Cell, Vol. 50, 573-584, August 14, 1987, Copyright 0 1997 by Cell Press

Yeast KEXl Gene Encodes a Putative Protease with a Carboxypeptidase B-like Function Involved in Killer Toxin and a-factor Precursor Processing

Aieksandra Dmochowska,’ Daniel Dignard,t Dominique Henning,t David Y. Thomas,+ and Howard Bussey* * Department of Biology McGill University Montreal, Quebec, Canada H3A 1Bl t National Research Council of Canada Biotechnology Research institute Montreal, Quebec, Canada H4P 2R2

Summary

The yeast KfXl gene product has homology to yeast carboxypeptidase Y. A mutant replacing serine at the putative active site of the KEXl protein abolished ac- tivity in vivo. A probable site of processing by the KEXl product is the C-terminus of the a-subunit of killer toxin, where toxin is followed in the precursor by 2 basic residues. Processing involves endoproteoiysis following these basic residues and trimming of their C-terminal by a carboxypeptidase. Consistent with the KEX7 product being this carboxypeptidase is its role in a-factor pheromone production. in wild-type yeast, KEX7 is not essential for a-factor production, as the fi- nal pheromone repeat needs no C-terminal process- ing. However, in a mutant in which a-factor production requires a carboxypeptidase, pheromone production is KEXl-dependent.

introduction

in higher eukaryotes, secreted proteins including pep- tide hormones and neurotransmitters are synthesized as larger precursors and released in mature form by the ac- tion of specific processing proteases (Steiner et al., 1980; Douglass et al., 1984; Lynch and Snyder, 1986). Many of these biologically active peptides are flanked within a precursor molecule by pairs of basic amino acids; en- doproteolytic cleavage after these residues is an initial step in precursor maturation. Further processing often re- quires removal of the basic amino acids by a carboxypep- tidase B-like enzyme. A metalioprotease showing speci- ficity toward C-terminal arginine or lysine residues was purified from bovine adrenal chromaffin granules (Fricker and Snyder, 1983), and recently the gene encoding this protease (named carboxypeptidase E or enkephaiin con- vertase) has been cloned (Fricker et al., 1986).

The analogous proteolytic maturation of precursor mol- ecules also occurs in the yeast Saccharomyces cerevi- siae. The a-factor pheromone and Kl killer toxin are the best characterized examples of secreted proteins derived from such processed precursors. Yeast a-factor is a pep- tide pheromone of 13 amino acids involved in the mating response of haploid cells. it is encoded by two genes: MFal and MFa2 (Kurjan and Herskowitz, 1982; Singh et al., 1983). The a-factor coding sequence occurs repeti-

tively, with either four or two copies within the respective 165 or 120 amino acid precursors encoded by these genes. Biologically active pheromone peptides are sepa- rated in the precursors by LysArgGiuAia(Glu/Asp)AiaGiu- Ala spacers. Liberation of the pheromone requires the activity of three proteoiytic enzymes: an endoprotease recognizing a pair of basic residues, a dipeptidyi amino- peptidase capable of removing N-terminal GiuAia repeats, and a carboxypeptidase responsible for cleaving C-ter- minal basic residues (see Figure 1). The first two enzymes have been identified as the KfX2 and the STE13 gene products, respectively, and both genes have been cloned (Julius et al., 1983, 1984b). A candidate for the carboxy- peptidase has been suggested on the basis of biochemi- cal evidence (Achstetter and Wolf, 1985).

The set of processing proteases required for secretion of killer toxin (a small protein secreted by yeast carrying linear Ml double-stranded RNA) is less weli-character- ized. Mature killer toxin consists of two subunits: a and 8, which are separated within a precursor by a giycosyiated y region (Bostian et al., 1984; Skipper et al., 1984; and see Figure 1). Additionally, the a-subunit is preceded by a 44 amino acid leader, the first 26 residues of which are a sig nai peptide removed during entry into the endopiasmic reticulum (Loiie and Bussey, 1986). In addition to the sig- nal peptidase, at least three other proteoiytic events are necessary to release mature toxin from the precursor: cleavage between the leader remnant and the a-subunit (at the P2 site) to generate the authentic N-terminus of the a-subunit, cleavage between the a- and y-peptides, and cleavage between the y- and f3-subunits (see Figure 1). The last cleavage occurs after LysArg residues of the y-peptide and is probably carried out by the KEX2-coded protease. The fact that killer toxin is not secreted from kex2 mutants harboring the Ml dsRNA (Leibowitz and Wickner, 1976; Bussey et al., 1983) is consistent with this scheme. Enzymes responsible for the first two cleavages have not been identified.

To broaden our knowledge about the processing of precursors of secreted proteins in yeast, we have focused on the role of the KEX7 gene product in killer toxin produc- tion. Mutations in this gene lead to failure to process the protoxin (Wickner and Leibowitz, 1976; Bussey et al., 1983) and, like kex2 mutations, result in a killer-minus phenotype. We expected that the KEX7 gene would en- code a protease involved in releasing the a-subunit from the precursor molecule. An endoproteoiytic, chymotryp- sin-like activity was proposed for this protein based on in- hibitor studies (Bussey et al., 1983) and a cleavage site in the protoxin was postulated (Bostian et al., 1984). To ex- plore this possibility, we decided to clone the KEX7 gene and to characterize the encoded protein. Nucleotide se- quencing revealed a surprising feature of the gene prod- uct, namely extensive homology with carboxypeptidase Y, including identity with residues at the active site. We have also found that kexl- null mutations are pieiotropic, af- fecting maturation of not only the killer toxin, but also the

Cell 574

KEX? dbo.tc endoprote.se KEX 2

I 1 I 1 I I 1

1 1 a III t b

I t SP PZ

I

N-termna, proco,,ng

dlpptl+l omlnopept0da.e 1

SE13 . STE 13

!

t J

13x1 b lkzl

t KEXI? I

I I

4

mDf”re B-,or,n

13x1 l?q El a I n

KEXI “DtUre ~-pheromone KEX,

I C-termmol procerrmg

corbc.xyprplldore B-Iok octl”lll I

(311 lzz a

matYre .-pheromone mot”le e-,0X,”

Figure 1. Scheme of Proteolytic Processing of Killer Toxin and a-Factor Precursors in Yeast

The initial KEX2-encoded endoprotease cuts, which occur after pairs of basic residues (shown as pairs of thin lines), are shown at the top. This step is followed in a-factor with N-terminal maturation by the STE73-encoded diaminopeptidase, which cleaves Glu/Asp Ala residues. C-terminal matu- ration is shown at the bottom, mediated by the KEX&encoded carboxypeptidase, which removes basic residues from the internal a-pheromone precursors and from the a-toxin subunit of killer. Note that the terminal repeat of a-factor (shown on right) does not require this KfX&dependent maturation. Also indicated for the killer precursor are the signal peptidase site (SP) and a P2 site at the N-terminus of the a-subunit, following ProArg 44.

active a-factor mating pheromone. We offer here experi- mental support for the proposal that the KEX7 gene prod- uct acts as a carboxypeptidase S-like processing protease in the maturation of these precursors in yeast.

Results

Isolation of the KEXl Gene A kexl mutant, carrying Ml dsRNA but phenotypically killer-minus (K-) (strain DGYlGa, Table l), was trans- formed with a yeast genomic library based on vector YCp50. Transformants were screened for the secretion of active killer toxin as described in Experimental Proce- dures. Three positive colonies were found among 35,000 tested. All 3 killer-plus (K+) transformants carried an iden- tical plasmid with a 10.3 kb insert capable of complement- ing the kexl mutation. The restriction map of the insert in plasmid pAD17 is presented in Figure 2. Southern blot analysis confirmed the presence of the cloned fragment in yeast genomic DNA (data not shown). Deletion analysis and subcloning indicated that a 3.1 kb Hindlll fragment was sufficient to complement the kexl mutation (see Fig- ure 2). This fragment encoded a 2.3 kb mRNA transcript, as detected by Northern blot hybridization (data not shown).

