Proc. Natl. Acad. Sci. USAVol. 86, pp. 5693-5697, August 1989Biochemistry

Adenylate cyclases in yeast: A comparison of the genes fromSchizosaccharomyces pombe and Saccharomyces cerevisiae

(cyclic AMP/fission yeast/evolution)

YURIKO YAMAWAKI-KATAOKA, TATSUYA TAMAOKI, HYE-RYUN CHOE, HIDEHo TANAKA,AND TOHRU KATAOKADepartment of Cellular and Molecular Physiology, Harvard Medical School, 25 Shattuck Street, Boston, MA 02115

Communicated by Howard Green, April 17, 1989

ABSTRACT A Schizosaccharomyces pombe gene encodingadenylate cyclase has been cloned by cross-hybridization withthe Saccharomyces cerevisiae adenylate cyclase gene. The pro-tein encoded consists of 1692 amino acids and has adenylatecyclase activity that cannot be activated by the Sa. cerevisiaeRAS2 protein. Sc. pombe cyclase has a high degree ofhomology(..60%) with the catalytic domain of Sa. cerevisiae cyclaseprecisely mapped by a gene-deletion analysis. A 2540%identity is observed throughout the middle segments of "1000residues of both cyclases, large parts of which are composed ofrepetitions of a 23-amino acid motif similar to those found inhuman glycoproteins, Drosophila chaoptin, and ToU gene prod-uct. However, a segment corresponding to the NH2-terminal620 residues of Sa. cerevisiae cyclase appears lost from Sc.pombe cyclase, and the COOH-terminal 140 residues are notwell conserved between the two yeast species. Deletions involv-ing the COOH-terminal residues of Sa. cerevisiae cyclase causeloss of activation by the RAS2 protein. These results suggestthat Sc. pombe cyclase may have lost the ability to interact withRAS proteins by the loss of a regulatory site.

Adenylate cyclase has been shown to play essential roles inregulation of cellular metabolism by catalyzing the synthesisof a second messenger, cAMP. A guanine nucleotide-binding(G) protein called Gs mediates hormone-dependent activationof mammalian adenylate cyclase (for review, see ref. 1).However, in the yeast Saccharomyces cerevisiae, adenylatecyclase is regulated by a different set ofG proteins, RAS1 andRAS2, which are homologs of mammalian ras oncoproteins(2, 3). It was shown that mammalian ras proteins couldactivate yeast adenylate cyclase but neither mammalian rasnor yeast RAS proteins could regulate mammalian adenylatecyclase (3-5). Although the precise mode of interactionbetween RAS proteins and yeast adenylate cyclase is un-known, regulatory proteins of adenylate cyclase appear tohave somehow switched during the course of evolution.Specifically, it was shown that the cAMP concentration ofthe fission yeast Schizosaccharomyces pombe cells was notaffected by the disruption or mutational activation of its soleras gene, suggesting that its adenylate cyclase is not regulatedby ras proteins (6). Sa. cerevisiae adenylate cyclase com-prises 2026 amino acids in which the COOH-terminal 417residues, called the catalytic domain, retains a Mn2+_dependent cyclase activity (7). The remaining portion of theNH2 terminus was proposed to be essential for the RASprotein- and GTP-dependent activation in the presence ofMg2+ (7-9). In this report, we describe the primary structureof Sc. pombe adenylate cyclase.* Sequence comparison withSa. cerevisiae adenylate cyclase reveals four segments dis-

playing varying extents of homology. The functional signif-icance of these segments is discussed.

MATERIALS AND METHODSCell Strains, Growth Media, and Transformation. A Sa.

cerevisiae strain, T50-3A (MATa, leu2, his3, trpl, ura3,cyrl-2) was described (7). A Sc. pombe strain 972h-s wasobtained from D. Beach, Cold Spring Harbor Laboratory.Culture media for yeast cells and method of yeast transfor-mation were as described (2-4, 10).DNA and RNA Preparation and Hybridization Analyses.

Isolation of yeast genomic DNA and Southern blot hybrid-ization were done as described (4). The low-stringency hy-bridization was performed at 50TC in 1 M NaCl, and washingof the filters was done in 0.15 M NaCl at 50TC. Preparationof poly(A)+ RNA from Sc. pombe cells and Northern blothybridization were done as described (7, 11, 12).

