The Maltose Permease Encoded by the MAL61 Gene of Saccharomyces

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The Maltose Permease Encodedby the MAL61 Gene of Saccharomyces cerevisiae Exhibits Both Sequence and Structural Homology to Other Sugar Transporters

Qi Cheng and Corinne A. Michels

Department of Biology, Queens College and the Graduate School of the City Uniwersity of New York, Flushing, New York 11367 Manuscript received November2 1, 1988 Accepted for publication August 10, 1989

ABSTRACT The MAL61 gene of Saccharomyces cerewisiae encodes maltose permease,a protein required for the transport of maltose across the plasma membrane. Here we report the nucleotide sequence of the cloned MAL61 gene. A single 1842 bpopen reading frame is present within this region encodingthe 6 14 residue putative MAL61 protein. Hydropathy analysis suggests that the secondary structure consists of two blocks of six transmembrane domains separated by an approximately 71 residue intracellular region. The N-terminal and C-terminal domains of100 and 67 residues in length, respectively, also appear to be intracellular. Significant sequence and structural homology is seen between theMAL61 protein andthe Saccharomyces high-affinity glucose transporter encodedby the SNF3 gene, theKluyueromyces lactis lactose permease encoded by the LAC12 gene, the human HepG2 glucose transporter and the Escherichia coli xylose and arabinose transporters encodedby the xylE and araE genes, indicating that all are members of a family of sugar transporters andare related either functionally or evolutionarily. A mechanism for glucose-induced inactivation of maltose transport activity is discussed.

ALTOSEfermentation in the Saccharomyces indication that the gene encoding maltose permease M yeasts is initiated by the transport of the disac- mapped to any of the MAL loci came from the iden- charide across the plasma membrane. This transport tification of a MALI-linked temperature-sensitive is carried out by maltose permease and the process is maltose transportmutation (GOLDENTHAL,COHEN the ratelimiting step in fermentation. An understand- and MARMUR1983). All of the MAL loci have been ing of the mechanisms controlling maltose transport cloned and structurally and functionallycompared is therefore fundamental to an understanding of the (FEDEROFFet al. 1982; NEEDLEMANand MICHELS factors regulating maltose fermentation. 1983; CHARRON,DUBIN and MICHELS1986; CHAR- The Saccharomyces maltose uptake system is an RON and MICHELS 1987; CHARRONet al. 1989). The inducibleactive transport system (HARRIS and MAL loci are all highly sequence-homologous, exhib- THOMPSON196 1; OKADAand HALVORSON1964; DE iting only a few restriction site polymorphisms. Each KROON and KONINGSBERGER1970; SERRANO1977). locus is a complex locus containing three genes re- SERRANO(1977) reports that this transport is inde- quired for maltose fermentation: GENEs 1, 2, and 3 pendent of intracellular ATP levels but is coupled to (NEEDLEMANet al. 1984). We have established a two the electrochemical gradient of protons. That is, malt- digit numbering system in order to distinguish the ose transport occurs via a proton symport system. As GENE 1, 2 or 3 functions mapping to the different has been seen in the glucose and galactose transport MAL loci. The first digit indicates the locus position systems of Saccharomyces, the maltose transport sys- and thesecond the GENE function (NEEDLEMANet al. tem exists in botha high and a low affinity form 1984; CHARRONand MICHELS1987, 1988). Thus, the (BISSON and FRAENKEL 1983a,b,1984; RAMOS, SZKUTNICKAand CIRILLO1989; BUSTURIAand LA- MAL61 gene is the GENE 1 function mapping to the MAL6 locus. GUNAS 1985). The basis of the differencebetween the two forms of these sugar transporters is not under- Transcription of GENEs 1 and 2 is induced by stood. maltose and repressed by glucose (NEEDLEMANet al. Saccharomyces strains ableto fermentmaltose carry 1984). That GENE 2 encodesmaltase is inferred from any one of five MAL loci: MALI, MAL2, MAL3, MAL4, the identification of an allele of the MAL12 gene (that and MAL6 (reviewed by BARNETT1976). The first is, GENE 2 of the MALI locus) that encodes a temperature-sensitive maltase (DUBINet al. 1985). GENE The publication costs of this article were partly defrayed by the payment of page charges. This article must therefore be hereby marked “advertisement” 1 encodes maltose permease. This conclusion is based in accordance with 18 U.S.C. $1734 solely to indicate this fact. on several lines of evidence reported by Y. S. CHANG,

