Vol. 161, No. 2

Nucleotide Sequence of the Extracellular Glucoamylase Gene STAIin the Yeast Saccharomyces diastaticus

ICHIRO YAMASHITA,* KATSUYUKI SUZUKI, AND SAKUZO FUKUI

Department of Fermentation Technology, Faculty of Engineering, Hiroshima University, Shitami,Higashi-Hiroshima 724, Japan

Received 21 June 1984/Accepted 18 November 1984

The complete nucleotide sequence of the extracellular glucoamylase gene STAI from the yeast Saccharomycesdiastaticus has been determined. A single open reading frame codes for a 778-amino-acid protein whichcontains 13 potential N-glycosylation sites. In the 5'- and 3'-flanking regions of the gene, there are strikingsequence homologies to the corresponding regions ofADHI for alcohol dehydrogenase and MAT6d for matingtype control in the yeast Saccharomyces cerevisiae. The putative precursor begins with a hydrophobic segmentthat presumably acts as a signal sequence for secretion. The presumptive signal sequence showed a significanthomology to that of Bacillus subtilis a-amylase precursor. The next segment, of ca. 320 amino acids, containsa threonine-rich tract in which direct repeat sequences of 35 amino acids exist, and is bordered by a pair ofbasic amino acid residues (Lys-Lys) which may be a proteolytic processing signal. The carboxy-terminal halfof the precursor is a presumptive glucoamylase which contains several peptide segments showing a high degreeof homology with a-amylases from widely diverse organisms including a procaryote (B. subtilis) and eucaryotes(Aspergillus oryzae and mouse). Analysis of both the nucleotide sequence of the STAI gene and the amino acidcomposition of the purified glucoamylase suggested that the putative precursor is processed to yield subunits Hand Y of mature enzyme by both trypsin-like and chymotrypsin-like cleavages.

The glycosylated extracellular glucoamylase of the yeastSaccharomyces diastaticus plays a role in the first step ofstarch fermentation (degradation of starch to glucose) (23).Knowledge of its primary structure is one of the prerequi-sites for the complete understanding of the structure-func-tion relationship and may provide some information on

different enzymatic behaviors to starch between gluco-amylase and ot-amylase. So far, the sequence of only onea-amylase from a fungus, Aspergillus oryzae, has beencompletely determined by classical protein sequence analy-sis techniques (21). However, the cloning of a number ofa-amylase genes has allowed derivation of the primarystructure of the corresponding enzyme from the DNA se-

quence (9, 20, 25).In the accompanying paper (24), we cloned the extracel-

lular glucoamylase gene STAJ. The essential region (2.5kilobases) of the STA1 gene was large enough to code ca. 700amino acids. Considering molecular weights of protein moi-eties of subunit H (41,000) and subunit Y (3,400) of theenzyme (23), posttranscriptional or posttranslational proc-essing or both must occur. We suggested that the STAI genemay have evolved from an unidentified intracellularglucoamylase gene (6) in the yeast Saccharomyces cerevis-iae by the acquisition of a signal sequence for secretion andprobably a promoter sequence for transcription at the sametime.As a further step in understanding the expression and

evolution of the STAI gene in molecular terms, the completenucleotide sequence of the gene has been determined. Thesequence data suggest that the extracellular glucoamylase isfirst synthesized as a large precursor protein which consistsof a putative signal peptide for secretion, a threonine-richtract, and a functional domain of the enzyme, followed bymaturation to subunits H and Y by both trypsin-like andchymotrypsin-like cleavages.

* Corresponding author.

MATERIALS AND METHODSDNA sequence analysis. DNA sequence analysis was car-

ried out by the method of Maxam and Gilbert (15).Purification and amino acid analysis of glucoamylase.

Glucoamylase secreted from cells of a strain (5106-9A)carrying the STAI gene was purified as described previouslyby us (23). The purified enzyme was dissociated to subunitsin the presence of sodium dodecyl sulfate. The subunitswere separated by gel filtration as described previously (23).Protein samples (5 to 50 pug) were hydrolyzed in evacuatedtubes for 24 and 48 h at 110°C in 0.5 ml of 6 N HCl. Theamino acid analysis was carried out with a Jasco amino acidanalyzer.