Cloning, Sequencing, and Deletion-Mutagenesis. Construc-tion of a cDNA library from Sc. pombe poly(A)+ mRNA wasdone (13) by using pUC18 as a vector. Fragments of the Sc.pombe gene were cloned into M13 vectors and subjected toDNA sequencing (14). Protein sequences were compared bythe DNA/PROTEIN sequence analysis program (InternationalBiotechnologies). Expression of Sc. pombe cyclase in Sa.cerevisiae was done using the vector pAD-1 (obtained fromJ. Nikawa, Gunma University, Gunma, Japan) (see Fig. 4A).YRp7-ADCJ-CYRJ (15), which over-expressed the complete

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FIG. 1. Structure and Northern (RNA) blot analysis of thespCYRI gene. (A) Restriction maps of the genomic spCYRI gene (a),ofthe sequenced region (b), and ofthe cDNA clones (c). The hatchedregions represent the protein-coding sequence. Abbreviations usedare as follows: B, BamHI; C, Cla I; E, EcoRI; and H, HindIll. (B)Five micrograms of poly(A)+ RNA from Sc. pombe 972h-s wasfractionated in 1% agarose gel containing formaldehyde. A Northernblot of the gel was hybridized with the 32P-labeled EcoRI-HincIIfragment of the spCYRI (corresponding to the amino acid positions1341-1502) and autoradiographed. RNA size markers were obtainedfrom Boehringer Mannheim.

Abbreviation: p[NH]ppG, guanosine 5'-[(3,y-imido]triphosphate.*The sequence reported in this paper has been deposited in theEMBL/GenBank data base (accession no. M24942).

5693

The publication costs of this article were defrayed in part by page chargepayment. This article must therefore be hereby marked "advertisement"in accordance with 18 U.S.C. §1734 solely to indicate this fact.

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Proc. Natl. Acad. Sci. USA 86 (1989) 5695

Sa. cerevisiae adenylate cyclase gene (CYRJ) from the yeastalcohol dehydrogenase I (ADCI) promoter, was cleaved withCla I and then digested with the BAL-31 exonuclease. DNAfragments carrying deletions were used to reconstruct theYRp7-ADCI-CYRI plasmids having deletions. The plasmidswere designated as YRp7-ADCI-CYRJ(x-x), in which x'srepresented the ranges of deletions in amino acid positions.Antibody Preparation and Western' Blot Analysis. A poly-

peptide of Sa. cerevisiae adenylate cyclase corresponding tothe amino acid positions 335-596 was expressed as a fusionprotein with Escherichia coli p-galactosidase in E. coli JM101by using pUR292 (16). The fusion protein was purified byabsorption and elution from Sepharose 4B coupled withanti-/3-galactosidase antibody (Promega) and used to immu-nize rabbits. For Western (immunologic) blotting 50 ,ug ofmembrane proteins was electrophoresed on 5% SDS/poly-acrylamide gels and transferred to nitrocellulose filters (17,18). Detection of Sa. cerevisiae adenylate cyclase proteinwas done by using the anticyclase antibody according to themanufacturer's (Promega) instructions.Assay of Adenylate Cyclase Activity. Yeast cells were grown

to the density of6-10 x 106 cells per ml, harvested, and lysedby shaking with glass beads using a Mini Beads-Beater(Biospec Products, Bartlesville, OK). Crude membrane frac-tions were prepared, and 30 ,g of proteins was assayed foradenylate cyclase activity essentially as described (2, 3, 7).Where indicated, 5 ,g of purified RAS2 protein (3) waspreincubated with 5 Al of 1 mM guanosine 5'-[3,8y-imido]-

triphosphate (p[NH]ppG) and added to the reaction mixtureas described (3, 7, 15).

