: 1057-1064Crossref PubMed Scopus (47) Google Scholar). In this report, we have mapped a minimal region of CAP responsible for its N-terminal function and analyzed the molecular mechanism for its association with adenylyl cyclase. The S. cerevisiaestrains used are listed in Table I. Replacement of the chromosomal CAP gene with its N-terminal deletion mutant CAPΔN-1 was carried out as described previously (20Shima F. Yamawaki-Kataoka Y. Yanagihara C. Tamada M. Okada T. Kariya K. Kataoka T. Mol. Cell. Biol. 1997; 17: 1057-1064Crossref PubMed Scopus (47) Google Scholar). The resulting yeast strain expresses only the C-terminal segment of CAP corresponding to residues 369–526 under control of the yeast ADC1 promoter. Yeast cells were grown in YPD (2% Bacto-peptone, 1% Bacto-yeast extract, 2% glucose) or yeast synthetic medium (0.67% yeast nitrogen base, 2% glucose) with appropriate auxotrophic supplements. Genetic manipulations of yeast cells were performed as described previously (21Rose M.D. Winston F. Hieter P. Methods in Yeast Genetics: A Laboratory Course Manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY1990Google Scholar). Transformation into yeast cells was carried out with lithium acetate (22Ito H. Fukuda Y. Murata K. Kimura A. J. Bacteriol. 1983; 153: 163-168Crossref PubMed Google Scholar).Table IYeast strains used in this studyStrain1-aStrains SP1, FS1, TK161-R2V, and YPB2 were described previously (3, 5, 20, 25).GenotypesSP1MATa his3 leu2 trp1 ura3 ade8 can1FS1MATa his3 leu2 trp1 ura3 ade8 can1 cap::pCAPΔN-1TK161-R2VMATa his3 leu2 trp1 ura3 ade8 can1 RAS2Val-19TK161-R2V(CAPΔN)MATa his3 leu2 trp1 ura3 ade8 can1 RAS2Val-19 cap::pCAPΔN-1YPB2MATa his3 leu2 trp1 ura3 ade2 canR gal4 gal80 LYS2::GAL1-HIS3 URA3::gal41–17 mers(3X)-cyc1tata-lacZYPB2(CAPΔN)MATa his3 leu2 trp1 ura3 ade2 canR gal4 gal80 LYS2::GAL1-HIS3 URA3::gal41–17 mers(3X)-cyc1tata-lacZ cap::pCAPΔN-11-a Strains SP1, FS1, TK161-R2V, and YPB2 were described previously (3Toda T. Uno I. Ishikawa T. Powers S. Kataoka T. Broek D. Cameron S. Broach J. Matsumoto K. Wigler M. Cell. 1985; 40: 27-36Abstract Full Text PDF PubMed Scopus (705) Google Scholar, 5Kataoka T. Powers S. McGill C. Fasano O. Strathern J. Broach J. Wigler M. Cell. 1984; 37: 437-445Abstract Full Text PDF PubMed Scopus (271) Google Scholar, 20Shima F. Yamawaki-Kataoka Y. Yanagihara C. Tamada M. Okada T. Kariya K. Kataoka T. Mol. Cell. Biol. 1997; 17: 1057-1064Crossref PubMed Scopus (47) Google Scholar, 25Bartel P.L. Chien C.-T. Sternglanz R. Fields S. Hartley D.A. Cellular Interactions in Development: A Practical Approach. Oxford University Press, Oxford1993: 153-179Google Scholar). Open table in a new tab A plasmid, pAD-GST-CAP (18Yanagihara C. Shinkai M. Kariya K. Yamawaki-Kataoka Y. Hu C.-D. Masuda T. Kataoka T. Biochem. Biophys. Res. Commun. 1997; 232: 503-507Crossref PubMed Scopus (12) Google Scholar), was used to express the full-length CAP in yeast as a fusion protein with GST under control of the ADC1 promoter. Various deletions were introduced into the CAP gene by cleavage of pAD-GST-CAP with suitable pairs of restriction endonucleases and resealing by T4 DNA ligase with a linker oligonucleotide, 5′-CTAGTCTAGACTAG-3′, bearing stop codons in all reading frames, between. The resulting plasmids were designated as pAD-GST-CAP-(x–x)s, where x–x represents the range of the expressed CAP polypeptides in amino acid positions. A 5′-terminal 107-base pair fragment corresponding to residues 1–36 of CAP, CAP-(1–36), was amplified by PCR (23Saiki R.K. Scharf S. Faloona F. Mullis K.B. Horn G.T. Erlich H.A. Arnheim N. Science. 