To prove that the cloned DNA contained the KEX7 locus, two types of experiment were performed. First, the pAD17 insert was hybridized to a nitrocellulose blot of yeast chro- mosomes separated by agarose gel, pulse-field elec- trophoresis (Carla and Olson, 1985). A strong positive sig-

nal was obtained for chromosome VII. This result is consistent with genetic mapping of the kexl locus to the left arm of chromosome VII (Wickner and Leibowitz, 1976). Weaker hybridization to other chromosomes was also ob- served, due to the presence of repetitive elements within the insert (unpublished results). Second, the original loca- tion of the cloned fragment within the yeast genome was determined by genetic analysis. The complementing DNA fragment, disrupted by insertion of the LEUP marker (see Figure 2, disruption 1, and Experimental Procedures), was introduced into the genome of the wild-type strain (DGY7a) using the one step gene replacement method (Rothstein, 1963). The resulting K- mutant (DGY7a-4) was crossed with a series of kexl mutant strains (421,411,519, see Table 1) to examine its complementation group. In all cases the diploids had a K- phenotype. Analysis of 17 tetrads derived from the cross with strain 421 showed a 4:0 segregation for the kex-:kex+ phenotype, demonstrat- ing close linkage of the disrupted locus and the kexl allele. These results taken together indicated that integration of the cloned fragment into the yeast genome occurred at the KEX7 locus and that we have cloned the KEX7 gene.

Null Mutations in the KEX7 Locus Do Not Have a Lethal Effect The cloned KfX7 gene was disrupted by replacing a 1.5 kb fragment of the coding sequence with a DNA fragment carrying a LEU2 or UftA3 marker (see Experimental Procedures and Figure 2). Introduction of the disrupted copy into the yeast genome was initially found to be

Protein Processing by Yeast KfX7 Protease 575

Table 1. Yeast Strains

Strain Genotype Source

DGY16a DGY7a DGY7a-4

Sc25k Sc25d

Sc25k-13

5026 Sc25k-13-I 5

421 411 519 1315

11D 4A ADY411

ADY41 l-9-5 S6 XMSL-12b M171-lc

MATa his3A200 ura352 ade2-70 kexl-7 [KIL-kl] MATa his Ural leu2-3, 2-772 trp7 [KIL-kl] MATa his ural leu2-3, 2-7 72 trpl kex7::LEUP [KILkl] MATa adel /eu2-3, 2-172, ura3-52 [KIL-kl] MA Ta/MA Ta ade l/adel leu2-3, 2- 7 72/leu2-3, 2-7 72 ura3-52/ura3-52 [KIL-kl] MATa adel /eu2-3, 2-l 12 ura3-52 kex7::LEUP [KIL-kl] MATa met73 leu2 trp5 cyh2 aro2 lys5 ade5 MATa lys5 aro2 met73 ura3 kexl::LEU2 [KIL-kl] MATa lys2 erg7 kexl-4 MATa argl thrl kexl-6 MATa leu2 lys7 kexl-8 MATaIMATa uraWura3 kexl::LEU/kexl::LEU2 adel/ADEl leu2-3, 2-772/LEU2 am2/AR02 met73/MET73 [KIL-kl] MATa ura3 leu2 his3 [KIL-ko] MATa ura3 trpl leu2 [KIL-ko] MATaIMATa ura3/ura3 leGYleu2 WplfTRPl his3/H/S3 [KIL-ko] MATa ura3 leu2 his3 kexlWRA3 [KIL-ko] w-r MATa i/v 9 arg9 oral sstl [KIL-kl] MATa wad his3 leu2 sst2

Diploid of Sc25K

Spore from cross of 5026 x Sc25k-13

Reed Wickner R. Wickner R. Wickner Diploid of Sc25k-13-15 x Sc25K-13

Diploid of 11D x 4A

Spore from ADY411 following kexl disruption D. Rogers (Lolle et al., 1964) M. Whiteway M. Whiteway

troublesome because of the presence of an ARS se- quence immediately adjacent to the 3’ end of the KEX7 gene.

The disruption of the KEX7 locus was first obtained in a haploid strain in which direct detection of the killer phenotype was possible. The Hindlll fragment, carrying

PAD17 SCALE

H H BanHI H H SamHI H I I I w

\

Wild Type

Disruption 1

Disruption 2

\ \ SCALP

Barn"1 XhoI ClaI HlndIII I I I

I T&A

HmdIII SalI

ATG

LEUZ *amfi* XhoI HindIII

I I I I TAA

Hind111 Sal1 LEU2 XhoI HindIII

Disruption 3

Figure 2. Structure of Chromosomal DNA in the KEX7 Region from Wild-Type and kexl- Disrupted Strains

The 10.3 kb yeast DNA fragment contained in pAD17 is shown at the top, with the functional 3.1 kb and Hindlll KEX7 fragment indicated by the heavy line. The position of the KfX7 open reading frame in this fragment is indicated on the second line. The structure of the two kex7::LEUZ disruptions and the kexl:tURA3 disruption and their relationship to the wild-type open reading frame are shown below and further discussed in Experimental Procedures. Abbreviations are as follows: H, Hindlll; N, Nhel; S, Sall.

Cell 576

AGGAT;ATTTTGTTACGTGAGGMTTGCG*GCTTT

TTGGCT~TMCTTTCMGTTTTGTCACCMCATTTATATTTG GTGGMTATTTTGATMTATAGG-CATTA

A~GCGATTTCTTATCATTGTCTTCM;TCCCAT~TA~TT GAAAMGCTTGCTGMGCMACTTTGGTTAMGAGTACCTTGGCTAT~G~TACCGT~GAGAT-G~CCTGMTAG _- -I __

ATG TTT TAC i,,T AGG TGG CiC GGA KG TGh CTA CCC ATG iCT GCT TTA Ah AGG .&TC TCi GTA TCC CTT tCG TCA TCT GiG GAG TX WA Met Phe Tyr Asn Arg Tr&, I,=” Gly Thr Trp LB” Ala “et Ser Ala Le” Ile Arg Ile Ser “al Ser 1.e” Pro Ser Ser Gl” Gl” Tyr Ly,

GTC GCA TAT ciG CTG TTG &A GGG TTA TCi GM GTG CCA & CCT TCA AiT ATT CCA Ck ATG CAT GCT &C CAT ATT CiT TTA CGT TCC "al Ala Tyr Gl" I,=" I,=" Pro Gly Le" 9=r Glu "al Pro Asp Pro S=r AS,, Ile Pro Gln ,,=t Hi, Ala Gly His IL= Pro Leu Arg Ser

GM cxf GcG GAT GM C*G G*T AGC TCT GAC TTG GIG TAC TTT TTT TGG .& TTT rcc MT MC GAC TCT MT GGC MT GTC GA.C CGT ccc Gl" A.)p Ala Asp Gl" Gin Asp S=r Ser Asp La" Cl" Tyr Phe Phe Trp Lys Phe Thr Asn As,, Asp S=r Asn Gly As,, "al asp Arg Pro