RESULTSCloning of Sc. pombe Adenylate Cyclase Gene. A Southern

blot of Sc. pombe DNA digested with EcoRI was hybridizedwith a probe covering the catalytic domain of Sa. cerevisiaecyclase (a Pvu II-Cla I fragment) (7) at reduced stringency(see Materials and Methods). Two bands, 14 and 4 kilobases(kb) in size, were detected (data not shown). These EcoRIfragments were cloned by hybridization screening from aDNA library in which EcoRI partial digests of Sc. pombeDNA were cloned into A phage vector EMBL3. The 14-kbEcoRI fragment was obtained in a clone containing threeEcoRI fragments of 5.6, 0.18, and 14 kb and analyzed further.By sequencing 5.3 kb surrounding the hybridizing sequence(see Fig. 1A), we found a single long open reading framedesignated spCYRI that could encode 1692-amino acid res-idues as shown in Fig. 2. The 4-kb EcoRI fragment was alsocloned and found by DNA sequencing not to have significanthomology with the Sa. cerevisiae CYR] gene. Because Sc.pombe genomic genes often contain introns, we isolatedcDNA clones from a Sc. pombe cDNA library by hybridiza-tion screening with an internal 5-kb BamHI fragment of thespCYRI. The structure of the cDNA clones depicted in Fig.L4 was identical with that of the corresponding part of thespCYRI, although the cDNAs obtained were split at internalEcoRI sites, presumably due to incomplete treatment with

SP 1 MDQSKRLLKSAV-PNPPEHFKTGISWLDDLDEKDDDSATSVNYDIPEITE 334 NFYLLLIQFNTERILLPHEQPCIIFERLLSLF-GCKVTSD*1* IIII* *II 11111* * *** 11* *11 1 1 *111 1 11 *11

SC 624 KSYTKKFTSSSVNMNSPDGAQS--SGLLLQDEKDDEVECQLEHYYKDFSD 718 NFQISLKVGKLSKILRPTSKPILI-ERKLLLNNGYRK-SD

SP 417 LEVIPVKIYPYA-HELISLNVSHNLSLDLPLDFMERCVKLKRLDISNNLRSPRGKP-IT1* 1* 11 11 * 111*1*1 *11 * 1*1 * 11

SC 800 RNMDLTTPPIIFYQHT-SEIESLDVSNNANIFLPLEFIESSIKLLSLRMV-NIRASKFPSNITKAYKLVS11 1*11 * * * 11 111 * * * 11 11 *

SP 485 RNDIYELDPLIFSGLSRNSLKELNIANNKLFFLP-HSTRYLVNLTYLALSYNNFVT-FPLIITELSQLET

SP 553 LNFSHNLLSQISSKIGSLVKLKHLYLQFNDLSNRLPQEIGLLKNLETIDLSYNI *I* * III1 11 1*1 11 1111 *111 1

SC 897 LELQRNFIRKVPNSIMKLSNLTILNLQCNEL-ESLPAGFVELKNLQLLDLSSN

Sp 690 VNYNHFTSISDAISAMQNLKYLSCTNCEMSYVSPNLGKLKHLVHLDLHANNIKIFPEEVWQVSSLKVVNLSSNI*1 * I1 * *11 * 1* 11 1111 111* * *** *1*111SC 1071 LNQNNLTRLPQEISKLTKLVFLSVARNKLEYIPPELSQLKSLRTLDLHSNNIRDFVDGMENL-ELTSLNISSNA

SP 809 EE-LYLVDNRLGNDCFTALEYFKCLKVLNLSYNYLTEIPSKFFQNFSDLK-H---------- SGNELANLSISSTAQVL-LETLYANGNRLSSFPKNEA-** *** 111111111 1111* I 111 11 * * 11 *

SC 1178 DDAMWPLFNCFVN------ LKVLNLSYN ---------- NFSDVSHMKLESITELYLSGNKLTTLSGDTVLKWSSLKTLMLNSNQMLSLPAELSN

SP 895 LSKSLRFLDISTNNLQNLAVEKAEKKSLTKLPQLEYLNLSGNTWF - RFSEHE-DTNFTK-SYLKNLKFLSIMDLNTKFSNAPSDVLNHFIQRNSPQP-11 1* 1*1 * 111 111 1* * * *11 *11* * * *l *

SC 1256 LSQLSVF-DVGANQLKYNISNYHSDWNWRNNKELKYLNFSGNRRFEIKSFISHDIDADLSDLTVLPQLKVLGLMDVTLNTTKVPDENVN-FRLRTTASII

SP 992 NILRYGVCGYLSRSIPVISACELVVNNFLHPQSSLYCVLDSDISA - GKNN-RVLKFVYDNLASCLAHEINAADSSSEQICNALRSFLRLNKKLGNVI*1111 1* I1 1*1 I*** *11 * * * **I 111 111 111 * **