1985; 230: 1350-1354Crossref PubMed Scopus (6659) Google Scholar) using suitable oligonucleotide primers and, after cleavage with BamHI and SmaI in the primer sequences, cloned into pAD-GST to produce pAD-GST-CAP-(1–36). Similarly, DNA fragments encoding various C-terminal polypeptides of adenylyl cyclase were amplified by PCR using suitable primers and cloned into pAD-GST to produce pAD-GST-CYR1-(y–y), where y–y represented the range of the expressed adenylyl cyclase polypeptide in amino acid positions. Specific amino acid substitution mutations were introduced into adenylyl cyclase by the gapped duplex method using suitable mutagenic oligonucleotides (24Kramer W. Fritz H.-J. Methods Enzymol. 1987; 154: 350-367Crossref PubMed Scopus (83) Google Scholar). The mutant genes were used to replace the corresponding wild-type genes in the expression plasmids. YEP24-ADC1-CYR1-(1–40, 1769–2026) and YEP24-ADC1-CYR1-(1–40, 606–2026), which expressed adenylyl cyclase carrying internal deletions of residues 41–1768 and 41–605, were identical to YEP24-ADC1-CYR1-(Δ41–1768) and YEP24-ADC1-CYR1-(Δ41–605), respectively, described previously (8Suzuki N. Choe H.-R. Nishida Y. Yamawaki-Kataoka Y. Ohnishi S. Tamaoki T. Kataoka T. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 8711-8715Crossref PubMed Scopus (100) Google Scholar, 19Wang J. Suzuki N. Nishida Y. Kataoka T. Mol. Cell. Biol. 1993; 13: 4087-4097Crossref PubMed Scopus (26) Google Scholar). The various mutant CAPgenes were transferred to pGBT9 or pGBT10 vector (25Bartel P.L. Chien C.-T. Sternglanz R. Fields S. Hartley D.A. Cellular Interactions in Development: A Practical Approach. Oxford University Press, Oxford1993: 153-179Google Scholar) for expression of the corresponding polypeptides as GBT fusions in yeast. Similarly, the various mutant CYR1 genes were transferred to pGAD-GH (25Bartel P.L. Chien C.-T. Sternglanz R. Fields S. Hartley D.A. Cellular Interactions in Development: A Practical Approach. Oxford University Press, Oxford1993: 153-179Google Scholar) for expression as GAD fusions. The resulting plasmids were designated as pGBT-CAP-(x–x) or pGAD-CYR1-(y–y), respectively. The reporter yeast strain YPB2(CAPΔN) was cotransformed with pGAD-CYR1-(y–y) and pGBT-CAP-(x–x), and the resulting Trp+, Leu+-transformants were assayed for β-galactosidase activity by a filter assay as described (25Bartel P.L. Chien C.-T. Sternglanz R. Fields S. Hartley D.A. Cellular Interactions in Development: A Practical Approach. Oxford University Press, Oxford1993: 153-179Google Scholar). A DNA fragment corresponding to CAP-(1–77), was subjected to an error-prone PCR to introduce random mutations as described before (26Gram H. Marconi L.-A. Barbas III, C.F. Collet T.A. Lerner R.A. Kang A.S. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 3576-3580Crossref PubMed Scopus (255) Google Scholar). The amplified fragments were cleaved with BamHI and SmaI present in the PCR primers, cloned into matching cleavage sites of pGBT10, and examined for interaction with pGAD-CYR1-(1879–2026) by the yeast two-hybrid assay as described above. The CAP mutants that failed to interact with CYR1(1879–2026) were characterized by DNA sequencing and transferred to pAD-GST for expression as GST-fusion proteins in yeast cells. Yeast FS1 was transformed with a combination of either YEP24-ADC1-CYR1-(1–40, 1769–2026) or YEP24-ADC1-CYR1-(1–40, 606–2026) and one of the pAD-GST-CAP-(x–x) plasmids bearing various mutations. In another series of experiments, SP1 was transformed with pAD-GST-CYR1-(y–y) bearing various mutations. The resulting transformants were grown to a density of 1 × 107 cells/ml, harvested by centrifugation, and disrupted by shaking with glass beads in buffer C (50 mmMES, pH 6.2, 0.1 mm MgCl2, 0.1 mmEGTA, 2 mm dithiothreitol, 10% glycerol) as described previously (9Minato T. Wang J. Akasaka K. Okada T. Suzuki N. Kataoka T. J. Biol. Chem. 1994; 269: 20845-20851Abstract Full Text PDF PubMed Google Scholar, 19Wang J. Suzuki N. Nishida Y. Kataoka T. Mol. Cell. Biol. 1993; 13: 4087-4097Crossref PubMed Scopus (26) Google Scholar). Crude membrane fraction was prepared by centrifugation of the homogenate at 27,000 × g for 80 min. GST-CAP-(x–x) or GST-CYR1-(y–y) protein was solubilized from the crude membrane fraction with buffer C containing 1% Lubrol PX, 0.5 m NaCl, and 1 mmphenylmethylsulfonyl fluoride, adsorbed onto glutathione-Sepharose resin, and eluted with 20 mm glutathione as described (27Smith D.B. Johnson K.S. Gene (Amst.). 1988; 67: 31-40Crossref PubMed Scopus (5028) Google Scholar). Proteins bound to the GST-fusion proteins, which were co-eluted from the resin, were subjected to Western immunoblot detection of CYR1 or CAP by using specific antibodies: rabbit polyclonal antisera for a 15-amino acid synthetic peptide of the C terminus of adenylyl cyclase (anti-CYR1CT) or for the full-length CAP (anti-CAP), respectively, as described previously (19Wang J. Suzuki N. Nishida Y. Kataoka T. Mol. Cell. Biol. 1993; 13: 4087-4097Crossref PubMed Scopus (26) Google Scholar). A rabbit polyclonal antiserum for GST (anti-GST) was used for detection of GST fusion proteins. Survival of yeast cells after heat shock treatment at 55 °C for 5 min was examined by a replica plating method as described previously (3Toda T. Uno I. Ishikawa T. Powers S. Kataoka T. Broek D. Cameron S. Broach J. Matsumoto K. Wigler M. Cell. 1985; 40: 27-36Abstract Full Text PDF PubMed Scopus (705) Google Scholar, 19Wang J. Suzuki N. Nishida Y. Kataoka T. Mol. Cell. Biol. 1993; 13: 4087-4097Crossref PubMed Scopus (26) Google Scholar). SDS-polyacrylamide gel electrophoresis and Western immunoblot analysis were performed as described (28Laemmli U.K. Nature. 1970; 227: 680-685Crossref PubMed Scopus (205531) Google Scholar, 29Towbin H. Staehelin T. Gordon J. Proc. Natl. Acad. Sci. U. S. A. 1979; 76: 4350-4354Crossref PubMed Scopus (44644) Google Scholar). The ECL immunodetection system (Amersham Pharmacia Biotech) was used for signal development. Previous experiments had already mapped the N-terminal function of CAP to residues 1–168 (12Gerst J.E. Ferguson K. Vojtek A. Wigler M. Field J. Mol. Cell. Biol. 1991; 11: 1248-1257Crossref PubMed Scopus (116) Google Scholar) and the CAP-binding site of adenylyl cyclase to residues 1879–2026 (19Wang J. Suzuki N. Nishida Y. Kataoka T. Mol. Cell. Biol. 1993; 13: 4087-4097Crossref PubMed Scopus (26) Google Scholar). To further delineate the binding sites, we introduced various deletion mutations into CAP and adenylyl cyclase as described under “Experimental Procedures.” Interactions of the various CAP mutants with CYR1-(1879–2026) and of the various CYR1 C-terminal mutants with CAP were examined by employing the yeast two-hybrid system (Fig. 1 A). As an indicator strain, we used YPB2(CAPΔN), whose chromosomal CAP gene was replaced by its N-terminal deletion mutant CAPΔN-1 in order to exclude the possibility that endogenous CAP complexed with an otherwise negative GAD-fusion CAP mutant may serve as a bridge to yield a positive interaction with the GBT-fusion CYR1. Formation of such a CAP dimer had been reported before (16Zelicof A. Protopopov V. David D. Lin X.