Genetics 123: 477-484 (November, 1989) 478 andQ. Cheng C. A. Michels R. A. DUBIN,E. PERKINS,C. A. MICHELSand R. B. lumbia University College of Physicians and Surgeons,New NEEDLEMAN (unpublishedresults). Point mutations in York; the profiles were made using the algorithmdeveloped by KYTE and DOOLITTLE(1 982) andutilized the values of the MAL61 gene as well as a deletion/disruption of EISENBERG(1 984)with a 2 1 -residuewindow. the MAL61 gene completely abolish maltose transport activity. Transformation of these mutant strains with RESULTS high copy plasmids carrying the MAL61 gene leads to up to a tenfold increase in maltose permease activity Sequence of the MAL61 gene and the proposed as compared to the single-copy parental strain. Most secondary structure of the deduced protein: Figure significantly, the integration of a fragment carrying 2 presents the sequence of the DNA fragment con- the yeast URA3 gene into the coding regionof MAL61 taining theMAL61 gene starting at theScaI site shown near the N-terminal end results in a low level consti- in Figure1 andextending to the right for 2000 tutive transcription of MAL61 and in a low level basepairs. A single large open reading frame is ob- constitutive synthesis of maltose permease. GENE 3 served with the AUG codonof the N-terminal methi- encodes the MAL activator and the product of this onine located 105 base pairs from the ScaI site. No gene is a cysteine-zinc finger protein (CHANG et al. other large open reading frames are observed in any 1988; KIM and MICHELS 1988; SOLLITIand MARMUR of the five other reading frames. The orientation of 1988). this single 1842 basepair open reading frame is con- This report presents the sequence of the MAL61 sistent with the size of the maltose inducible transcript gene. Analysis of the deduced aminoacid sequence of of the MAL61 gene (2.0 kbp) and with the direction the proposed MAL61 protein indicates that it is an of transcription of the MAL61 gene as reported in integralmembrane protein. Additionally, MAL61 NEEDLEMANet al. (1984). Construction of a MAL61- protein shows significant homology to several other lacz fusion at the EcoRI site near the N-terminal end sugar transport proteins fromyeast and other species. of the coding region supports the conclusion that the This homology is seen both on thelevel ofthe primary AUG codon indicated as the translation initiation can sequence and on thelevel of secondary structure. function as such in Saccharomyces (J. LEVINE,L. TANOUYEand C. A. MICHELS,unpublished results). A MATERIALSAND METHODS consensus “TATA” sequence is located at position -89 to -94. The sequence of the open reading frame Sequencing: Figure 1 shows a restriction endonuclease predicts a 67,174 dalton protein of 614 amino acid map of the MAL61 gene. Sequencing was done according to the method of SANGER,NICKLEN and COULSON(1977). residues. The region was divided into threefragments: the PstI-EcoRI Figure 2 also depicts the positions of twelve postu- fragment containing the MAL61 upstream sequences and lated hydrophobic transmembrane domains. Each of the 5’-end of thegene; the 1.7-kb EcoRI-Sal1 fragment the twelve postulated 2 1-residue transmembrane do- containing sequences internal to the MAL61 gene; and the mains has an average hydrophobicity value of greater SalI-Hind111 fragment containing the 3’-end of the gene. Each of these was then sequenced by acombination of than 0.42. As in the SNF3 protein,no signal sequence methods. Nested deletions within the MAL61-insert frag- is seen at the N-terminal end of the MAL61 protein ments were constructed with the fragment cloned into the and thefirst predicted transmembrane domainbegins M 13 sequencing vector mpl8 using exonuclease 111 and at residue 100, suggesting that the N-terminal 100 these were sequenced using the universal primer (MESSING amino acid residues lie on the cytoplasmic face of the 1983; HENIKOFF 1984).Gaps were filled by using oligonucleotide primers identical to known sequences. Nested dele- plasma membrane. The overall secondary structure tionswere also constructed using Ba131 todegrade the of the MAL61 protein thus appears to consist of two MAL61-insert fragment cloned in the plasmid vector blocks of six transmembrane domains separatedby an pBR325. For sequencing, these deletions were subcloned approximately 7 1 residue intracellular region. Both into the M 13 sequencing vectors. Sequencing of the second the 100 N-terminal residues and the 67 C-terminal strand was carried out using the 3.6-kb BglII-Hind111 fragment containing the entire MAL61 gene cloned into the residues are predicted to lie on the cytoplasmic face M13vector mp19.This was sequenced with a series of of the plasma membrane. Although two potential N- oligonucleotide primers complementary to known MAL61 linked glycosylation sites are foundat Asn-15 and sequence. Asn-27, these may notbe modified since they lie Computer analysis: Sequence data were analyzed using within the proposed cytoplasmic N-terminal region. the programs of IntelliGenetics, Inc. of Palo Alto, Califor- nia. Alignment of the MAL61 protein sequence with several This remains to be determinedsince it has been shown other transport proteinsequences was carried out using the that tunicamycin inhibits the synthesis of the maltose GENALIGN program. GENALIGN is a copyrighted soft- transport system inSaccharomyces (LAGUNAS,DEJUAN ware product of IntelliGenetics, Inc.; the program was de- and BENITO1986). Work is now in progress in our veloped by HUGOMARTINEZ of the University of California laboratory to test the predicted membrane topology at San Francisco. The hydropathy plots shown in Figure 4 comparing MAL61 and SNF3 proteins are thegift ofJOHN of the maltose permease. CELENZA,LINDA MARSHALL-CARLSONand MARIAN CARL- Homology of MAL61 protein to other sugar trans- SON of the Department of Genetics and Development, Co- porters: Comparison of the deduced sequence of the Sequence of Maltose Permease 479