SC 13 54 NGMRYGVADTLGQRDYVSS.K.RDVTFERFRGNDDECS LCLHDSKNQNADYGHNISRIVRDIYDKIL--IRQLERzYGDETDDNIKTALRFSFLQLNKEINGML

SP 1088 HYDLRKSSEGDVDSN-YVTTMNISEKGYSMDSSCLDIGVSIILVYVRDTRAFVANVGTSMAIMSTRNDSEPTTLSVMHDVYNRDEIRRIVDSCGFI-SGE11 ** 11 1* 1 1*1 II 1I1**l1

SC 1452 NSV ---DNGADVANLSYADL--------------LLSGACSTVIYIRGKKLFAANLGDCMAILSKNN-GDYQTLTKQHLPTKREEYERIRISGGYVNNGK

SP 1186 IKSTT--TRAIGRLSQFPGVQAVPYVNVQYLSELNEFIILANQEFWSVLSKRTVIDVVRGNRHSPLLASTKLRDYAIAYGAEKNVLVVIVELNGLFEENS* *11*1 1 *11*1I1* 1 *1*1 1 *'111* 1 1 11 1*1 1*111 I****I I1

SC 1534 LDGVVDVSRAVGFFDLLPHIHASPDISVVTLTKADEMLIVATHKLWEYMDVDTVCDIARENSTDPLRAAAELKDHAMAYGCTENITILCLAL---- YEN-

SP 1284 LNFNQLRGDEKTLAISEKNDNMSFVQDLPDDSSLARMNREVSPPKGCIAMVFTDIKNSTLLWERHPIAMRSAIKTHNTIMRRQLRATGYEVKTEGDAFM**II111 11 I1 1* *I**II1**I*III 1 *11111111 11 111 1lI|l*llIIII 11.11111 Il11111111111SC 1629 _-NOLRF----TLN---KNSLMIRRSTF-EDTTLRRLOPESISPPTGNLAMVFTDIKSSTFLWELFPNAMRTAIKTHNDIMROLRIYGGYEVKTEGDASP 1384 VCFQTVPAALLWCFSVQLQLLSADWPNEIVESVQ-GRLVLGSKNE-VLYRGLSVRIGVNYGVTVSELDPITRRMDYYGPVVNRTSRVVSVADGGQIAVSA

II IIIlll11ll11111111I11 *1 **l11111*l**I Il111*III111II*II* 11 11111111*1SC 1721 VAFPTPDSGLTWCLSVOLKLLDAOWPEEIT-SVODGCOVTD-RNGNIIYOGLSVRMGIHWGCPVPELDLVTORMDYLGPMVNXAARVOGVADGGOIAMSS

SP 1482 EVVSVLNQLDS--ETMS----SEKTN-------- VNEMEVRALKQIGYIIHNLGEFKLKGLDTTEMISLVYPVQLQGRLERLIK---SRSLGTPTALPET* * I* 1* 111* 11 INl* 1I****11 I I I 1* 11 1

SC 1819 DFYSEFNKIMKYHERWKGKESLKEVYGEEIIGEVLEREIAMLESIGWAFFDFGEHKLKGLETXELVTIAYPKILASRHEFASEDEQSK-LINETML---

SP 1565 QTYTPVRSRSNSLR---PMLARLS ------ DSKSVHGEEGGSGKRSVSSLRNVSPSESTGGYEGCIFDDQQYQLLY-E-LCERLEDHAAILHGFPEPPPC

SC 1915 -----FRLRVISNRLESIMSA-LSGGFIELDSRT ----EGSYIKFNPKVENGIMQSISEKD----------ALLFFDHVITRIESSVALLHLRQQR--C

SP 1654 DTGLAAPVNQAEEYSLFYRLTLR--IENTIYCVSQMLGHTG*11 * ** I I1I* * 11*

SC 1994 -SGLEICRN--D------ KTSARSNIFNVVDELLQMVKNAKDLST

FIG. 3. Protein sequence comparison between Sc. pombe (SP) and Sa. cerevisiae (SC) adenylate cyclases. The amino acid sequences ofregions of Sc. pombe and Sa. cerevisiae cyclases found to have homology are shown. Vertical lines represent identities, and asterisks representconservative amino acid substitutions. Dashes indicate insertions introduced for the best alignment. Underlined is the range of the catalyticdomain of Sa. cerevisiae cyclase described in text.