RESULTS AND DISCUSSION

Nucleotide sequence of the STAI gene and a functionaldomain. Figure 1 shows the complete nucleotide sequence ofa 2,753-base-pair-long DNA fragment containing the struc-tural gene for the yeast extracellular glucoamylase in whicha unique open reading frame was identified from nucleotide-9 to nucleotide 2334. Nucleotide 1 is A of the first ATGencountered in this frame. The corresponding peptide se-

quence consisting of 778 amino acids is also shown in Fig. 1.It yields a molecular weight of 83,700 which is ca. twofoldlarger than that measured for the purified protein: the sum ofthe molecular weights of protein moieties of subunits H andY is ca. 44,400 (23). The putative protein carries 13 potentialN-glycosylation sites, Asn-X-Thr (or Ser) (19), and consistsof three characteristic regions, i.e., a signal peptide forsecretion, a threonine-rich tract in which direct repeatsequences of 35 amino acids exist, and a functional domaincarrying subunits H and Y of mature glucoamylase (Fig. 1).The carboxy-terminal half of the putative protein is precededby a pair of basic amino acid residues (Lys-Lys) at position357 to 358 (Fig. 1) which may be a proteolytic processingsignal to yield a functional domain of glucoamylase carryingsubunits H and Y. For many mammalian polypeptide hor-

567

JOURNAL OF BACTERIOLOGY, Feb. 1985, p. 567-5730021-9193/85/020567-07$02.00/0Copyright C 1985, American Society for Microbiology

568 YAMASHITA, SUZUKI, AND FUKUI

BamHI/Sau3A -80 -60 -40 -20GA CCTAAACTAA ACCTATAAAA AGCACCCTAT TCATCAGTTA TAATCTCTTG TCATGTTGT`G GTTCTAATT AAAATATACT

ATG GTA GGC CTC AAA AAT CCA TAT ACG CAC ACT ATG CAA AGA CCA TTT CTA CTC GCT TAT TTG GCTC CTT TCG CTT CTA TTT AAC TCA GCTMet val gly leu lys asn pro tyr thr his thr met gln arg pro phe leu leu ala tyr leu val leu ser leu leu phe asn ser ala1 10 20 30

BamHIT1¶G GGT TTT CCA ACT GCA CTA GTT CCT AGA C-GA TCC TCC TCT AGC MC ATC ACT TCC TCC GGT CCA TCT TCA ACT CCA TTC AGC TCT GCTleu gly phe pro thr ala leu val pro arg gly ser ser ser ser ile thr ser ser gly pro ser ser thr pro phe ser zer ala

40 50 60Hindl II

ACT GM AGC TTT TCT ACT GGC ACT ACT GTC ACT CCA TCA TCA TCC AM TAC CCT GGC AGT AAA ACA GM ACT TCT GTT TCT TCT ACA ACCthr glu ser phe ser th gly thr thr val thr pro ser ser ser lys tyr pro gly ser lys thr glu thr ser val ser ser thr thr

70 80

DIRECT REPEATGAA ACT ACC ATT GTT CCA ACT ACA ACT ACG ACT TCT GTC ATA ACA CCA TCA ACA ACC ACT ATT ACC ACT ACG GTT TGC TCT ACA GGA ACAglu thr thr ile val pro thr thr thr thr thr ser val ile thr pro ser thr thr thr ile thr thr thr val cys ser thr gly thr

AC TCT GCC GGT GM ACT ACT TCT GGA TGC TCT CCA MG ACC ATT ACA ACT ACT GTT CCA TGT TCA ACC AGT CCA AGC GM ACC GCA TCGasn ser ala gly glu thr thr ser gly cys ser pro lys thr ile thr thr thr val pro cys ser thr ser pro ser glu thr ala ser

130 140 150

GAA TCA ACA ACC ACT TCA CCT ACC ACA CCT GTA ACT ACA GTT GTC TCA ACC ACC GTC GTT ACT ACT GAG TAT TCT ACT AGT ACA AM CMglu ser thr thr thr ser pro thr thr pro val thr thr val val ser thr thr val val thr thr glu tyr ser thr ser thr lys gln

160 170 180

BstEIIGGT GGT GM ATT ACA ACT ACA TTT GTC ACC AAA MC ATT CCA ACC ACT TAC CTA ACT ACA ATT GCT CCA ACT TCA TCA GTC ACT ACG GTTgly gly glu ile thr thr thr phe val thr lys asn ile pro thr thr tyr leu thr thr ile ala pro thr ser ser val thr thr val

-00 ~~~~~~~~~210-, r DIRECT REPEAT

ACC MT rrC ACC CCA ACC ACT ATT ACT ACT ACG GTT TGC TCT ACA GGA ACA MC TCT GCC GGT GM ACT ACC TCT GGA TGC TCT CCA MGthr maphe thr pro thr ,=, ile thr thr thr val cys ser thr gly thr asn ser ala gly glu th,r th ser gly cys ser pro lys

230 240PvuII

ACT GTC ACA ACA ACT GTT CCT TGT TCA ACT GGT ACT GGC GAA TAC ACT ACT GAA GCT ACC GCC CCT GTT ACA ACA GCT GTC ACA ACC ACCthr val thr t thr val pro cys ser thr gly thr gly glu tyr thr thr glu ala thr ala pro val thr thr ala val thr ,L thL

in m ~~~~~~~~~~~~~~~~~~~~270

GTT GTT ACC ACT GM TCC TCT ACG GGT ACT MC TCC GCT GGT MG ACG ACA ACT AGT TAC ACA ACA MG TCT GTA CCA ACC ACC TAT GTAval val thr thr glu ser ser thr gly thr asn ser ala gly lys thr thr thr ser tyr thr thr lys ser val pro thr thr tyr val