TTA ATT ATA +GG TTA MT GiT CGA CCC GGi TGC TCT TCC iTG GAT GGT G;C TTG GTC GAi TCC GGC CCT ;TT AGG GTG MT TCA G.,C GGT IX" Ile Ile Trp IS" AS,, Gly Gly Pro Gly Cys Ser Ser "et Asp Gly Ala Le" "al ClU Se= Gly Pro Phe Aq "al As,, S=r Asp Gly

-240

-120

1 1

91 31

191 61

271 91

361 121

151 151

511 181

631 ATT TTG MC &T MC AM T-iT TCA MG AT; GAC GGG GAT iCA TAC GAC TiG MG GCG CTi TTG ATT GGT iAC GGT TGG ATT GAC CCC MT 211 Ile LB" As,, His Asn Lys Phe S=r Lys Ile A,p Gly Asp Thr Tyr A=p Leu Lys Ala L=u I.=" Ila Gly Asn Gly Trp Ile Asp Pm Asn

721 241

611 271

901 301

991 331

1081 ATT G.&T CAT kG ,.AG GM T& ACT MT AGC GTT GGA ACT i&A TTG TCT FiT CCT ATT TCA MG CCA TCT iTC CAT TTA T& CCT GGT CTA 361 Ila Asp His Trp Lys Glu Cyl Thr Asn .9=r "a1 Gly Thr Lyr La=" .S=r Asn Pro Ile S=I Lyr Pro Ser Ile His Le" Ix" Pro Gly Leu

1171 391

1261 AM TGG GGT k,, ATA MG Gci TTT AGC GA;. GAT GCT GTT iCG TTC GAT TiG ATC CAT AAA TCG MG AGT iCA GAC GAC A& GM GM TTT 421 Ly?l Trp Gly Cly Ile Lyr Cly Phe S=r ASP Asp Ala "al Ser Phe Asp Trp Ile Hi. LyS .S=r Lys .?.=I Thr Asp Asp S=r Glu Gl" Phe

1351 451

1411 481

1531 CM GAT GCT ACT ACT GM A& GGT WT W.6 CCA AAA GM iAC CTC GM GiG GAA GM CAi GM GCG CAG ihT GAG GM G&, MG GM AAA 511 Gin Asp Ala Thr Thr Glu Ser Gly &.p Lys Pro Lys Glu Asn LB" Glu Glu Cl" G1" Cln Gl" Ala Gin Asn G1u Glu Gly Lys Glu my,

1621 GM GGC MT i?.A GAT AM GiT GGC GAT GA+ GAT MC GAC iAT GAT GAC GiC GAT GAA GA; GAT CM MC &C GAG GGC GiC 'SAC GAT GAT 541 Glu Gly Asn Lys ASP Lyr A=p Gly A=p Asp Asp Asn Asp A?m ASP Asp Asp A=p Glu I\=p A,p Hi. Asn Ser Gl" Gly ASP Asp Asp As‘,

1711 571

lSO1 GM GAG a GAG GTA GM GM TTT GCA GAA GAG ATT TCA ~TG TAT .4m tic m GCG GTA GTA GTT xc ATC GTT AC* TTT TTG ATA GTG 601 Glu Gl" Gl" G1" "al Gl" Cl" Pho Ala Gl" Glu Ila Ser Met Tyr Lys His Lys Ala "al "al "al Thr Ile "al Thr Phe I.=" 11.x "al

1891 GTT TTA GGA iTT TAT GCG TAT GAT AGA AGi GTG AGG AGA .iAA GCG CGT CiC ACA ATT TTi GTT WT CCA iAT AAT AG., CiA CAT WC AGT 631 "al Lau Gly "al Tyr Ala Tyr A.p Arq Arg "al Arg Arg Lys *la Arg His Thr 11s L=u "al Asp Pro Asn Asn Arg Cl" His Asp Ser

1981 661

2071 GC* ccc ATA AGT AGC ACT TCT MC - GCA GGA TCT AM TTG IrM ACT MG - MG AAC MG TAT ACT AGC CTT CCG MC ACT GM ATC 691 All Pro Ile Ser S=r Thr S=T AS,, Lys Ala Gly S=r Lys Leu Lys Thr Lys Lys Lys Lys Lys Tyr Thr Ser LB" Pro ..sn Thr Glu Ile

2161 721

2271

2391

2511

AM. CTT TAT &A MT GM GiG TCC TGG ATi TCC Au GGC ;;AT CTT TTA T’iT ATC GM CAi CCT ACT GGT ACT GGG TTT TkT GTC GM CM Lys Le" Tyr Le" Am Gl" Gly S=T Trp Ile S=r Lys Gly ASP L=u La" Phe Il= ASP Gin Pro Thr Gly Thr Gly Phe S=r "al Glu Gin

MT AM GAG GM GGT AM a& GAC AM AAC - TTT GAC GM GAC CTA GM GAT GTG KC MG CAT TTT ATG GAT TTT CTG WG MC TAT Am, Lys A?rp Gl" Gly Lys Ile Asp Lys Alln Lyr Phe Asp Gl" Asp LB" Glu Asp "al Thr Lys His Phe "at Asp Phe Leu Gl" Aa, Tyr

TT~ MG ATT TTT cc+. GM G~C CTC ACT AGG u.4 ATC ATA C~TA TCG GGT GM AGT T*C GCT GGC CM T*C ATA CC* TTC TTT GCC MT GCA Phc Ly, Ile Phe Pro Cl" Asp I,=" Thr Arg I.ys Ila Ile Leu Ser Gly G1" S=r Tyr Ala Gly Gin Tyr IL= PLO Phe Phe Ala Asn Ala

ACA CAG TCC ;TA TCG TAC CiT CCG TTT GCi RTG GAG MG iA.% CTG ATT GiT GM AGC MC CCC MT TTC iAA CAC TTA A&G MC GCA CAC Thr Gln S=r Ia" S=r Tyr LB" Pro Phe Ala Met Glu Lys Lys Ia=" Ile Asp Gl" S=r Aon Pro Am, Phe Lya His I.=" Thr Am Ala Hi.

GAG MT TGC iAG MT CTG Ai, MC TCT GCk AGT ACA GAT &,G GCA GCC CAT TTT TCG TAT CAG GM TGT GAG MT ATT TiA MC CTT CTA Gl" Asn Cys Cl,, As,, leu Ile Asn S=r Aila .9=r Thr Asp Glu Ala Ala His Phe S=r Tyr Gin Gl" Cys Gl" Asn Ila LB" Asn I,=,, Leu

TTG TCT TAT iCC AGG GM T;T TCG CM Mb GGA AICA GCG &'tT TGC TTG AiC ATG TAT &AC TTC MT TTA iAA GAT AGT TAT CCA TCT TGT Mu Sar Tyr Thr Mg G1" S.=r Sar Gin Lys Cly Thr Ala Asp Cy8 Leu Am, "et Tyr Asn Phe Asn Le" Lyr ABE S=r Tyr Pro Ser Cys

GGT ATG PAT iGG CCG AM CAT .,TT TCC TTT GTC AGT AM TTC TIT AGC AC.8 CCT GGT GTT ATT GAT TCG TTG CAT CTT GAT TCT GAT AM Gly Mat Asn Trp Pro I,y, Aq, Ile Ser Pha "al Ser Lys Phe Ph= S=r Thr Pro Gly "11 Ile Asp Ser IA" His LB" Alp S=r Aq, Ly,

CTT GM AGT &A ATA GAG AiT GTC TTG TTi MT GGT GAC . MC TTG AiT TGT MT MC MG GGC GTA iTA GAT ACT Ai, GAC MT CTA Le" Glu S=r Gly Ile Gl" Ile "al I,=" Ph= Asn Gly Asp Alp Lell Ile Cys A=n Am, Lys Gly "al LB" Asp Thr Ile Asp Am, Ia"