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EcoRI methylase upon cloning. Therefore the spCYRI genehad no intron in its coding sequence. The molecular size ofmRNA derived from the spCYRI gene was determined as -6kb (see Fig. 1B), which approximately corresponded with thelength (5.5 kb) of sequence represented in the cDNA clones.

Structure of the spCYRI Gene Product and Its Homology withSa. cerevisiae Adenylate Cyclase. The nucleotide sequence ofthe spCYRJ gene and the deduced amino acid sequence areshown in Fig. 2. The protein sequence was compared with thatof Sa. cerevisiae adenylate cyclase (7), and an alignment thatshowed the best match is shown in Fig. 3. The result indicatedthat the spCYRI product consisted of four segments havingdifferent degrees ofhomology with Sa. cerevisiae cyclase. Thehighest homology (-60%o) was seen between amino acidpositions 1313 and 1540 of the spCYRJ product and 1650 and1890 of Sa. cerevisiae cyclase. The middle part ofthe spCYRJproduct (positions 334-1313) was -25-40% homologous withthe corresponding part of Sa. cerevisiae cyclase, althoughshort stretches of nonconserved regions were interspersed. Alarge portion of this segment was composed of repetitions ofa 23-amino acid motifvery similar to that seen in Sa. cerevisiaecyclase (7) (see Fig. 2). The motif was PXXaXXLXXLXX-LXLXXNXaXXa (P, proline; L, leucine; N, asparagine; a,aliphatic amino acids; and X, any amino acids). As reported,this motif has striking similarity to repetitive motifs identifiedin human glycoproteins and Drosophila proteins (7, 19-23). Asegment corresponding to the NH2-terminal 620 residues ofSa. cerevisiae cyclase appeared lost from the spCYRl prod-uct, whereas =280 residues (positions 50-334) of the spCYRIproduct did not have a corresponding sequence in Sa. cere-visiae cyclase. The COOH-terminal segments of Sa. cerevi-siae cyclase (positions 1891-2026) and of the spCYRI product(1540-1692) had <25% identity with each other, and introduc-tion of many insertions were required for their alignment. Asseen in Sa. cerevisiae cyclase, the spCYRI product had notypical nucleotide-binding consensus sequences (24) and noparticularly hydrophobic segment.The spCYRI Gene Encoded Adenylate Cyclase. The striking

homology with the catalytic domain of Sa. cerevisiae cyclasestrongly suggested that the spCYRI gene encoded adenylatecyclase. To prove that, we constructed two plasmids, pAD-spCYRJ-1 and pAD-spCYRI-2, which could express thespCYRI product starting from the amino acid position 85 or1, respectively, under the ADCJ promoter (see Fig. 4A). Theplasmids were transformed into the Sa. cerevisiae strain,T50-3A (7), which has a temperature-sensitive adenylatecyclase, the cyrl-2 mutation (25). Leu+ transformants wereobtained at the nonpermissive temperature 34°C on selectionplates for leucine auxotrophy supplemented with 1 mMcAMP, which had been shown to rescue the cyrl-2 mutant atthe nonpermissive temperature (25). The transformants werestreaked on the cAMP-containing plates, replica-plated onplates with and without cAMP, and incubated at 34°C. Asshown in Fig. 4B, the spCYRI plasmids could suppresstemperature sensitivity of T50-3A without cAMP. The colo-nies were grown at 34°C in the presence of cAMP, andmembrane fractions were isolated and assayed for adenylatecyclase activity. The result shown in Table 1 indicated thatT50-3A cells having the spCYRI expression plasmids gaineda low but significant adenylate cyclase activity in the pres-ence of Mn2+ or Mg2+. These data lead us to conclude thatthe spCYRI gene encoded adenylate cyclase. However, wecould not detect any activation of the Sc. pombe adenylatecyclase activity by adding the purified RAS2 protein andp[NH]ppG at the concentrations sufficient to activate Sa.cerevisiae cyclase by 50-fold (see Table 1). No activity wasdetected in soluble fractions. Because the relatively lowactivity and loss of activation by the RAS2 protein might bedue to degradation or improper modification ofthe Sc. pombecyclase protein in Sa. cerevisiae cells, crude membrane was