290___300

PvuIITTT GAC mTT GGC AAG GGC ATT CTC GAT CM AGC TGC GGC GGT GTA TTT TCA MC MC GGC TCT TCG CM GTG CAG CTG CGG GAT GTA GTCphe asp phe gly lys gly ile leu asp gln ser cys gly gly val phe ser asnas gly ser ser gln val gln leu arg asp val val

310 320 330

TTG ATG MT GGG ACA GTG GTA TAC GAT TCA MC GGC GCT TGG GAC AGT AGT GCG CTG GAG GAG TGG CTC CAG CGA CAG AM AAA GTT TCCleu met gly thr val val tyr asp ser asn gly ala trp asp ser ser ala leu glu glu trp leu gln arg gln lys lys val ser

340 350 360

ATC GM AGA ATA TTT GM MT ATT GGG CCC AGC GCC GTG TAT CCG TCT ATT TTG CCT GGG GTC GTG ATT GCG TCA CCA TCG CM ACG CATile glu arg ile phe glu asn ile gly pro ser ala val tyr pro ser ile leu pro gly val val ile ala ser pro ser gln thr his

370 380 390

CCA GAC TAC TTC TAC CAA TtG ATA AGG GAC AGC GCG TTG ACG ATA AAC AGT ATT GTC TCT CAT TCT GCG GAC CCG GCA ATA GAG ACG TTApro asp tyr phe tyr gln trp ile arg asp ser ala leu thr ile asn ser ile val ser his ser ala asp pro ala ile glu thr leu

400 410 420

mones (7, 16, 18) as well as yeast a-factor-a tridecapeptidemating hormone (11, 12)-a trypsin-like protease targetspairs of basic amino acids to yield a mature hormone. Thefunctional domain of glucoamylase contains several peptidesegments showing a high degree of homology to those ofa-amylases from widely diverse organisms including a pro-caryote, Bacillus subtilis (25), and eucaryotes, A. oryzae (21)and a mouse (9) (Fig. 2). The comparative study indicatesthat the glucoamylase shares an aspartic acid residue atposition 635 (indicated by an arrowhead in Fig. 1 and 2)which is highly conserved in diverse a-amylases and issuggested to function to cleave the a-1,4-glycosidic bond inA. oryzae a-amylase (14).Amino acid composition obtained from amino acid analy-

sis of the mature enzyme (subunits H and Y) and subunit Y,and that deduced from the nucleotide sequences (Table 1),indicated that the functional domain of glucoamylase isprocessed by both chymotrypsin-like and trypsin-like cleav-

ages following a tyrosine residue at position 702 and a lysineresidue at position 757, respectively, to yield mature subun-its H and Y (Fig. 1). Chymotrypsin-like and trypsin-likeactivities are found in a wide variety of organisms, includingyeast (4). It should be noted, however, that other proteolyticprocessing events may occur near the putative cleavagesites, because exact amino acid sequences of amino-terminaland carboxy-terminal regions of subunits H and Y are notyet known. Molecular weights of subunits H and Y deducedfrom the corresponding nucleotide sequences (Fig. 1) are ca.40,000 and 6,600, respectively. The deduced molecularweight of subunit H fits well with that (41,000) measured forthe purified subunit H (23). The deduced molecular weight ofsubunit Y is ca. twofold larger than that (3,400) measured forthe purified subunit Y (23). However, several lines ofevidence establish that the nucleotide sequence at position2107 to 2271 does code for subunit Y. (i) Amino acidcomposition of subunit Y determined by amino acid analysis

J. BACTERIOL.

NUCLEOTIDE SEQUENCE OF THE STA1 GENE IN YEAST 569

TTG CAG TAC CTG AAC GTT TCA TTC CAC TTG CAA AGA ACC AAC AAC ACA TTG GGC GCT GGC ATTleu gin tyr leu asnval ser phe his leu gin arg thrE3 asn thr lou gly ala gly ile

430 440SalI

GGA GAC CCT AAG IGG AAC GTC GAC AAC ACG GCT TTC ACG GAA CCT TGG GGT CGT CCT CAA AACgly asp pro lys trp asn val asp asn thr ala phe thr glu pro trp gly arg pro gin asn

460 470

GGT TAC ACT MC GSAT ACA GTIG GCT TTgly tyr thr asp thr val ala leu

450

GAT GGC CCT GCT CTTasp gly pro ala leu

CGA AGC ATT GCCarg ser ile ala

480

ATC TTA AAA ATC ATC GAC TAC ATC MG CAA TCT GGC ACT GAT CTG GGG GCC MG TAC CCA TTC CAG TCC ACC GCA GAT ATC TTT GAT GATile leu lys ile ile asp tyr ile lys gin ser gly thr asp leu gly ala lys tyr pro phe gin ser thr ala asp ile phe asp asp