AGC GGA TAC tiTG AM TAT GiT AGA MT TTi ACG TTT GTT iGC GTT TAT ,iT GCT TCT CAh ATG GTA CCC iTC GAT AM AiT TTA GTG AGT ST Gly Tyr "al Ly= Tyr Asp Arq Aan Leu ThZ Pha "al S=r "al Tyr Am, Ala S=r His ,,=t "11 Pro Phe Asp Lys Ii== L=u "a1 S=r

AGA GGC ATT ;TC GAT ATT TiC TCG MC GA; GTT ATG ATC iTT GAC MC MT GGG AM MT GTT ATG ATT iCT ACT GAC GiC GkT AGT GAT Arr~ Gly Ile "al Asp Ile Tyr Ser Asn A&q, "al "et Ila II= Asp Asn A=n Gly LyS ..=I, "al "ct Ila Thr Thr Asp ASP As,p 9.x As,p

MC GAT MC &tT CAT CM GiC GAT MT AA+ GM A&A CM ACT MT CM G& CTC GAG GA; AGT AG.4 CAT .& TCT TCA Gih TAT GA.4 CM ,..rp ASP Asp A,&, Asp Glu Asp As,p AS,, Asn Gl" Lys Gin S=r AS,, Gin Gly L=" Glu Asp S=r Arg His Lys S=r S=r Gl" Tyr Glu Gl"

CCC MT MG ACT, GTT TCA TiG GCA GAT GA; CTG GM AGC &A CTC GGG G&A GAA GAT GA;. TTA GAA CM &,T GAG CAG T&, GM GGC GGA Pro Asn Lys Thr "al Scr Trp Ala Asp Asp Le" Cl" S=r Gly LB" Gly Ala Gl" asp Asp I,=" Gl" Cln Asp Glu Gln Le" Gl" Gly Gly

GAT CM TCT ;TT GAG ATG AiT GAT TTT TAi CTTGCC-iUTATA+- TACAGAAAAATAGTTATMTACCTtGCACACGCiTTTCGTAGTiACAGCGACA~

Protein Processing by Yeast KEX7 Protease 577

Figure 4. Structure of the KEXI-Encoded Protein

The diagram at the top represents the hydropa- thy profile of the KEXI product (Kyte and Dcolit- he, 1982). The box structure below depicts the main structural features of the protein dis- cussed in the text. The putative N-terminal leader is folknved at amino acids 84-250 and 381-475 by domains with sequence homology to yeast CPV; the conserved serine region at the active site is indicated. An acidic domain spanning amino acids 508-811 is followed by a hydrophobic, potential membrane-spanning domain, residues 819-837. Potentially glyco- sylatable asparagine residues amino-terminal to the membranespanning domain are shown by circled Gs.

the KfX7 locus disrupted by the LEUP marker (see Figure 2, disruption 2), was used for transformation of recipient strain Sc25k. The resulting K- kexl- null mutant, Sc25k- 13, retained the same growth rate and mating capacity as the parental strain Sc25k. Furthermore, the disruption did not affect sporulation efficiency or spore viability as ascer- tained by sporulating a diploid that was homozygous for the disrupted KfX7 locus (strain 1315, see Table 1). The viability of the kexl- haploid cells indicated that the KEX7 gene does not have an essential vital function in yeast grown under the conditions tested. These results were confirmed by the disruption of the KfX7 locus in diploid strain ADY411. The successful experiment was performed using an Nhel-Clal fragment with the KEX7 gene dis- rupted by the URA3 marker (see Figure 2). The transfor- mants sporulated normally and segregated 2 URA+:2 ura- spores. UFlA+ spores were kex7- as judged by trans- formation with a plasmid carrying the killer toxin gene (~1342) and testing for the killer phenotype. Southern blot analysis confirmed that the URA+ kexl- spores contained a disrupted copy of the KEX7 locus (data not shown).

Nucleotide Sequence of the KEX7 Gene The nucleotide sequence of the KEX7 gene was deter- mined using the dideoxy method described by Sanger et al. (1977) and the strategy outlined in Figure 7. DNA sequence analysis revealed one long open reading frame uninterrupted by any splicing consensus sequences (Lang- ford and Gallwitz, 1983) (see Figure 3). Three AUG codons are present in the same reading frame at the beginning of

the putative coding region. lb identify the initiation codon, we determined the 5’ end of the KEX7 mRNA using the primer extension method (see Experimental Procedures). Transcription appeared to initiate at several sites, all up- stream of the first AUG (Figure 3). Five major transcription initiation sites were found at positions -67, -68, -69, -78, and -79, and 5 minor ones at positions -38, -51, -52, -95, and -114 (Figure 3). Such heterogeneity at the 5’end

of mRNAs is known for some genes in yeast (McIntosh and Haynes, 1986). Upstream of the transcription initiation sites are three candidates for a TATA box (sea Figure 3). The se- quenceTAG. . . TAGT. . . TTT, postulated as a transcription termination signal in yeast (Zaret and Sherman, 1982) is found downstream of the coding sequence (nucleotides 2227-2285). A putative polyadenylation site AATAAA (Fitz- gerald and Shenk, 1981) is also present at position 2198.

The KEX7 gene encodes a protein consisting of 729 amino acid8 with a calculated molecular weight of 82,238. The hydropathy profile determined according to Kyte and Doolittle (1982) demonstrates the presence of two hydro- phobic regions (Figure 4). The first, situated at the N-ter- minus, is a reasonable candidate for a signal sequence (Watson, 1984) capable of directing the KEX gene product into the endoplasmic reticulum. Furthermore, the se- quence surrounding the Ser 22 fulfills the rules (von Heijne, 1984) for a signal cleavage site. The second hydra phobic region, situated 100 residues from the C-terminus of the KEX7 protein, contains 18 amino acids and possibly functions as a membrane-spanning domain of the kind seen in many transmembrane proteins (Kyte and Doolittle,

Figure 3. Nucleotide and Derived Amino Acid Sequences of KEXI

The mapped positions of the five minor and five major transcription start points are underlined with 1 or 2 lines, respectively. Coordinates in the left margin indicate nucleotide and amino acid positions used in the paper, with nucleotide 1 corresponding to the first nucleotide of the initiation codon and amino acid 1 assigned to the first residue of the putative leader peptide of the KEX7 product. The black dots mark off blocks of 10 nucleo- tides. Ser 198, which is conserved at the active site of CPY is shown by an asterisk.

Cell 578

Figure 5. Comparison of Amino Acid Sequences of the KEXl Product and Yeast Carboxypeptidase Y

The amino acid sequence of KEX7 is shown on the upper fine starting at residue 64 of the sequence shown in Figure 3. Directly below it is shown the sequence of mature CPY, starting at residue 22. The sequences have been aligned to display regions of identity, shown by the boxes; Ser 146, containing active site region of CPY, is shown by a heavier box. Note the discontinuity following KEX7 residue 250 where a second region of homology starting at KEX7 residue 361 and CPY residue 314 is shown on the last 3 lines. The CPY protein sequence used in this comparison is that predicted from the CPY DNA sequence (Valls et al., 1967).

1982). Four potential glycosylation sites (AsnXSerTThr) are found throughout the amino acid sequence. Three are situ- ated on the N-terminal side of the putative membrane- spanning domain. Interestingly, also N-terminal to this hy- drophobic region is a tract of 105 residues very rich in aspartic and glutamic acids. In some places these nega- tively charged amino acids form stretches comprising up to 11 consecutive residues.