I MU61 I

Pv H2 Ps Sc R NANH2 S Pv R H3

I 1 kb i FIGUREI.-Restriction endonuclease map of the MAL61 gene of S. cereuiriae. The restriction endonuclease map of the 3.6-kb DNA fragment containing the MAL61 gene and its flanking sequence is showvn. The abbreviations used are: A. Aval; Rg, Bglll; H2, Hindll; H3. Hindlll; N, Ncol; Ps. PslI; Pv, Pvull; R, EcoRI; Sc, Scal.

-105 AGTACTCAGCATATAAAGAGACACAATATACTCCATACTTGTTGTGAGTGGTTTTAGCGTATTCAGTATAACAATAAGAATTACATCCAAGACTATTAATTAACT

Met LysGly Leu Ser Ser Leu Ile AsnArq Lys Lys Asp Arq Asn Asp ser His Leu ASP Glu Ile GluAsn Gly Val ASn Ala ThrGlu 30 1 ATG AAG GGA TTATCC TCA TTA ATA AAC AGA AAA AAA GAC AGG AACGAC TCA CAC TTA GAT GAG ATC GAG AATGGC GTG AAC GCT ACC GAA PheAsn Ser Ile Glu Met Glu Glu GlnGly Lys Lys Ser Asp Phe Asp leuSer His LeuGlu Tyr Gly Pro Gly Ser Leu Ile PI0 Asn60 91 TTC AACTCG ATA GAG ATG GAG GAG CAAGGT AAG AAA AGTGAT TTT GATCTT TCC CAT CTT GAG TACGGT CCA GGT TCA CTA ATA CCA AAC