490 500 510EcoRI

ATT GTA CGT TGG GAC CTG AGG TTC ATT ATT GAC CAC TGG MT TCT TCC GGA 'ImT GAT CTAile val arg trp asp leu arg phe ile ile asp his trp asn ser ser gly phe asp leu

520 530Pst I

ACT TTA CTG GTA CM CTG TCT GCA GTG GAC AGG TCG CTG TCG TAT TTT AAC GCC TCA GAP.thr leu leu val gin leu ser ala val asp arg ser leu ser tyr phe ala ser glU

550 560

TGG GAG GM GTC AAT GGC ATG CAT TTC TTTtrp glu glu val asn gly met his phe phe

540

CGG TCG TCT CCC TTT GTT GM GAA TTG CGTarg ser ser pro phe val glU glu leu arg

570

CAG ACA CGC CGG GAC ATC TCC AAG TTT TTA GTG GAC CCT GCG MT GGG TmT ATC MC GGC MG TAC MT TAT ATT GTT GAG ACA CCC ATGgin thr arg arg asp ile ser lys phe leu val asp pro ala asn gly phe ile asn gly lys tyr asn tyr ile val glu thr pro met

580 590 600

ATT GCC GAC ACA TTG AGA TCC GGA CTG GAC ATA TCC ACT TTA TTA GCT GCG MC ACC GTC CAC GAT GCG CCA TCT GCT TCC CAT CTT CCGile ala Asp thr leu arg ser gly leu asp ile ser thr leu leu ala ala asn thr val his asp ala pro ser ala ser his leu pro

610 620 630

TTC GAT ATC AAT GAC CCT GCC GTC CTG AAC ACG TTG CAC CAT TTG ATG TTG CAC ATG CGT TCGphe asp iie asn asp pro ala val leu asn thr leu his his leu met leu his met arg ser

4 640 650

AMT GCA ACG GGT ATT GCC CTG GGC CGG TAT CCT GAG GAC GTA TAT GAT GGA TAT GGC GTT GGC&la thr gly ile ala ieu gly arg tyr pro glu asp val tyr asp gly tyr gly val gly

670 680H -y--

TGT GCC GCT TCA ACA ACG CTT TAT CAG CTC ATT TAC AGA CAC ATC TCT GAG CAG CAT GAC TTGcys ala ala ser thr thr leu tyr gin 1eu ile tyr arg his ile ser glu gin his asp leu

700 710

AAC GCA TTT TGG AGC GAG CTG GTA TTC TCC MC CTC ACG ACT TTG GGA MT GAC GAA GGC TATasn ala phe trp ser glu leu val phe ser leu thr thr leu gly asn asp glu gly tyr

730 740y .o I

TIC AAT CAA ACC ATA CM AAA ATC TTC CAA CTA GCT GAT TCA TiC TTG GTC MG CTGphes gin thr ile gin lys ile phe gln leu ala asp ser phe leu val lys leu

760+20 +40 +60

ATA TAC CCC ATC MC GAT AGC TCC AM

ile tyr pro ile asp ser ser lys

660

GAG GGA AAT CCC TGG GTC CTG GCC ACGglU gly asn pro trp vai leu ala thr

690

GTT GTC CCA ATG MC MC GAT ¶lGT TCGval val pro met asn asn asp cys ser

720

TTG ATT TTG GAG TTC MT ACA CCT GCCleu ile leu glU phe asn thr pro ala

750

AAA GCC ACG TGGlys ala thr trp770

+80

+1GAA CAG ACG GGG MC TMglU gln thr gly asn stp

+100GTGAACA ATTTAACMA TACACAGGGT TTATGCAGGG TGCCCMCAC CTTACCTGGT CCTATACTTC ATTCTGGGAT GCCTATCAA TMGACMGA AGTTTTACAG

+120 +140 +160 +180 +200 +220AGTTTGTAGA CAAAAAAAAA TAAAAGAAAA GCGAGMGTA TACACMGTG TATTTCCTAG ATATTTACAT CMATATATA TATATATACT TATTTACAAA ACTCTGATAT

+240 +260 +280 +300 +320 SalITATAAATTMATTAGATACTA TGTCGGMCG TCCAGCCCAA CCACGTTTGC AGTTCTTTTC ACTTTCTCAT CCTGTGTCM CTTGTTGCCA GGATTGTATC TGTCGAC