Homology of the Protein Encoded by the KEX7 Gene to Carboxypeptidase Y The amino acid sequence of the KEX7 gene product was compared with known protein sequences using the NBRF protein sequence database. Significant homology was found with the carboxypeptidase Y (CPY)-a well-char- acterized yeast vacuolar serine protease (Hayashi et al., 1975; Martin et al., 1982; Svendsen et al., 1982; see Bred- dam, 1986 for a review). The region of homology com- prises two tracts. The first, of 196 residues, is situated in the N-terminal part of both proteins. It begins at residue 64 of the KEX7 protein and at residue 22 of mature CPY, or residue 134 of the preproCPY sequence (Valls et al., 1987) (see Figure 5). There is then a region of 131 residues in KEX7 with no homology and a second homology tract commencing at residue 381 of KEX7 and residue 314 of mature CPY that extends for 95 amino acids. The overall identity of both tracts (comprising 291 residues) is 390/o, but within two sequence blocks the similarity is over 50%. One conserved region in the KEX7 protein contains the se- quence GlyGluSerTyrAlaGly 201, which is identical in the active site of CPY and includes the active serine residue (Shaw and Wells, 1972). To establish whether this serine residue was essential for activity of the KEX7 gene prod-

uct, we generated a point mutation that changed serine to alanine by oligonucleotide site-directed mutagenesis (see Experimental Procedures). The mutated KEX7 gene, car- ried on plasmid pAD26, was introduced into a kexl- mu- tant, and transformants were tested for the killer pheno- type. No killer toxin secretion was detected. This result suggests that the KfX7 gene product functionally con- serves the active serine of CPY and strongly implies that the KEX7 protein is a serine protease.

Possible Sites of Action of the KEX7 Protease in the Killer Toxin Precursor Two regions of the killer protoxin were considered as can- didates for KEX7 protease cleavage sites: the P2 site, i.e., after the ProArg 44 residues of the leader; and the junc- tion between the a- and y-polypeptides (see Figure 1). To check whether cleavage at the P2 site depends on the KEX7 gene product, a kexl- mutant (ADY411-9-5) was transformed with a plasmid in which the 44 amino acids of the preprotoxin leader were replaced by the 17 residue signal sequence of yeast acid phosphatase. This plasmid (pL360-2) transforms wild-type nonkiller yeast (K-) to the killer phenotype (K+). In cells carrying pL360-2, signal se- quence cleavage is sufficient to generate the authentic N-terminus of the a-subunit of killer toxin. We reasoned that if the KEX7 protease acted only at the P2 site, yeast har- boring this construct would no longer require KEX7 for killer toxin maturation. The kexl- transformants, however. maintained a K- phenotype, demonstrating that the KEX7 gene is still required and that the P2 site of the killer pro- toxin (if used at all) is not the only KEX7 cleavage site.

The exact endoproteolytic cleavage site of the protoxin precursor at the junction of the a- and r-peptides was not

Protein Processing by Yeast KEX7 Protease 579

Figure 6. Effects of KEX7 on a-factor Activity

Yeast strains or extracts were spotted onto plates seeded with ssf2, a mutant strain supersensitive to a-factor. The halos are caused by inhi- bition of growth of the sst2 cells by the a-pheromone. See Experimen- tal Procedures for further assay details. (A) u-Factor expression in haploid strains. (1) Mafa KEX7 strain Sc25k. (2) Msta kex7::LfU2 disruptant Sc25k-13. (3) Mata kexl::LEU2 Sc25k- 13 transformed with KfX7 plasmid pAD17. (B) Diploid strains expressing a-factor from plasmids containing either a wild-type Ma7 gene, pJK6, or a mutation in the Ma7 gene defective in the last repeat of the coded a-pheromone, pJK6-4. These plasmids express a-factor from a GAL7 promoter. The diploid strains harboring them were grown in YEP medium with 2% galactose, and suspensions were spotted to the ssf2seeded plates in the same medium. (1) KEX7 strain Sc25d with pJK6. (2) homozygous &axl:tLEU2 disruptant strain 1315 with pJK6. (3)KEXl strain Sc25d with pJK6-4. (4)/rexl::LEU2 strain 1315 with pJK6-4. In vitro activation of a-factor precursors by carboxypeptidase B. (C) Synthetic aF-KR. (Top) Synthetic aFKR (0.4 pg). (Bottom) Same as top, but incubated with carboxypeptidase B (see Experimental Proce- dures). (D) Extract from kex7::LEUP diploid strain 1315 with pJK64 (the equivalent of 1 ml of cell culture medium; see Experimental Proce- dures for details). (Top) No treatment. (Bottom) Same as top, but in- cubated with carboxypeptidase B. (E) Extract from KfX7 diploid Sc25d with pJK6 (amount as for [D], but diluted l/100). Fop) No treatment. (Bottom) Incubated with carboxypeptidase B.

known. Based on the migration rate of the a-subunit on SDS-PAGE and the inhibition of killer toxin processing by the chymotrypsin inhibitor TPCK, Trp 130 was proposed as a possible cleavage site (Bostian et al., 1984). The Asn- ArgPro 133 sequence was also a plausible cleavage site

found in some hormone precursors (Schwartz, 1988). To precisely locate the a/~ junction, the amino acid sequence at the C-terminus of the a-subunit of the killer toxin was determined (Zhu et al., 1987). The a-polypeptide was found to extend beyond Trp 130 to a C-terminal residue at Ala 147. This residue is followed in the precursor by Arg- Arg 149, a candidate for a dibasic cleavage site recog- nized by the KEX2-encoded protease. These findings sug- gested the interesting possibility that the KEXl protease has a conserved carboxypeptidase function and removes basic residues from the C-terminus of the a-subunit fol- lowing a KEX2 endoprotease cut after ArgArg 149.

&8x7 Null Mutations Affect a-Factor Processing The hypothesis that the KEXl gene product has a carboxy- peptidase B-like activity raises the possibility that it func- tions in the maturation of the mating pheromone a-factor. The KEX2 endoproteolytic cleavage of the a-factor precur- sor occurs after LysArg residues and releases a prema- ture form of the pheromone (see Figure 1). The conversion of this propheromone to the mature a-factor requires sub- sequent action of dipeptidyl aminopeptidase A and, in the case of the internal pheromone repeats only, a further car- boxypeptidase B-like activity to remove the C-terminal dibasic residues. Therefore, if the KEXl gene product contains a carboxypeptidase B-like activity required for a-factor maturation, and if the presence of 2 basic residues at its C-terminus essentially removes the biologi- cal activity of the pheromone, then a mutation within the KEXl locus should lead to reduction in the level of mature a-factor secreted by MATa yeast. The expected level of the reduction would be about 75%, because the majority of the pheromone secreted from yeast cells is encoded by the MFal gene (Brake et al., 1983; Kurjan, 1985). This gene has 4 repeated units of the pheromone coding re- gion, with the 3 internal repeats requiring C-terminal maturation (see Figure 1).

We synthesized the a-factor peptide with the LysArg residues at the C-terminus, which we have called aF-KR (see Experimental Procedures). This aF-KR peptide had essentially no biological activity on MATa cells (see Figure 6C), but could be converted to a biologically active form by in vitro treatment with carboxypeptidase B (see Figure 8C). We then tested pheromone production in vivo, com- paring the l3vel of mature a-factor secreted by a wild-type strain (Sc25k) and its KEXl disrupted derivative (Sc25k-13, disruption 2 in Figure 2). The secretion of active phero- mone was monitored by the presence of zones of growth inhibition surrounding cells spotted on a plate seeded with a strain supersensitive to a-factor (sstl or ssf2 mu- tants) (Chan and Otte, 1982). As can be seen in Figure 6A, the kexl- mutant generates a zone slightly smaller than the wild-type strain and this zone can be restored to wild- type size if the kexl- mutant strain carries the plasmid pAD17 containing the KEXl gene.