A

LEU2 HPoo/Hpof LEU2 HpoI/HoPa2 )j circle 2 jjcircle

XboZ XboZ

/(AmpR Promoter (AmpR promoter

~~~ATG HindZOK!P Or! )

sCYRI

PS/I SpCYRI

BcAMP a b c d e f

g h i k

FIG. 4. Complementation of cyrl-2 mutation by various plas-mids. (A) Structures of plasmids pAD-spCYRI-1 (Left) and -2(Right), which express the spCYRI gene under the ADCO promoter,are shown. Restriction sites in parentheses mean the sites are lostafter ligation. (B) T50-3A cells transformed with the pAD-spCYRI-1(a), pAD-spCYRI-2 (b), pAD-1 (c), YRp7-ADCJ-CYRJ(1916-2026)(d), YRp7-ADCJ-CYRJ(1881-2026) (e), YRp7-ADCJ-CYRJ(1900-2026) (f), YRp7 (g), YRp7-ADCJ-CYRJ(1932-2026) (h), YRp7-ADCI-CYRI(1906-2026) (i), YRp7-ADCI-CYRI(1882-2026) (j), andYRp7-ADCJ-CYRJ(1890-2026) (k) were grown on suitable auxo-trophic selection plates with and without 1 mM cAMP for 2 days andphotographed.

prepared from Sc. pombe 972h-s cells and assayed foradenylate cyclase activity as shown in Table 1. The adenylatecyclase activity of the Sc. pombe cells was low comparedwith that of Sa. cerevisiae and unable to be activated byRAS2 protein plus p[NH]ppG or p[NH]ppG only.Mapping of Functional Domains of Sa. cerevisiae Adenylate

Cyclase. The NH2-terminal border of the catalytic domain

Table 1. Adenylate cyclase activity of various cell membranesAdenylate cyclase activity

Mg2+ +Mg2+ + p[NH]-

Host p[NH]- ppG +Plasmid cell Mn2+ Mg2+ ppG RAS2

pAD-spCYRI-1 T50-3A 1.74 0.14 0.12 0.12pAD-spCYRJ-2 T50-3A 0.72 0.10 0.11 0.10pAD-1 T50-3A 0.02 0.00 0.00 0.00None 972h-s 1.21 0.59 0.46 0.37None SP-1 9.8 0.19 1.32 4.3YRp7-ADCI-CYRI T50-3A 106.1 2.4 NT 117.5CYRI(1906-2026)* T50-3A 428.9 9.3 NT 5.2CYRI(1932-2026)* T50-3A 178.8 8.8 NT 11.7

All values presented are units of activity where one unit is definedas the production of 1 pmol ofcAMP per min per mg of protein. NT,not tested.*Abbreviation of the corresponding YRp7-ADCI-CYRI (deletion)plasmids.

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a b c d e f FIG. 5. Detection of adenylate cy-clase proteins from deletion mutantplasmids. Membrane fractions iso-lated from T50-3A cells harboringYRp7-ADCJ-CYRJ(1906-2026) (lane

20OWk-0 a), YRp7-ADCJ-CYRJ(1932-2026)(lanes b and c), YRp7-ADCJ-CYRJ(lane d), and YRp7 (lane f), and from awild-type Sa. cerevisiae strain SP1 (7)(lane e) were fractionated in 5% poly-acrylamide gel. The Western blot ofthe gel was subjected to detection ofthe Sa. cerevisiae adenylate cyclaseprotein as described. The molecularmass markers were myosin heavy

97ko chain and phosphorylase b. k, kDa.

had been mapped approximately to the amino acid position1609 (7). Comparison between the Sa. cerevisiae and Sc.pombe cyclase protein sequences suggested that the uncon-served COOH-terminal 140 residues might not be included inthe catalytic domains. Therefore, we introduced step-wisedeletions into the COOH-terminal part of Sa. cerevisiaecyclase by using exonuclease BAL-31. The deletions startedfrom a Cla I cleavage site at amino acid position 1959 andextended to the upstream sequence, and the resultant cyclasegenes bearing deletions were expressed under the ADCJpromoter. The plasmids were transformed into T50-3Astrain, and complementation of the cyrl-2 mutation wasexamined as described above for the spCYRJ gene. As shownin Fig. 4B, the deletions that extended further upstream thanamino acid position 1890 abolished the complementationability. Therefore, we concluded that the catalytic activityresided in the 280 residues (positions 1609-1890) of Sa.cerevisiae cyclase. Some ofthe cyrl-2 strains having deletioncyclase plasmids were grown at 34WC, and their membraneswere assayed for adenylate cyclase activity. As shown inTable 1, the deletion