-Y. Lustgarten V. Gerst J.E. J. Biol. Chem. 1996; 271: 18243-18252Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar). To our surprise, the shortest CAP construct carrying only the N-terminal 36 residues, CAP-(1–36), as well as the longer CAP-(1–66), CAP-(1–77), and CAP-(1–88) exhibited a positive interaction with the C-terminal region of adenylyl cyclase (Fig. 1 A). In contrast, CAP-(78–526) lacking the N-terminal region did not exhibit any interaction. On the other hand, the shortest fragment of CYR1 giving a positive interaction with CAP was 119 residues corresponding to positions 1898–2016 (Fig. 1 A). An N-terminal deletion up to position 1935 destroyed the activity to interact with CAP. Physical associations of the same sets of CAP and CYR1 mutants with their counterparts were also examined biochemically as shown in Fig. 1,B and C. The CAP deletion mutants were expressed as GST fusions from pAD-GST-CAP-(x–x)s in yeast FS1 cells harboring YEP24-ADC1-CYR1-(1–40, 1769–2026). The GST-CAP fusion proteins were purified by glutathione-Sepharose chromatography and examined for the bound CYR1-(1–40, 1769–2026) by immunoblotting with anti-CYR1CT antibody (Fig. 1 B). Similarly, proteins copurified with GST-CYR1-(y–y)s from yeast cells harboring pAD-GST-CYR1-(y–y)s were examined for CAP by anti-CAP antibody (Fig. 1 C). The results were in good agreement with those of the yeast two-hybrid analysis; the shortest CAP and CYR1 fragments that retained the activity to associate with their counterparts were CAP-(1–36) and CYR1-(1898–2016), respectively. These results indicated that residues 1–36 of CAP and residues 1898–2016 of adenylyl cyclase are sufficient for their mutual association. We examined the abilities of the CAP deletion mutants to confer heat shock sensitivity to TK161-R2V(CAPΔN) cells, which carried the CAPΔN-1 gene encoding the protein lacking its N-terminal function and thereby were made resistant to heat shock in the RAS2 Val-19 background. As shown in Fig. 2 A, expression of the shortest fragment, GST-CAP-(1–36), as well as other longer CAP N-terminal fragments was sufficient to restore the heat shock sensitivity in this yeast strain. As observed in the binding assays, both GST-CAP-(77–526) and GST only were found inactive. This result implied that the N-terminal 36 residues are functional in the Ras-adenylyl cyclase pathway. Heat shock sensitivity was also used for examining the CAP binding activity of various CYR1 fragments. It had been shown that overexpression of the CAP-binding region of adenylyl cyclase suppressed the RAS2 Val-19-dependent heat shock sensitivity presumably by competitive sequestration of CAP from the endogenous adenylyl cyclase (19Wang J. Suzuki N. Nishida Y. Kataoka T. Mol. Cell. Biol. 1993; 13: 4087-4097Crossref PubMed Scopus (26) Google Scholar). We examined the activity of the CYR1-deletion mutants overexpressed from pAD-GST-CYR1-(y–y) to suppress the heat shock sensitivity of TK161-R2V (Fig. 2B). Again, the result was in good agreement with that of the CAP-binding assays,i.e. CYR1-(1898–2016) was the shortest fragment exhibiting this activity. The mutually interacting regions, CAP-(1–36) and CYR1-(1898–2016), were searched for a peculiar sequence motif hinting at the mechanism of their interaction. The search identified tandem repetitions of a heptad motif αXXαXXX (where α and X represent a hydrophobic amino acid and any amino acid, respectively; for reviews, see Refs. 30Cohen C. Parry D.A.D. Trends Biochem. Sci. 1986; 11: 245-248Abstract Full Text PDF Scopus (301) Google Scholar and 31Lupas A. Trends Biochem. Sci. 1996; 21: 375-382Abstract Full Text PDF PubMed Scopus (1008) Google Scholar) in both residues 13–30 of CAP and residues 1916–1930 of adenylyl cyclase (Fig. 3 A). If the heptad repeat motif is taken as that of a leucine zipper LXXXXXX (32Landschultz W.H. Johnson P.F. McKnight S.L. Science. 