AspAsn Asn Glu Glu ValPro Asp Leu Leu Asp Glu Ala Met GlnAsp Ala LYS GluAla ASP Glu SerGlu Arq Gly Met ProLeu Met 90 181 GATAAT AAT GAAGAA GTCCCC GAC CTT CTCGAT GAA GCTATG CAG GAC GCC AAA GAG GCAGAT GAA AGT GAG AGGGGA ATGCCA CTC ATG

Thr Ala LeuLys Thr Tyr Pro Lys Ala AlaAla Trp Ser Leu Leu Val Ser Thr Thr Leu Ile Gln Glu GlyTyr Asp Thr Ala Ile Leu 120 271 ACAGCT TTG AAG ACA TATCCA AAA GCTGCT GCT TGG TCA CTA TTA GTT TCC ACA ACA TTG ATT CAA GAG GGTTAT GAC ACA GCC ATT CTA

Gly Ala PheTyr Ala LeuPro Val PheGln Lys Lys Tyr Gly Ser Leu Asn Ser Asn ThrGly Asp Tyr Glu Ile Ser Val SerTrp Gln 150 361 GGA GCTTTC TAT GCC CTG CCT GTT TTT CAA AAA AAA TATGGT TCT TTGAAT AGC AAT ACA GGA GATTAT GAA ATT TCA GTT TCC TGG CAA

Leu Ile Met Ala LeuPhe Phe Leu Ala Ala Phe Ile Phe Ile LeuTyr Phe Cys Lys SerLeu Gly Met Ile AlaVal Gly Gln Ala Leu210 541 CTGATC ATG GCG TTG TTC TTT TTAGCG GCT TTC ATTTTC ATT CTG TAT TTT TGC AAG AGTTTG GGT ATG ATT GCC GTG GGA CAGGCA Tl'G CysGly Met ProTrp Gly Cys Phe Gln Cys Leu Thr Val Ser Tyr Ala Ser Glu Ile CysPro Leu Ala LeuArq Tyr Tyr LeuThr Thr 240 631 TGT GGTATG CCA TGG GGT TGT TTC CAA TGTTTG ACC GTT TCT TATGCT TCT GAA ATTTGT CCT TTG GCC CTA AGA TACTAT TTG ACG ACT

TyrSer Asn Leu Cys Trp Thr Phe Gly Gln Leu Phe Ala Ala Gly Ile Met LysAsn Ser Gln Asn Lys Tyr Ala Asn Ser Glu Leu Gly 270 721 TATTCT AATTTA TGT TGG ACG TTC GGT CAA CTTTTC GCTGCT GGT ATT ATG AAA AATTCC CAG AAC AAA TATGCC AAC TCA GAA CTA GGA

TyrLys LeuPro Phe Ala Leu Gln Trp Ile Trp Pro Leu Pro Leu AlaVal Gly Ile Phe Leu Ala Pro GluSer Pro Trp Trp Leu Val 300 811 TAT AAG CTACCT TTT GCTTTG CAG TGG ATC TGG CCC CTTCCT TTG GCGGTA GGT ATT TTT TTGGCA CCA GAG TCTCCA TGG TGG CTG GTT

LysLys Gly Arq Ile AspGln Ala Arq Arq Ser Leu Glu Arq Ile LeuSer Gly Lys Gly Pro Glu LysGlu Leu Leu Val Ser Met Glu330 901 AAA AAA GGA AGG ATTGAT CAG GCG AGG AGATCA CTT GAA AGA ATATTA AGT GGT AAA GGA CCC GAG AAA GAA TTACTA GTG AGT ATG GAA

Leu Asp Lys Ile LysThr Thr Ile GluLys Glu Gln Lys Met SerAsp Glu Gly Thr Tyr Trp Asp Cys Val Lys Asp Gly Ile AsnArq 360 991 CTCGAT AAA ATC AAA ACTACT ATA GAA AAG GAG CAGAAA ATG TCT GAT GAA GGA ACTTAC TGG GAT TGT GTG AAA GAT GGT ATT AAC AGG