FIG. 1. Sequence of the STA1 gene. Shown is the complete sequence of a contiguous 2,753-nucleotide region encompassing the STAIstructural gene and over 400 nucleotides of flanking DNA. The sequence is shown only for the mRNA-coding (i.e., plus) strand of the gene.The numbers above the sequence indicate the number of nucleotides, in each direction, from the A in the translation-initiating ATG. Thenumbers below the sequence denote the amino acid number, starting with 1 at the methionine. Direct repeat sequences and the putativeregions encoding subunits H and Y are indicated. A pair of basic amino acid residues (Lys-Lys) and potential N-glycosylation sites are boxed.Threonine residues in the threonine-rich tract are underlined. A potential mRNA-splicing signal, 5'-TACTAACT-3', beginning fromnucleotide position 837 is also underlined. The aspartic acid residue at amino acid position 635 is marked with an arrowhead. Restriction sitesare also indicated above the nucleotide sequence.

is in good agreement with that deduced from the correspond-ing nucleotide sequence (Table 1). (ii) It is evident thatsubunit Y contains a tyrosine residue which can be iodinatedwith the aid of chloramine T (23). The putative subunit Ycontains a tyrosine residue at position 741 (Fig. 1). (iii)Subunit Y is a glycoprotein (23). The putative subunit Ycontains two asparagine residues at position 731 and 752which are potential N-glycosylation sites (Fig. 1). (iv) SubunitY, separated from the native enzyme, is hydrophobic, sinceit is insoluble in water (23). Of the amino acid residues of theputative subunit Y, 40% are hydrophobic (Fig. 1).The STA1 gene contains an mRNA-splicing signal se-

quence (5'-TACTAACT-3') (13) beginning at position 837(Fig. 1) where STAI mRNA might be spliced under the

restriction that frame shift does not occur, otherwise stopcodons appear frequently. It is under investigation whetherthe STAI gene contains an intron.The 5'- and 3'-flanking regions of the STAI gene. There is a

striking sequence homology between the 5'-flanking regionof the yeast ADHI gene (3) and that of the STA1 gene (Fig.3). The sequence 5'-TATAAA-3' (position -67 to -62) ofthe STAI gene is in good agreement with the "Hogness-box" structure (5'-TATAAATA-3') which is found in the5'-flanking region of many eucaryotic genes (3) and isimplicated to function as a part of the eucaryotic RNApolymerase recognition site. The nucleotide sequence (posi-tion -59 to -39) of the STAI gene is highly analogous to thataround one of two 5'-capping sites of the ADHI mRNA

VOL. 161, 1985

570 YAMASHITA, SUZUKI, AND FUKUI J. BACTERIOL.

I H359 4J U

S VSIERI FENIGPS AV Y PSILPGV VIASP SQl' if P)U)YFYQ WIPR D S A l'TIN S IVSHSA DPAIE TL LQYLNVS FHA -T-ADWR--SI --LLTDRIA -T-G-TrT-WTN -ADQ KY C G GTW QG-1 DK -D-IQGMG-TMS QYDPHTQYGRTA- -HL--WRWVDI-KECER-LA-NGFA--Q- --PNENIVV -S-SHPW -E-Y Q p I -YK---R--NE-EFRD M VP - SD----_--------------------K--G---- V---N----- - -----T----- -

B LT- - SIKS DGF -I-A -KIIIE-PDDG--G -QFW PNITNTSAEI Q- -E

SAMSMPB

450

LQ RT NN T LG A G IGYTNDTV ALGDPK WN V DNT AF TEPWGR PQN D GP A LRSIAI LKIAIWI -P V-AQ-PQDC -Y-DA -- - Y - QT - IYSLN - -Y - T-DDLKA-S- - -HERGM

-R -N -VG-RIYV -AVI -HMCG-G -QAGQSS- C -SYFN-N-R- F -G VPY-G FT - -- -------- ---- ----- A- NP--T--- - ---L----- E- - - ---- - W

---DSAS-DAAYA-YMD-- -SNY -HSIRS --- T -LHA--WSF --LKHNMK -IHD - G- T--QTSP

500S IDYI KQ SGT DLGA K YPFQ ST ADI F D DI VRW DLR FI I D HPWA YLMY -WANHM _y - --G S - V -YSV -KPFSSQ -Y - -PFCFMS -F -W-;CTA-- - IENYQDAA -VRDCRL- G LL -LALEK - Y--TKVA--MNHL -D-GVAGFRL-ASK-M-PMP----N--N-E-D--N--Y - --N--- - - -- ------ - ------------- ------------A-----RMP ----N--N -E-D--N--Y ---N -A--

B -NQ- -E -NQ - - --M,SNWYWL- -PT -YQI-NRY-GTEQE --EMCAAAEEYG -K-I - -AVI

S NSS G FD L WEEV NGM HF F IA IQ-YED QTVE -C -L GD - TVSLPDLDT -MS -DIKAIL -KLHN-NTK-F SQ - -R- -I-QEVIDN4P ----V------------ -- - --_