1988; 240: 1759-1764Crossref PubMed Scopus (2513) Google Scholar), adenylyl cyclase has one more repeat unit in residues 1931–1937. These heptad repeats enabled us to predict formation of α-helices that are wound around each other to form a superhelix, the coiled-coil structure (30Cohen C. Parry D.A.D. Trends Biochem. Sci. 1986; 11: 245-248Abstract Full Text PDF Scopus (301) Google Scholar, 31Lupas A. Trends Biochem. Sci. 1996; 21: 375-382Abstract Full Text PDF PubMed Scopus (1008) Google Scholar). This was also supported by calculation of the probability of adopting a coiled-coil conformation using the computer program COILS (33Lupas A. Methods Enzymol. 1996; 266: 513-525Crossref PubMed Google Scholar). Residues 11–33 of CAP were predicted to have more than 95% probability of forming a coiled-coil, whereas the probability for the other portion of CAP was almost 0. To further analyze the molecular mechanism for the CAP-adenylyl cyclase interaction, we introduced random mutations into residues 1–77 of CAP by an error-prone PCR and carried out a yeast two-hybrid screening for mutants that became defective in associating with adenylyl cyclase as described under “Experimental Procedures.” Out of 208 clones analyzed, 43 showed up negative in interacting with CYR1-(1879–2026) and were subjected to DNA sequencing to identify the nature of mutations. 33 clones carrying a stop codon or more than two mutations within residues 1–36 were discarded, and CAP-(1–77) inserts of the remaining 10 clones, whose mutations are shown in Table II, were transferred to pAD-GST for expression as GST-fusion proteins in the yeast FS-1. GST-CAP-(1–77) proteins derived from all 10 clones were expressed in a similar amount and solubilizable from the membranes by Lubrol PX, but those from five clones (HS92, HS113, HS143, HS155, and HS197) were found to have lost the ability to attach onto glutathione-Sepharose resin (data not shown), suggesting gross alterations in their conformations, and, therefore, were excluded from further analyses. The remaining five clones were examined for physical association with a deletion mutant CYR1 protein that retained the Ras-responsive adenylyl cyclase activity (8Suzuki N. Choe H.-R. Nishida Y. Yamawaki-Kataoka Y. Ohnishi S. Tamaoki T. Kataoka T. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 8711-8715Crossref PubMed Scopus (100) Google Scholar) (Fig. 4 A). The results clearly indicated that GST-CAP-(1–77) from the five clones HS10, HS27, HS139, HS205, and HS208 lost the ability to associate with CYR1-(1–40, 606–2026). Concurrently, the same CAP mutants lost the activity to confer heat shock sensitivity to TK161-R2V(CAPΔN) yeast cells (Fig. 5 A). Strikingly, four out of the five clones turned out to carry an amino acid substitution mutation at Leu-20, Leu-27, or Val-30, all of which corresponded to the key hydrophobic residues in the heptad repeats (Fig. 3 A). Moreover, the mutations were introduced in such a way that the hydrophobic residues were converted to neutral or hydrophilic residues (Table II). The other clone HS205 carried two mutations, T31P and Q34L. Although Gln-34 is located at the position in the heptad repeats corresponding to the key hydrophobic residue, it is presently unclear which of the two mutations is responsible for the effect.Table IIAmino acid substitutions found in CAP mutantsMutant CAP clonesMutations in residues 1–36HS10V30DHS27L20P, D29GHS92Q9LHS113T24AHS139L27SHS143T31AHS155Y6L, T7AHS197L16RHS205T31P, Q34LHS208L20P Open table in a new tab Figure 5Heat shock sensitivity of yeast cells expressing the mutated CAP and adenylyl cyclase. A, heat shock sensitivity of TK161-R2V(CAPΔN) yeast cells harboring pAD-GST (Vector), pAD-GST-CAP-(1–77) (Wild type), or pAD-GST-CAP-(1–77) carrying the indicated mutation was examined as described in the legend to Fig. 2 A.B, heat shock sensitivity of TK161-R2V cells harboring pAD-GST (Vector), pAD-GST-CYR1-(1822–2026) (Wild type), or pAD-GST-CYR1-(1822–2026) carrying the indicated mutation was examined similarly. The experiments were repeated three times, yielding identical results.