ArqArq Thr Arq Ile AlaCys Leu Cys Trp Ile Gly Gln CysSer Cys GlyAla Ser Leu Ile Gly Tyr Ser Thr TyrPhe Tyr Glu Lys 390 1081 AGA AGA ACG AGA ATAGCT TGT TTA TGT TGGATC GGT CAA TGC TCC TGT GGT GCA TCA TTA ATT GGT TAT TCA ACT TAC TTT TAT GAA AAA Ala GlyVal Ser Thr Asp Thr Ala PheThr Phe Ser Ile Ile Gln TyrCys Leu Gly Ile AlaAlaThr Phe Val Ser Trp Trp Ala Ser 420 1171 GCTGGT GTT AGC ACT GAT ACG GCT TTT ACT TTC AGTATT ATC CAA TAT TGT CTT GGTATT GCT GCA ACG TTT GTA TCC TGG TGG GCT TCA

LysTyr Cys Gly Arq Phe Asp Leu Tyr AlaPhe Gly Leu Ala Phe Gln Ala Ile Met PhePhe Ile Ile GlyGly Leu Gly Cys Ser Asp 450 1261 AAA TATTGT GGC AGA TTT GAC CTTTAT GCT TTT GGG CTGGCT TTT CAGGCT ATT ATG TTC TTC ATT ATC GGT GGT TTA GGA TGTTCA GAC

Thr His Gly Ala Lys Met GlySer Gly Ala LeuLeu Met ValVal Ala Phe Phe Tyr AsnLeu Gly Ile Ala ProVal Val Phe Cys Leu 480 1351 ACTCAT GGC GCT AAA ATGGGT AGT GGT GCT CTT CTAATG GTT GTC GCG TTC TTT TACAAC CTC GGT ATT GCA CCT GTTGTT TTT TGC TTA

ValSer Glu Met Pro Ser Ser ArqLeu Arq Thr Lys Thr Ile Ile LeuAla Arg Am Ala Tyr Asn Val Ile GlnVal Val Val ThrVal 510 1441 GTG TCT GAAATG CCG TCT TCA AGG CTA AGA ACCAAA ACA ATTATT TTG GCT CGT AAT GCT TACAAT GTG ATC CAA GTT GTA GTT ACA GTT Leu Ile Met Tyr GlnLeu Asn Ser Glu LysTrp Asn Trp Gly Ala LysSer Gly Phe Phe Trp Gly Gly Phe cys Leu Ala Thr Leu Ala 540 1531 -1CAA TTGAAC TCA GAG AAA TGGAAT TGG GGT GCT AAA[TCA GGC TTT TTC TGG GGA GGA TTT TGTCTG GCC ACT TTA GCT

TrpAla Val Val Asp Leu Pro Glu Thr Ala GlyArq Thr Phe Ile Glu Ile AsnGlu Leu Phe Arq Leu Gly Val ProAla Arq Lys Phe 570 1621 TGGGCT GTI' GTCGAT TTA CCA GAA ACCGCT GGC AGG ACT TTT ATT GAG ATAAAT GAA TTG TTT AGA CTT GGTGTT CCA GCA AGA AAG TTC

Lys Ser ThrLys Val AspPro Phe Ala Ala Ala Lys Ala Ala AlaAla Glu Ile Asn ValLys Asp ProLys Glu Asp LeuGlu Thr Ser 600 1711 AAG TCGACT AAA GTCGAC CCT TTT GCAGCT GCC AAA GCAGCA GCT GCA GAA ATT AAT GTT AAA GATCCG AAG GAA GATTTG GAA ACTTCT

G lu Sly Arq Ser Thr Pro Ser Val Ser Pro Thr Val Ser Val Arq AspSly Glu LysVal Asn *** *tt 614 1801 GTGGTA GAT GAA GGG CGAAGC ACC CCA TCTGTT GTG AAC AAA TGATTTTTTTAGCCAGTAGGTAGATCGGCGTTATTTAATTTTATTTTATATAA FIGURE2.-Nucleotide sequence of the MAL61 gene and predicted amino acid sequence of the gene product. The nucleotide sequence of the MAL61 gene is given starting at the upstream Scal site. Nucleotide numbers are on the left with the first base of the initiation codon as nucleotide +I. The amino acid residue numbers are shown to the right. Asterisks indicate the termination codons. Putative 21 residue nlembl.alle-spanningregions are boxed and shaded. The location of these is based on the algorithm of KYTE and DOOLIITLE(1982) using the hydropathy parameters of EISENRERG(1 984).