B -HT - --YAAIS- --K-IP-W- -GN

550TL LVQL SAV DR SL S YF NASERSSPF VE ELR QTRR DISK F-KD -V KNEWY -WVG --V-N- -ID G - -I-TV-HVQKD-

- GG - -- --N -YFG NG-- -- - -I KG- ---- ---

-QI -- - -DRW- -TQD--LGL-DW--VNR -HN-MAG -PEE

600S L V DPAN GF I N GKYNY IVETPM IAD TLR S GL DISTL LA AN TV HDAPS ASH LP F

A WPGYNKAAGVYCIGE-LD - ---YTCPYQ -VMD-VL--P-Y Y- LLN-FKS- - -SM-D -YNMI - --KS-C- D -TL- GT-MS -TEFKYGAKL -KVMRKWD - -K - -Y-K -WGEGWGL M ---R - - V -

MP ---------- -T- -RKW- - ------ -------- - ---- - - - -

B -S - - - -N -Q -F - -Q -G-H-W --N- -SSS - -INT- -K--

650S DI N D P AV LNT LHHL44LH MR SIYPI NDA V E -H-N -RF- - - T --IMS -- ----QRGHGAGG -SI- -FWDAR- YK-AVGF-LAHP -GF-RVMMP -- ------------S --- ------ M------------ -------

B -GRY --K -GAG - sQ - -C

H 70011S E GN PWV LATC A AS TTL YQLIYRHISEQHDA --D-ANRE -- W L -GYP -D --LY KMS K-VSI- -DS --GND- IC EHRWR - - -NMVAFR NMP ---T--A -T ------V - ----- - - ------- -

B - -N - - -T- --D-N --KAV-- -N -GP -DRR

S VF S N L TTLG N DEG YLILEFNTA -TYK -PY IKMD --I A MR K-TDG SQIVT --S -IMS TYCD-I -GDKVDG-CTG --VYV - - - -KAHF- - - -

MP ------ ------------R -N- - S - ------

B - - -T-- - - --RTEK- --V-

SSK N AT GIALG AY P EDV YDGYGV GIALA - -V-AFII LN D - -PII -AGQ - QH - A -

-- y -W -RNFQNGK --N -WV- PPNNN-KT--N ------- -Q- --I - ------V-

3 -LTGTI- -RSVAV-YP-D --KA -HV-L - - -KT --THSF

yLVV PM NNDC SN AFWSE L- IASANAIR - YAI- KPTG -

-- -GQ - FANWWDNDSNQVAFGRGNKGLIVF---DW - L--T-QIGLPAG--- - -S -----N------S---R-F-------- - --A---------

- -E - -SQ -E-EIQ -GK -Y -IM

750y

r P AFN Q T I QKKG - SGDSYTLSLSGASYTAGQ-L-EV-GCTTVTVGSDGNVPVPMAGGLPRVLYPTEKSAED- -

-R --V-

778S I FQ LA DSF LVKLKATWEQIl;NA --G - - IC SDSSMS -AI H -E - - -MP ---

B NLG-MLLATHLQEQ--S --F-Y -FFV- -

FIG. 2. Sequence homologies among the functional domain of putative glucoamylase precursor and oa-amylases from widely diverseorganisms. Amino acid sequences of the following amylases were aligned to maximize the homologies: S, the functional domain of putativeglucoamylase precursor from S. diastaticus; A, A. oryzae a-amylase (21); MS, mouse salivary gland a-amylase (9); MP, mouse pancreasa-amylase (9); B, B. subtilis a-amylase (24). To maximize the homologies, amino acid residues of B. subtilis ai-amylase are aligned in thefollowing order: 1-8 171-239 9-148 337-551 (numbers of anino acid residues start with 1 as the leucine which is the NH2-terminal amino acidof the mature amylase). The identical amino acid residues to above are indicated by bars. The conserved aspartic acid residue is marked withan arrowhead. The putative regions for subunits H and Y are also indicated.

T

NUCLEOTIDE SEQUENCE OF THE STAI GENE IN YEAST 571

TABLE 1. Amino acid composition of glucoamylaseHydrolysate DNA sequence

Aminoacid Subunits Subunit Subunits Subunit

H and Y Y H and Y YAsx 60 5 57 10Thr 27 3 26 4Ser 34 4 34 4Glx 36 7 29 7Pro ND NDa 22 2Gly 23 4 23 2Ala 29 3 28 2Cys ND ND 2 1Val 18 2 23 3Met 5 1 5 1Ile 23 1 32 3Leu 42 3 38 6Tyr 18 1 17 1Phe 20 3 19 4His 12 2 12 2Lys 10 2 8 1Arg 16 1 16 1Trp ND ND 8 1a ND, Not determined.