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Next, we examined the importance of hydrophobic residues in the predicted heptad repeats of adenylyl cyclase in association with CAP. The key hydrophobic residues Leu-1916 and Leu-1923 corresponding to the α-position (Fig. 3 A) were converted to Ser and to either Pro or Arg, respectively, by oligonucleotide-directed mutagenesis. The CYR1 C-terminal fragments carrying L1916S, L1923P, and L1923R mutations all lost the ability to associate with CAP as assayed by the yeast two-hybrid method (Fig. 4 B) or by the in vivobinding assay (Fig. 4 C). Overexpression of CYR1-(1822–2026) bearing the same mutations could not suppress theRAS2 Val-19-dependent heat shock sensitivity (Fig. 5 B). These results indicated that the hydrophobic residues of both CAP and adenylyl cyclase are indeed critical not only for their mutual association but also for their proper function in the Ras-adenylyl cyclase pathway and further supported the involvement of the coiled-coil mechanism for their interaction. We have shown that the N-terminal 36 residues of CAP are sufficient for association with adenylyl cyclase as well as for itsin vivo function in the Ras-adenylyl cyclase pathway. The CAP-binding site of adenylyl cyclase was mapped to a 119-residue segment near the C terminus. Close inspection of the primary sequences of the two mutual binding sites has identified typical heptad repeat motifs (αXXαXXX)n indicative of a coiled-coil interaction (30Cohen C. Parry D.A.D. Trends Biochem. Sci. 1986; 11: 245-248Abstract Full Text PDF Scopus (301) Google Scholar, 31Lupas A. Trends Biochem. Sci. 1996; 21: 375-382Abstract Full Text PDF PubMed Scopus (1008) Google Scholar) (Fig. 3 A). Furthermore, the presence of the coiled-coil in the CAP N terminus was predicted by the computer program COILS (33Lupas A. Methods Enzymol. 1996; 266: 513-525Crossref PubMed Google Scholar). Coiled-coils are composed of two, three, or four α-helices wound around each other to form a left-handed superhelix. This structure is found in a wide variety of proteins including cytoskeletal structural proteins, transcription factors, etc. (30Cohen C. Parry D.A.D. Trends Biochem. Sci. 1986; 11: 245-248Abstract Full Text PDF Scopus (301) Google Scholar, 31Lupas A. Trends Biochem. Sci. 1996; 21: 375-382Abstract Full Text PDF PubMed Scopus (1008) Google Scholar). Further, a number of proteins whose three-dimensional structures have been determined are found to contain coiled-coil segments, many of them very short. The presence of this structure in a protein can be predicted from an array of the typical heptad motif in its amino acid sequence, which is labeled a-b-c-d-e-f-g, where a and d are primarily hydrophobic, most frequently Leu, residues and form the helix interface, whileb, c, e, f, and g are hydrophilic and form the solvent-exposed surface of the coiled-coil. The association of the helices is stabilized by hydrophobic interactions at the side chains of the hydrophobic residuesa and d, which form an apolar stripe along one side of the helix, and additionally by ionic interactions at the side chains of the nearby charged residues. This is apparent in spinning wheel representations of the two possibly interacting segments CAP-(6–34) and CYR1-(1916–1940) (Fig. 3 B). The assignments of the a- and d-positions were supported by an amino acid sequence homology between CAP-(6–34) and Dirofilaria immitis paramyosin (34Limberger R.J. McReynolds L.A. Mol. Biochem. Parasitol. 