MAL61 protein to that of the SNF3 protein reveals transporter of Saccharomyces or a component of this an approximate 24% sequence homology (Figure 3). transport system (CELENZA,MARSHALL-CARLSON and The SNF3 geneencodes the high affinity glucose CARLSON1988). More impressive than the sequence 480 and Q. Cheng C. A. Michels

UAL6I DESER-C~PL~~A~-~T~P~-AA~U~LL~SIILI~EGIDIA--I-L~-AF~ALPVF~KKYGS~NSNTG HGTSNP3 :: I- sE----P-PquqsnunsIcvc-v~-v~vcc~L-~c~D~cL-~n---s-t-~snNyv~sn------nE---q P-SSK--KLTCRL~L~V-GCA~LG~L~~G~NTG-~I~AP~K~IEEFY~-~T~~~-----R LAC12 5: IaD A R E E v L L p c uOsiq y YBL y c 1. c F I T y L c A T na- 6 y 11 G ,\ L - - - ugs s -m-T E D A y LmyHa- - - - - XYLE I ””” n--NOquNssOIFsITLv---ATLccLt.FGynT-Avt sGTv--EsIN--T-v-----Fv ARAE It ------I~TPR~LRDTRR~~~~~~~--*IAIAGLL?~LD-I~~~~~AG--AL~F~------En TDHFV UAL6I 142 ~Y~l~VS u~~c~c~cr~~ca~vc~qv~ ~~DY~GNRYTLI~A-LF-FL------~---~ HCTSNF3 131 VAPNHD~~~~~~~~SI~VSFLSLGIFFG-AL~~ISDSYGRKPTIlFSTlPlP-----S--lG-NSL 52 YC~~ILPTTLTTL~SLS~AIF~~GG~IG-~~~~GLF~~RF~RR~~~L~~~LLAF---~-~-~-~L~GF LACI~ 109 ~-IN~S~GT-GLVFSI---F-SVC~ICGAPFYP-L~~YKC-RKPAI------I.IGCLGV~~C-~I~ XYLEARAE 4456 AP~NLSESAANSLLGFCVASALIGC~IG~GAL~YCSSRFCRRDSI.~I~AVLFFISG~‘GS-~\UPEI.GF“”L-Ts---RL~Euvvssn~LGIIIG-*LIHGYLSFRLc~~ysLn~c~~LFvLcsxcs-~----~~ o o wv-g HAL61HGT 193 F IO? P CaLG W I - A V G ------q~~c~n~u~c~qc~~~~~~~~c~~~~~~~~~s~cu~~c SNF3 189 ~VGAG-CITLLI--VC---R----VI-SGlGlGAISAVVPLI~~EATllKSLRGAIlSI?~UAITUG-L 113 ~SKLGKSFE~LI-L-C~--~I”--TI-GITCGLITGYYP~Y~GE~~PTAFRGA~G~LH LGIVVCIL LAC12 160 SS~TTT~-ALI~-GC~~-R-----UFVAFFA~lANAAAPTY~AEVAPAlll.RGK~’A~~LY~LYS\’G-S XYLE l10 TS-INPI~STYPVYLACYVPEFVIYRIIC IGVCLASU~SPUYIAELAPAlllRGK~VSFN~FAllFG ARAE tlo rs-----veuL~-~-ARv---v-----~~~~v~~~sy~~pL~LsEuAsENvncuuxsn~q~uv~LG--oyl UAL61 252 - FOGI n K NW--mA - N S E L G Y K L P F U~P~~I~APESPUWLVKKGRIDARRS-LEm SNF3 245 LVSSAV---S CTHARNDaS--YRIPI z HCTLAC12 ;iz - IA~~--F---GLDSI~CRKDL~PLLL$I~~IPALL~~I~LPF~P~~PRFLLI~R~EE~RAK~~L-~?vussFL*1cnFsLPrspRyYvL~L~uO-O-s IVAAFSTYC-T~--~NFP~~KAFKI~LYL-a ~X~PCLYCIFGV~IPE~PRVLVGVGREEEARE~IIKY XYLE 177 L-~~YCVNYFIARSG~AS~L~TD~~RY~FA~~~~P~~I.LFL~LLYT~PE~PR~L~~RGK~EAEGI-L-R ARAE 161 -I~LAFLSDTAFS--~S-CN---~RA~L~~LALPA~LLIIL~~FLP~~PR~LA~KGR~I~AEE~-L-Rm%QEXD on UAL61 315 ILSGKCPEK~L~SUELDKIK-TTIEKEQOMSDEOTYWDCVK o-----”G~N~RR~R~~~~~~q~~; SNF3 306 FLRG-V~VHDSGLLEELVEIKATYDYEASFCSSNFINCFISSKSRPKq-TL-R~FTGIA HGT 230 KLRG---TAD--~THDL~E~KEE~R~~~RE~K~TILELFR~PAYR--~-P~-L---IA~~L~L~q~s LACIZ 281 HLNC-DRTH-PLLD~E~AEIIE~F~GTDL~~PLE~L~-~R~-LFRTR~~RY-~A~L~~L~A~-F~~F~[I XYLE 242 KI~C-----NTLAT~AV~EIK-HSLDHGR-~og $3gn- 0TG~RL------L~FGVCVI\~--I~\~~ISIF~~~F\’ ARAE220 nO---RDTsOuAREELNE-I-nEsL-----OII--LKqccuALFKxNRNv-n~AvFLcnL~qAnqq!! MAL61SNP3 ;E ~::::.:::~f‘,;:rr~:8::;r::f~~E::::~:~4E:’:::::~’rl::~:::~::~~:.::~;~I~::::p HCT 286 CINAVF1YSTSIF--EKACV--~qPVIATIGSGIVNT~F~V~LFVVERACRRT~HLIG-~GUA--- LAC12 343 GNNVCSYY-LPTULR~VCUKSVSLNVL~Sl:\~YSl~‘TUlSSl~~~AFFlDKlGRRE~:FL-GSlSG-AALAW XYLE 292 GINVVLYYAPEVFKTLC~--STDI~LL~T~VGVISLTFVLAI~T~DKFCRKP~~IIGA~-G~A--- *RAE 273cnmI~n~YAPRIFKnAC-FT~~~qqn~~~LvvcL~pn~~x~v~~vDu~cnupALK~cpsv-n~--- F!% OF MAL61 441 FllGG~CsD~-8GA~-UGSGALLHVVAPPYNL~P~V~CLVSE~PSSRLR~-~--IILA---RN n HGTsw3 429 I~~nFxv~x--vCcsL-uT~~uvn~~Fx~~Fs~Iuccvvuv~sAEL~pLc\~nsKc~A~c~~~ 346 -GCAI~UTIALA--LLEQLPUYSYLSIV IPGPVAFFE~GPGPIPU?IVAELFSqGPRPAAlAVAGFSR LAC12 408 LTCLSl---COARYEOTKKKSASNGAL\,~lYLFGGlFSFA~TPSqS~YSTEVS~NL~RSK--A--qLLAF--YTQAPCIVALLSU~-VA~~SU~PVCUVLLSEIFPSAIRGKALAIAV~A~W I] XYLE 354 I C U F SUT- - - ARAE 336 L~TL~LG~~L~~FDN~TA~~GL~~L~~G~T~~~IAGYA~~AAP~~~IL~~EI~PL~~RDFGIT~~TIT8 MAL61 500 AY~VI~~-V~,VLI~I~LNSEKU~WGAKSGPFUGG~CL~ILAUAVVDLPETAGRTFIEINELFRLGVP HGTSNF3 494411 RYL~NFICALITPIIVDTG~HTSSLCAKlPPlUGSL~A~CVlVV?LIVYETKG~TLEElD~--YtKSN~TSWFIVG~CP~Y-~-----~~LCCPIIPIIPTVLLILP~IFT~~K~~ETKGRTFDEI---A~GFR~Pm LAC12469 N-F~VSGVA-~FVN~FATPKAUK~IKYUFY\~~YVF~DIFEFI\~IYFFF\’ETKGRSLEEL-EVVFEAPS XYLE 417 ~LAIYFVS~TFP~~DKNS~LVAHFHSGF~~~IY~C~GVLAALF~~KFV~ETKGKTLEEL-E------ARAE 404 ~UVSN~lICA~P----LTLLDSlGAAGIF-ULYTAL~I~FVGlIFULI~ETKSVTLEllt-E------CP MAL61 567 ~FKST~PFFA~KAAAAEI~VK... bm SNF3 560 STCVVS-- PKFUKDIRERA-LKFQ ... HGT 470 CGASqSDKTPEELFHPLGADSPV LAC1 2 534 &?---LUEPETKKTqqTATLPRK-AS-~qAPLOqVRATLVqRU.. . XYLE 476 0 ARAE 459 -RK---LUA-GEKLRNI--GI0 FIGURE3.-Sequence and structural homology among MAL61 protein and othersugar transporters. Amino acid sequences of the MAL61 protein, the high affinity glucose transporter of Saccharomyces encoded by SNF3 gene (CELENZA,MARSHALL-CARLSON and CARLSON 1988). the lactose permease of K. lactis encoded by the LAC12 gene (CHANGand DICKSON1988). human HepG2 glucose transporter (MUECKLERet al. 1985) and the Escherichia coli xylose and arabinose transporter (MAIDENet al. 1987) are shown using standard single-letter amino acid symbols. The proteins are aligned so as to maximize identity to the MAL61 protein sequence. Gaps (indicated by dashes) are introduced to optimize the alignment. Identities with the MAL61 protein are boxed. Shaded regions indicate the putative transmembrane regions in the MAL6I. SNFS (CELENZA,MARSHALL-CARLSON and CARLSON 1988), human HepG2 (MUECKLERet al. 1985), and AraE (MAIDENet al. 1987) protein sequences. Amino acids are numbered on the left.