cyclases failed to be activated by theRAS2 protein and p[NH]ppG even though they retained theMn2+-dependent activity. Loss of the activation did notappear to be ascribed to degradation of the mutant cyclaseprotein as shown in Fig. 5, where the mutant cyclase proteinswere detected by Western blot analysis using an antibodyagainst Sa. cerevisiae cyclase. The T50-3A strain havingYRp7-ADCJ-CYRl overproduced the complete cyclase pro-tein of the estimated Mr of 220,000, which coincided with thepredicted value. The cyclase proteins detected in the cellshaving the mutant plasmids were found to be proportionallysmaller depending on the extent of the deletions.

DISCUSSIONThe gene encoding adenylate cyclase was cloned from thefission yeast Sc. pombe to trace the evolutionary path ofadenylate cyclase from Sa. cerevisiae to Sc. pombe. Incontrast to Sa. cerevisiae cyclase, Sc. pombe cyclase is notlikely to be regulated by RAS proteins as suggested by Fukuiet al. (6) and confirmed in this study. Therefore we think thestructural comparison of the two cyclases might give insightinto a possible switch in regulatory proteins of adenylatecyclase during evolution. To further the understanding ofadenylate cyclase regulation, we have been dissecting the Sa.cerevisiae cyclase protein into separate functional domains.The catalytic domain is mapped to 280 residues near theCOOH terminus and corresponds exactly to the only regionhighly conserved between cyclases of the two yeast species.However, much less homology is seen in the remaining parts.Only a moderate degree of conservation exists in the middlesegments of 1000 residues between Sa. cerevisiae and Sc.pombe adenylate cyclases. About 600 residues of thesesegments mainly consist of repetitions of a 23-amino acid

motif, which has a homology to 24-amino acid repetitivemotifs of human leucine-rich a2 glycoprotein (19), plateletglycoprotein lb (20, 21), Drosophila chaoptin (22), and theToll gene product (23). Previously we suggested that therepetitive sequence in Sa. cerevisiae cyclase might be in-volved in membrane anchoring (7). However, the cyclasemutants that have lost the whole repetitive region are stillrecovered in membrane fraction upon expression in yeast(our unpublished data). Therefore, the function of the repet-itive sequence is still unknown.We have been trying to map a domain of Sa. cerevisiae

adenylate cyclase that is responsible for interaction with RASproteins. Preliminary mapping showed that a deletion of theNH2-terminal 1317 residues abolished the activation byRAS2 protein (7). At this point we still do not know whetherthe NH2-terminal 620 residues, found to be deleted from Sc.pombe cyclase, are indispensible for interaction with RASproteins. In this paper we found another segment at theCOOH-terminal region of Sa. cerevisiae cyclase that appearsindispensible for interaction with RAS2 protein. Which ofthetwo segments (or both) is directly responsible for interactionwith RAS proteins remains uncertain. In any case the twoproteins, especially the COOH-terminal one, are much lessconserved in Sc. pombe cyclase than the catalytic domain.Therefore, in Sc. pombe adenylate cyclase the loss of con-servation of the regulatory regions might correlate with theloss ofinteraction with RAS proteins. So far nothing is knownabout the regulation of Sc. pombe adenylate cyclase, espe-cially regarding its regulatory G protein.

We are grateful to Mr. Samiy Nasrollah and Mr. MasahiroShirakawa for purification of the RAS2 protein used. The researchwas supported by grants from the National Institutes of Health andthe Lucille P. Markey Charitable Trust.

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