1990; 38: 271-280Crossref PubMed Scopus (40) Google Scholar), which was found by BLASTP search (35Altschul S.F. Gish W. Miller W. Myers E.W. Lipman D.J. J. Mol. Biol. 1990; 215: 403-410Crossref PubMed Scopus (68368) Google Scholar) of GenBankTM entries. Residues 16–29 of CAP (Fig. 3 A) shared nine identical residues with residues 213–226 of paramyosin (LAQQLEEARRRLED). This match made it possible to predict positions a–g of each residue of CAP from the proposed α-helical structure of paramyosin (36McLachlan A.D. Karn J. Nature. 1982; 299: 226-231Crossref PubMed Scopus (352) Google Scholar). A further proof for the coiled-coil interaction came from the studies on mutations of CAP and adenylyl cyclase, which abrogated the interaction. Strikingly, the three residues of CAP (Leu-20, Leu-27, and Val-30) and two residues of adenylyl cyclase (Leu-1916 and Leu-1923) that were identified to be essential for the interaction based on these mutational studies are all hydrophobic and located at positiona or d. These results strongly support the notion that the coiled-coil mechanism forms a molecular basis for the CAP-adenylyl cyclase interaction. At present, it is impossible for us to predict from the amino acid sequences how many strands of CAP and adenylyl cyclase contribute to the formation of the coiled-coil superhelix. It is also impossible to predict the relative orientation, parallel or anti-parallel, of the strands of CAP and adenylyl cyclase and how individual pairs of the residues from each strand are formed, both of which are primarily determined by polar and ionic interactions between residues flanking the hydrophobic core (30Cohen C. Parry D.A.D. Trends Biochem. Sci. 1986; 11: 245-248Abstract Full Text PDF Scopus (301) Google Scholar, 31Lupas A. Trends Biochem. Sci. 1996; 21: 375-382Abstract Full Text PDF PubMed Scopus (1008) Google Scholar). Elucidation of these structural features awaits determination of the three-dimensional structure of the CAP-adenylyl cyclase complex. The heptad repeat structure is well conserved in CAP homologues identified in other organisms including Schizosaccharomyces pombe and mammals, although the N-terminal function of the budding yeast CAP is not conserved among them (37Kawamukai M. Gerst J. Field J. Riggs M. Rodgers L. Wigler M. Young D. Mol. Biol. Cell. 1992; 3: 167-180Crossref PubMed Scopus (95) Google Scholar, 38Matviw H., Yu, G. Young D. Mol. Cell. Biol. 1992; 12: 5033-5040Crossref PubMed Scopus (62) Google Scholar, 39Zelicof A. Gatica J. Gerst J.E. J. Biol. Chem. 1993; 268: 13448-13453Abstract Full Text PDF PubMed Google Scholar, 40Vojtek A.B. Cooper J.A. J. Cell Sci. 1993; 105: 777-785PubMed Google Scholar). This suggests that in those organisms CAP may establish a coiled-coil interaction at its N-terminal short segment with a certain protein to exert a function that is presumably different from that of the Ras-adenylyl cyclase pathway. The identification of such a CAP-interacting protein may reveal a novel function of CAP in addition to its C-terminal cytoskeletal function, which is known to be conserved between yeasts and mammals (37Kawamukai M. Gerst J. Field J. Riggs M. Rodgers L. Wigler M. Young D. Mol. Biol. Cell. 1992; 3: 167-180Crossref PubMed Scopus (95) Google Scholar, 38Matviw H., Yu, G. Young D. Mol. Cell. Biol. 1992; 12: 5033-5040Crossref PubMed Scopus (62) Google Scholar, 39Zelicof A. Gatica J. Gerst J.E. J. Biol. Chem. 1993; 268: 13448-13453Abstract Full Text PDF PubMed Google Scholar, 40Vojtek A.B. Cooper J.A. J. Cell Sci. 1993; 105: 777-785PubMed Google Scholar). We thank X.-H. Deng for skillful technical assistance and A. Seki and A. Kawabe for help in preparation of this manuscript.

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