In an additional test, we examined the effect of the kexl- mutation in a strain in which the region coding for the last pheromone repeat in the MFal gene was mutated

Cdl 580

to render it functionally inactive. To do this, we introduced a deletion into the plasmid pJK6, carrying the Mfal gene. The deletion resulted in replacement of the 5 C-terminal amino acids of the last a-factor repeat (GlyGlnProMetTyr) by 10 amino acids of the tetracycline resistance gene from the pBR322 part of the vector (ArgArgGluAlaGlyTrp- ProSerProLeu) (see Experimental Procedures). This mu- tation eliminated the authentic C-terminus of the last pheromone unit and resulted in a peptide that was unlikely to have a-factor activity. The liberation of mature active a-pheromone from such a mutated precursor should be strictly dependent on a carboxypeptidase B-like activity. The mutated plasmid (pJK6-4) was introduced into diploid kexl- and KEX7 strains (1315 and Sc25d, respectively). In the diploid cell type the expression of chromosomal copies of the MFal and MFa2 genes is repressed (Sprague et al., 1983). Transcription of the MFa7 gene in plasmid pJK6-4 occurs under the control of the GAL7 promoter, which replaces the intrinsic Mfal promoter in this construct. The zone assay revealed that active a-factor was secreted from a galactose-grown wild-type diploid, carrying plas- mid pJK6-4, but not from the homozygous kexl- mutant (Figure 6B). This result is consistent with and strongly supports the hypothesis that the KEX7 protein is involved in the carboxypeptidase B-like step of a-factor precursor maturation.

If the kexl- null mutant is defective in a carboxypepti- dase B-like activity, it will produce and could secrete aF- KR, the inactive propheromone with C-terminal LysArg residues. If this is the case, then the dibasic residues should be removed in vitro by carboxypeptidase B treat- ment, generating biologically active pheromone, as was found with the synthetic aF-KR. We checked for possible activation by carboxypeptidase B of material secreted by the kexl- mutant. The medium from the culture of the kexl- strain 1315, carrying plasmid pJK6-4, was passed through a Bio-Rex 70 column as described in Experimen- tal Procedures. The resultant column eluate had 100 times less pheromone activity than the equivalent eluate from a wild-type strain, as determined by a zone test on the a-factor supersensitive mutant sst2. Both extracts were treated with carboxypeptidase B and spotted onto a seeded plate of strain sst2. Significant enhancement in activity was observed for the extract obtained from the kexl- mu- tant, but no effect was seen in the case of the wild-type strain (Figures 6D and 6E).

Discussion

The identification of proteases involved in vivo in precur- sor processing in multicellular eukaryotes remains an elu- sive goal. In the single celled eukaryote yeast, the combi- nation of genetics and molecular biology has enabled several proteases to be unequivocally associated with specific processing events (Julius et al., 1983, 1984b; Am- merer et al., 1986; Woolford et al., 1986). These process- ing events are of general interest as they are common to the processing of precursors in all eukaryotic cells. To ex- ploit the yeast system further, we studied the KEX7 gene,

which is known through mutants to be involved in the pro- cessing of the killer toxin precursor.

Homology with CPY Cloning and sequencing of the KEXl gene revealed a do- main in the protein with significant homology to the yeast vacuolar serine protease CPY. The homologous region conserved the CPY Ser 146, which is essential for the pep- tide bond hydrolysis reaction (Breddam, 1986) providing a case for the KEXl product being a protease. To extend the analysis beyond homology to the functional level, we made a site-specific mutation replacing the presumed ac- tive site serine with alanine in the KEX7 product. Replace- ment with an alanine residue should completely remove the activity of the protein if it functions in this way. When introduced into yeast, this Ser 198 to Ala 198 mutation failed to complement the disruption phenotype.

Yeast CPY is a serine carboxypeptidase, a protease with homology to some plant carboxypeptidases (Breddam, 1986) but it is at best only distantly related to the exten- sively characterized classic serine endoproteases like tryp- sin and chymotrypsin. Consequently, far less is known about the structure of CPY, and we are unable to predict from the homology whether the KEX7 protease is a car- boxypeptidase; although we present below some indirect evidence that this is the case. It is of interest that CPY is inhibited by TPCK (Hayashi et al., 1975) although the essential histidine residue involved has not been identi- fied. His 214 and His 470 in KEX7 are conserved in CPY, and the observed inhibition by TPCK of in vivo processing and secretion of killer toxin may well be caused by inhibi- tion of the KEX7 product.

Other Domains of the KEXl Product Studies with set mutants suggest that the KEX7 product acts on the killer precursor at the Golgi stage in the yeast secretory pathway. Conditional set mutants, which block at the endoplasmic reticulum at the restrictive tempera- ture, do not process the killer precursor, while sec74 mu- tants, which block secreted proteins in the Golgi, do pro- cess the killer precursor (Bussey et al., 1983). Similar results have been found with the KEX2-encoded en- doprotease on the a-factor precursor (Julius et al., 1984a). There is a potential amino-terminal signal sequence for directing the KEX7-encoded protein to the yeast endoplas- mic reticulum, a series of potential Asn-linked glycosyla- tion sites, and a potential membrane-spanning region to- ward the C-terminus for possible anchoring of the protein to a membrane location. In fact, the overall organization of the KEX7 protein structure bears a considerable resem- blance to the KEX2 protein product, which, as well as the above, has a polybasic tract following the membrane- spanning region (Fuller et al., 1986). In addition, in the KEX7 product there is a polyacidic tract. Such polyacidic regions are found in other proteins (e.g., in the RAD6 prod- uct of yeast, Reynolds et al., 1985) but are of uncertain function. A speculation is that in the KEX7 product, the polyacidic region might be involved in activation of the protein as it encounters increased acidity in the Golgi

Protein Processing by Yeast KEXI Protease 581

compartments (Kelly, 1987). Such activation could be structural, for example, a change to an a-helical form of the polyacidic domain in acidic conditions, or might in- volve release of bound metal ions necessary for protease function.

Function in Killer and a-Pheromone Precursor Processing Having demonstrated that the KfX7 product is likely a pro- tease, we wished to determine its site of action in the killer protoxin and to deduce its cleavage specificity. As out- lined above (Figure l), possible cleavage regions included the P2 site at the leader/a junction and a site at the a/r junction. Deletion of the P2 site, using an acid phospha- tase signal sequence construction, did not abolish the re- quirement for a functional KEX7 product, suggesting that it was not the cleavage site involved. To find the actual pro- cessing site at the a/r junction required determination of the C-terminus of the a-polypeptide (Zhu et al., 1987). The salient point from this work was that the C-terminus of the a-polypeptide is at Ala 147 of the protoxin, which is fol- lowed by 2 basic residues in the precursor sequence. This structure suggests a processing pathway widespread among eukaryotic precursor proteins, namely an endo- protease cleavage after the dibasic residues followed by a trimming back of the dibasic residues by a carboxypep- tidase B-like activity to generate the mature C-terminus of the polypeptide. The endoprotease responsible is prob- ably the KfX2 product, known to be involved in killer pro- toxin processing, and the KfX7 product is a candidate for the carboxypeptidase B-like activity.