ASTAI

ADH1

STA1

ADH1

-80TTTGC TTC CTAAACTAAACCTA]***** ** ** * ** **1

TTTGCCGCTTTGCT ATCAAG TAT-130

(indicated by the arrows in Fig. 3). It seems likely that theSTA1 mRNA starts within this conserved region. A potentialbinding region for the 3' end of 18S rRNA (10) to thepresumptive STAJ-mRNA exists at position -16 to -10. Webelieve that the ATG codon shown in Fig. 3 is the initiationcodon of the STAI gene for the following reasons. (i) Thesequence 5'-TATAC-3', immediately upstream from theinitiation codon of the ADHI gene,' is also found in thecorresponding region of the STA1 gene (Fig. 3). (ii) The ATGis the first ATG codon encountered in the unique openreading frame of the STAI gene (Fig. 1). (iii) Most yeastgenes that have been sequenced contain an A residue threenucleotides before the initiation codon (12); the STAI genedoes too.The 3'-flanking region of the STAI gene shows significant

sequence homologies to those of the ADHI (3) and theMATcY2 (2) genes (Fig. 3). The sequence 5'-AATAAA-3' andits analogous sequences are frequently found in the 3'-flanking regions of many yeast genes (3). Such sequences arealso found at six positions (+15 to +20, +89 to +94, +129 to+ 134, +183 to + 188, +221 to +226, and +225 to +230; fiveof six sequences are underlined in Fig. 3) in the 3'-flankingregion of the STAI gene. Another interesting feature is that

-60 -40rAAAAA GCACCCTATT CATCAGTTATAA**** ~**** **** ** ** .****

rAAA -- 72 -- GCACAATATTTCA AGCTATAC-40 4

3' 5'AUUA CU--18S rRNA

-20 1TCTCTlTCAT; GTGGTTCTAAMlG AAAATATACT AT<

** * * ***

CAAGCATACAATCAACTATCTCA TATACA ATG4 -20 -6 1

B +100 +120 +140

STA1 AA*-AAGAAGTTTTACA GAGTTG EgcMAAAAAAAATAAAAGAAAAGCGAGAA******* * *** *** * ** *** ** ** ** ****** **** **

ADHI C GAA TTTCTTATGA T7j~TTTTTATTATTAAATAAGTTATAAAA AAAATA AGT+20 +40 +60

STAl

ADK1

MATa2

+160 +180 +2 00ACMCAAGTGT; CCTMhATTTACATCAAATATATATATATATACTTA@|******* ** * *** * *** * ***

GTATACA AA T RAAGTGACTCT.AGGT TTT***** * * +80 ** **** * * ****************

ACA AA A TATATATCII;TGI ATATATATATATATA+20 +40

+220 +240 +260STAl ACAAAACTC17GATAT TA A7FRT TAC~CGGAACGTCCAGCCCACCAC

* **** * *** * **.* ** * ** * * ** **

MATat2 CGCA AAAATAC ATA AACAATCAACCCTCTCCTCAGAC ACTAC+60 +80

STAI GEGCAGTT***** * *

MATO.2 TAAGATGTTTGTAACT+100

FIG. 3. The 5'- and 3'-end homologies of STAI and other yeast genes, ADHI and MATa2. (A) The 5'-flanking regions of STAI and ADHI.Nucleotide residues that are identical in both sequences are marked by asterisks. For clarity, 72 nucleotides (-122 to -51) of the ADHIsequence are deleted. The sequence 5'-TATAAA-3' is underlined. The 5' ends of the ADHI mRNA are marked by arrows. Thecomplementarity between the nucleotide sequence (-16 to -10) of STAI and the 3' end of the 18S rRNA is indicated. (B) The 3'-flankingregions of STAI, ADHI, and MATa2. Nucleotide residues of ADHI and MATa2 that are identical to those of STAI are marked by asterisks.The sequence 5'-AATAAA-3' and its analogs are underlined. The structure 5'-TAAG(TATA or TAG)--- TAG (or TATGT)---TTT-3' is boxed.

VOL. 161, 1985

572 YAMASHITA, SUZUKI, AND FUKUI

Glucoamylase (S. diastaticus)

m- ** * * * *m aaamm . mm-.V * *---*MetValGlyLeuLysAsnProTyrThrHisThrM4etGlnAtggProPhheL@uLeuAlATyrateuVa auSerL@uLe4Ph@nSerfAla LeuGlyPheProThrAlalauValProArgGly

C C C C CTTTTCCCCCC C S S S S S SSS SS C C C C C C C C C C C T T T T

,-am7lase (B. subtilis)

* * * * * a * *w*-"-*----* - P * m* m!--- * * *

II ii H H H C C C C C C C S S S S S S S S S S S S C C C C C C H H H H H H T T T T

Killer toxin (S. cerevisiae)

*U **U * - * *U -E-S- *m-*m m m*E-S- v *- *-UMetThrLysProThrGlnValLeuValArgSerValSerIleLeuPhePheSleThrILeuLeuHis[euValValAla LeuAsnAspValAlaGlyProAlaGluThrAlaPro

C C C C C S S S S S S S S S S S S S S S S S SSSC C C C C C CC Cc V C c

-factoi (S. cerevisiae)

MetArgPheProSer I lePheTthrAlaValLeuPheAlaAlaSerSerAlaLeuAlaAla ProValAsnThrThrThrGluAspGluThrAlaGln