If the KfX7 product functions in this way, it may act in an equivalent way in the processing of prepro-a-factor. Previous work had shown that while kex2 mutants failed to secrete processed a-pheromone and were thus sterile as a-cells, kexl a-cells were able to mate (Leibowitz and Wickner, 1976). Examination of the structure of the pre- pro-a-factor gene indicates, however, that while KEX2 is necessary for a-pheromone production, there is only a partial requirement for a carboxypeptidase B-like activity. This is because only the internal a-factor repeats require a carboxypeptidase for mature processing (see Figure 1). We tested for this partial phenotype in kexl- disrupted a-cells and found that they did indeed make less active a-pheromone. An assumption here was that the aF-KR propheromone was less active than the mature phero- mone. This was tested by synthesis of the peptide, and it was found that the aF-KR was approximately 200-fold lower in specific activity than synthetic a-pheromone. Parenthetically, it should be mentioned that the prohor- mone a-factor*, containing an unprocessed N-terminus, which accumulates in sre73 mutants (Julius et al., 1983), also has a lower specific activity than the mature phero- mone. Using the above information, a more sensitive test of KEX7 function in a-pheromone processing was de- vised. We mutated the final pheromone-coding repeat of the MFal gene to give a nonfunctional a-pheromone. Pheromone production from such a construct became es- sentially dependent upon a KEX7 gene product. In an ad-

ditional test we showed in vitro activation by carboxypepti- dase B of material secreted by a kexl- mutant, but no such activation of material from a KEX strain. This result indicates that some pheromone precursor was specifically present in the /@xl- disruption strain that was activat- able by carboxypeptidase B and strongly suggests that this was the aFKR propheromone. The more stringent test of isolation and characterization of this aF-KR peptide specifically from kexl- disrupted strains remains to be made.

Our results demonstrate a role for the KfX7 product in the processing or action of both the a-pheromone and the killer toxin precursors that is consistent with it being a car- boxypeptidase B-like protease, although we stress that we have not shown this directly. Thus the formal possibility re- mains that the KfX7 product, itself a serine protease and likely a carboxypeptidase, activates some other protease that then acts directly as a carboxypeptidase on the precursors. Another possibility that we cannot dismiss is that a-factor and killer processing require additional pro- teases for unidentified reactions on the precursor se- quences and that the KfX7 product fills such a role. In the case of the a-pheromone precursor, this possibility seems particularly unlikely, as the only obvious remaining uniden- tified protease function is the carboxypeptidase B-like activity. We favor the simplest explanation of our data, which is that the KEX7 product is a carboxypeptidase B-like enzyme acting directly on the killer and a-factor precursors following the KfX2cleavage event, and we are currently attempting to purify the KfX7 product to test the specificity of its proteolytic action. A membrane- associated carboxypeptidase B-like activity, termed ysc a, (Achstetter and Wolff, 1985) that is a candidate for the KfX7 product has been identified. The identification of the KfX7 activity, if confirmed, completes the earlier classic work on functioning of the KfX2 and STEM gene products (Julius et al., 1983, 1984b) and offers the first instance in which the complete protease-processing pathway has been delineated for a eukaryotic precursor. With regard to processing of the killer precursor, our knowledge is less complete, and characterization of the signal peptidase and the protease responsible for the cleavage following ProArg 44 remains to be accomplished. The present study indicates the functional order KEX2, KEX7 in the process- ing pathway. The reverse order was suggested earlier on the basis of toxin precursor stabilities (Bussey et al., 1983). The reason for the relative stability of the precursor in kexl mutants compared with kex2 mutants is unknown, although it is consistent with the observation that TPCK (which probably inhibits the KfX7 product) also stabilizes the toxin precursor and prevents toxin secretion (Bussey et al., 1983). Possibilities include interaction of the KEX products or selective degradation of the toxin precursor. Relative precursor stability is an indirect measure of pro- cessing protease action, especially since the precursor is not stable and there is no significant toxin or precursor material secreted in either of the kex mutants.

The possible role of the KEX7 product on other pro- cessed proteins in yeast is of some interest. The only

Cell 582

known other function is in the secretion of active K2 killer toxin, derived from another dsRNA viral based toxin precursor gene that is not closely related to the Kl killer system (Reed Wickner, personal communication; Hannig and Leibowitz, 1985). Disruption of the KEX7 gene is not lethal in yeast, and normal mitotic growth occurs in haploid and diploid cells, as do the processes of meiosis, sporulation, and spore germination under the conditions we employed. Most carboxypeptidase B-like KEX7 func- tions would depend upon a prior KfX2-dependent cleav- age, and as KfX2 disruptions are also viable (Fuller et al., 1986), our result is perhaps not surprising. We think it probable that the KEX7 protease has other, as yet undis- covered, functions in yeast and would point out that the effect on the yeast a-pheromone precursor is a subtle quan- titative one that would be overlooked without a detailed knowledge of the system. Yet the KEX7 effect that in- creases the amount of pheromone produced approxi- mately 4-fold may have dramatic effects on mating effi- ciency in natural situations.

There is a ubiquitous requirement for a carboxypepti- dase B-like activity in processing of peptides from precur- sors in higher eukaryotes. Is such a function provided by a KEX7 homolog? The only well-described p&ease thought to act as a processing carboxypeptidase is the carboxypeptidase E or enkephalin convertase (Fricker and Snyder, 1983; Fricker et al., 1986), a serine protease unrelated at the amino acid sequence level to the K/IX7 product. The evidence that carboxypeptidase E performs the enkephalin maturase function in vivo is indirect; the enzyme is present in high concentration in the appropriate compartment of hormone-producing cells, for example, in the chromaffin granules of cells of the adrenal medulla, and will carry out the carboxypeptidase B-like reaction in vitro. However, it has not yet been possible to genetically relate mutations in the gene for this protease to defects in peptide processing, and the authenticity of its in vivo role awaits definitive demonstration. This leaves open the pos- sibility that some KfX7-like protein is involved in peptide processing in other eukaryotes.

At a practical level, yeast is finding application for the production of proteins of commercial importance, some of these (for example insulin) require processing by both the KEX2 and the KEX7 gene products. High level production of such proteins may also require the overproduction of the appropriate processing proteases; indeed, there is evidence that STf73 product can be limiting (Julius et al., 1983) and some evidence that C-terminal trimming of in- sulin chains is not completed in yeast (Thim et al., 1986). The identification and cloning of these protease coding genes makes feasible the possible construction of such hyperprocessing yeast strains.

Experimental Procedures

Yeast Strains and Genetic Techniques Strains of S. cerevisiae are listed in Table 1 Growth conditions were as described previously (Bussey et al.. 1982). Standard techniques were used for diploid construction, sporulation. and tetrad dissection (Sherman et al.. 1979)

Transformation Yeast transformation with the YCp50 library was carried out usmg the spheroplast method of Hinnen et al. (1978); otherwise, the lithium ace- tate technique was employed (ho et al , 1983). Transformation of E. co11 was performed as described by Hanahan (1983).

DNA Purification and Recombinant DNA Techniques Plasmid DNA was purified from E. coli as previously described (Boone et al., 1986). Yeast DNA was Isolated according to Davis et al. (1980). Ligation, Klenow polymerase, and mung bean nuclease treatment were performed according to Maniatis et al. (1982). Southern blot hy- bridization was carried out as described by Southern (1975). Northern blot hybridization was performed according to Thomas (1980). DNA fragments were labeled by nick translation (Mackey et al., 1977).

Plasmids Vector YCp50 (Kuo and Campbell, 1983) and the yeast genomic library constructed by M. Rose were generously provided by B. Futcher. Plas- mid pAD17 is described in this paper. Plasmid pL360-2 was provided by T Vernet. Plasmid pFL44 was a gift from F. Lacroute and is a 2 pm DNA-based, 4.4 kb, yeast-E. coli shuttle vector with URA3 and Ap’ markers and the pUC19 polylinker in a IacZ fragment. Plasmid pAD81 contains the KEX7 gene on the 3.1 kb Hindlll fragment inserted into the pFL44 Hindlll site. Plasmid pAD82 is a derivative of pAD81 with the Sacl-Pstl fragment of the polylinker deleted by SacllPstl digestion fol- lowed by mung bean nuclease and Klenow polymerase treatment to create blunt ends. Plasmid pAD26 is described below. Plasmid pJK6 was a kind gift from J. Kurjan. This is a YCp50-based plasmid with the MFa7 coding sequence behind the GAL7 promoter, inserted into the EcoRI-Sal1 sites of the vector. Plasmid pJK6-4 was constructed as fol- lows. A 1.5 kb Sspl-Sal1 fragment containmg the GAL7 promoter and MFa7 coding sequence was isolated from plasmid pJK6, partially digested with Neil, made blunt-ended with Klenow polymerase. digested with EcoRl, and ligated into the EcoRI-Nrul sites of YCp50

Disruption of the KEXl Locus The structure of the three disrupbons used in this work are shown In Figure 2. insertion of the LEUP marker was obtained by introducing a 2.8 kb Sal1 fragment with the LEUP gene (derived from plasmid pYF91) into the Sall site within the KEX7 gene (see Figure 2); strain DGY-7a-4 contains such a disruption. To obtain a deletion of the coding region of the KEX7 gene, the 1.5 kb Sall-Xhol fragment was replaced by a 1.9 Xhol-Sall fragment containing the LEUP marker. Alternatively. Ncol-Xhol fragment of the KEX7 gene (1.5 kb) was replaced by a 1 1 kb Bglll fragment carrying the URA3 marker (isolated from plasmid pFL44), usmg Klenow polymerase to generate blunt ends.

One step disruptions (Rothstein, 1983) with the LEUP marker were obtamed through the use of a Hindlll fragment for transformation of the recipient yeast strains. The disruption with the URA3 marker was ob- tained using the Nhel-Clal fragment.

In the text of the paper we have designated disrupttons of KfX7 as kexl-: full genotypes are indicated in Table 1.

DNA Sequencing Subclones of suitable restrictnon fragments were made in M13mp8 and M13mp19 vectors (Yannisch-Perron et al., 1985). Sequencing was by the dideoxy method (Sanger et al., 1977) and was determined for both DNA strands. The strategy employed is summarized in Figure 7. DNA primers were either the universal Ml3 sequencing primer or primers synthesized to be complementary to parts of the KEX7 DNA sequence

Site-Directed Mutagenesis Oligonucleotide-dIrected mutagenesis was carried out as described by Zoller and Smith (1982), using ssDNA of Mldmpl8 with the 1.1 kb BamHI-Sal1 fragment of the KEX7 gene and the mutagenic ollgonucle- otide 5’.ATCGGGTGAAGCTTACGCTGGCC-3’. The mutation was de- tected by the presence of a newly created Hindlll site and confirmed by sequencmg of the mutated site and the surrounding region. The mu- tated Sall-Baml fragment was cloned into Sall-BamHI site of plasmld pAD82, resulting in plasmid pAD26.

Mapping of Transcript Initiation The 5’ end of the KEX7 mRNA was mapped using two synthetic

Protein Processing by Yeast KEXI Protease 563

loobp

PH SE SK X b-4 H

Figure 7. DNA Sequencing Strategy for KEX Gene

Restriction sites used for sequencing the KEX7 gene (see Figures 2 and 3 for details) are indicated above the heavy line, and the relevant fragments are shown by the plain arrows. Arrows starting with open cir- cles indicate fragments sequenced following initiation with synthetic KEXI internal primers (see Experimental Procedures). Abbreviations are as follows: P, Pstl; H, Hindlll; S, Sall; E, EcoRI; B, BamHI; K, Kpnl; X, Xhol.

primers, PEl (5’-TGAGATCCTTATTAAAGCAGA-37 and PE2 (5’-TAAAG- GAATATGGCCAGCATG-3’) complementary to nucleotides 40-61 (PEI) and 154-174 (PE2) of the extended ORF. The oligonucleotides (2 pmol) were labeled at their 5’ end using W polynucleotide kinase and an ex- cess [32P]ATP The labeled primer (2 pmol) was annealed with total yeast poly(A)+ RNA (10 rg) at 50°C in PIPES buffer (0.4 M NaCI, 100 mM PIPES [pH 6.41). Primer extension was with AMV reverse transcrip tase (10 U) in 50 mM Tris-HCI (pH 82), 10 mM DTT, 6 mM MgCIz, and 500 VM of each dNTP at 42OC for 1 hr. The sample was phenol- extracted and run on a sequencing gel alongside a dideoxynucleotide sequencing run of the KEXl gene primed with the same primer.

Tests for the Kilter Toxin and a-Factor Secretion The killing zone plate assay for the killer phenotype using sensitive strain S6 was described previously (Lolle et al., 1964). a-Factor secre- tion was monitored as follows: a 2 ~1 sample of a cell suspension from 1 ml of a logarithmically growing culture at h nm 0.9-1.0 (ml x 10’ cells per ml), centrifuged and resuspended in 50 ul of YEPD medium, was spotted onto a tester strain (sst7 or ssf2), which was seeded in 1% YEPD medium pH 3.5 (Chan and Otte, 1982).

Partial Purification of a-Factor and Carboxypeptldass B Digestion Yeast were grown in 100 ml of minimal medium as described previously (Julius et al., 1963). The culture medium was passed through Bio-Rex 70 resin, and the retained material was eluted according to Ciejek et al. (1977). The eluate was brought to pH 4.0 with ammonium hydroxide, lyophilized, and suspended in 100 ~1 of water. Carboxypeptidase B treatment was performed as described by Wolff et al. (1962).

Synthesis of Synthetic aF-KR The uF-KR peptide of sequence NHTTrpHisTrpLeuGluLeuLysProGly- GluProMetTyrLysArg-COOH was synthesized on an Applied Bio- system (ABI) Model 430A peptide synthesizer. T-Boc amino acids and standard ABI protocols were used. The peptide was cleaved from its support with HF, desalted, and lyophilized. Purification was achieved on a reverse phase semipreparative column (Vydac Protein and Pep- tide C16, 10 x 250 mm) with 0.1% TFA/H& and a 0.1% TFA/CH$N gradient. Amino acid analysis and sequence analysis showed that the purified peptide may have up to 4% of aF-KR with a missing Trp at the N-terminus.

Acknowledgments

We thank Charles Boone, Antony Cooper, Thierry Vernet, and Malcolm Whiteway for discussions and help in various stages of this work, and Denis Banville for advice on 5’ mapping. Thanks also to Deirdre Greene for technical assistance; to Hannah Klein for yeast chromo- some blots; to Vivian McKay and Robert Fuller for KEX2 information; to Janet Kurjan for pJK6; to the National Biomedical Research Founda- tion, Washington, D.C., USA, and Universitb de Montreal for access to their sequence databases; and to Anne-Marie Sdicu and Lucy Byrne

for preparing the manuscript. This work was supported in part by NSERC strategic and operating grants. Issued as NRCC #26305.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 16 U.S.C. Section 1734 solely to indicate this fact.

Received April 16, 1967; revised May 22, 1967

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