C C C C C S S S S S H HHHHH HH C C C C C C C H H H H H H

Acid phosphatase (S. cerevisiae)

* U* *-*E-PU*** -*** *U- A*a

MetPheLysSerValValTyrSer IleLeuAlaAlaSerLeuAlasnAIla GlyThr IleProLeuGlyLysLeuAlaAspValAspLys

C C C C S S S S S C H H H H H T T T T C C C C C C C T T T T

FIG. 4. Structural similarity of the putative signal peptide for secretion of the yeast glucoamylase precursor with other secretory signalpeptides from B. subtilis and S. cerevisiae. Shown are the putative signal peptides for secretion of S. diastaticus glucoamylase, B. subtilisa-amylase (25), S. cerevisiae killer toxin (4), S. cerevisiae a-factor (12), and S. cerevisiae acid phosphatase (1). Identical peptides in S.diastaticus glucoamyla$e and B. subtilis a-amylase are underlined. Basic, acidic, and hydrophobic amino acid residues are indicated by stars,open triangles, and closed squares, respectively. Possible secondary structures are indicated as follows: H, a-helix; S, 13-sheet; T, ,-turn; andC, random coil. Proposed cleavage sites for signal peptidases are indicated by arrowheads.

the structures, 5'-TAAG (TATA or TAG) --- TAG (orTATGT) --- TTT-3', are found in three regions (+91 to + 165,+149 to +205, and +232 to +268, boxed in Fig. 3). Such a

sequence is suggested to have a role in transcription termi-nation and polyadenylation in yeast (26). It should be notedthat the alignment of two signals (5'-AATAAA-3' and 5'-TAAG (TATA or TAG) --- TAG (or TATGT) --- TTT-3') inthe 3'-flanking region of the STAI gene is closely similar tothose found in the 3'-flanking regions of the ADHi andMATa2 genes (Fig. 3).

Presumptive signal sequence for secretion. The amino-ter-minal segment of ca. 30 amino acids in the presumptiveglucoamylase precursor (Fig. 1) resembles signal sequencesfound in a wide variety of secretory protein precursors (22).The putative signal peptide shows a remarkable sequencehomology to that ofB. subtilis a-amylase precursor (25) (Fig.4). Furthermore, the central hydrophobic segments of thetwo signal sequences allow the formation of a stable 1-sheetconformation, calculated by using the method of Chou andFasman (5) (Fig. 4). Emr and Silhavy reported that a stablea-helical conformation in the central hydrophobic region ofthe LamB signal sequence plays a key role in its secretoryfunction (8). However, their finding is not applicable to theputative STAJ signal sequence or to those of other secretoryproteins, e.g., heat-labile enterotoxin of Escherichia coli (17)and killer toxin (4), a-factor (12), and acid phosphatase (1) ofS. cerevisiae. Central hydrophobic segments of E. coliheat-labile enterotoxin (17) and yeast killer toxin exhibit astable 1-sheet structure, and those of yeast a-factor and acidphosphatase exhibit a 1-sheet conformation followed by an

a-helical one (Fig. 4). By analogy with other secretoryproteins, especially with B. subtilis a-amylase precursor

(25), we expect that the STAJ signal peptide is cleaved afterthe alanine residue at position 30. However, it seems likely,as in the case of a-factor signal peptide (11), that removal ofthe STAI signal sequence is not essential for production ofmature glucoamylase, since the other proteolytic processingmust occur as described.

Evolutionary origin of the STAI gene. Finally, we wouldlike to discuss the evolutionary origin of the extracellularglucoamylase gene. As described in the accompanying paper(24), S. cerevisiae contains DNA segments (Asta, sl, and s2)homologous with the STAI gene. Nucleotide sequence anal-ysis of the STAI gene strongly suggests that the Asta DNAcodes for an intracellular glucoamylase that plays a key rolein spore maturation in yeast (6), because the correspondingDNA sequence of the STAI gene codes for the functionaldomain of the extracellular glucoamylase (Fig. 1). Theextracellular glucoamylase gene may have evolved from theintracellular glucoamylase gene (Asta DNA) by the acquisi-tion of both the sl DNA, which may code for the threonine-rich tract, and the s2 DNA, which may be an origin of boththe promoter sequence for transcription and the signalsequence for secretion of the extracellular glucoamylasegene. The mechanism of evolution of the STAI gene wasdiscussed in detail in the accompanying paper (24).

ACKNOWLEDGMENTS

We are very grateful to A. Toh-e for helpful discussions andencouragement. We thank I. Utazu for help with her excellent DNAsequencing techniques. We also thank S. Oka and H. Okai for aminoacid analysis.

This work was supported in part by grants from the Ministry ofEducation, Science and Culture of Japan.

J. BACTERIOL.

NUCLEOTIDE SEQUENCE OF THE STA1 GENE IN YEAST 573

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