homology is the structural homology between these 1987;CELENZA, MARSHALL-CARLSON and CARLSON two proteins. Figure 4 depicts the hydropathy plots of 1988; CHANC and DICKSON1988). Homologous se- both MAL6 1 and SNFS proteins. One clearly sees the quences are seen in bothhydrophobic membrane twelve proposed transmembrane domains organized spanning domains as well as in hydrophilic regions. into two blocks of six each. The spatial distribution of The proposed secondary structure of thesesugar these domains is so similar that the plots are nearly transporters is also remarkably similar and this is perfectly superimposable. Both proteins contain an illustrated in Figure 3. Comparison of the MAL61 hydrophilic N-terminal domain of similar size. While protein to the E. coli lactose permease and to the yeast the MAL61 protein, like the SNFS protein, contains plasma membraneATPase (encoded by the PMAl an hydrophilic C-terminal domain, this domain is sig- gene) reveals little, if any, sequence homology even nificantly smaller in the MAL61 protein. though all are proton transporters (SERRANO 1977; Equivalent sequence andstructural homology is KABACK1983; SERRANO,KIELLAND-BRANDT and FINK 1986). seen to other sugartransport proteins from other species. Figure 3 aligns the amino acid sequence of the MAL61 protein with those of the SNFS protein, DISCUSSION the human HepG2 glucose transporter, the Kluyvero- The results presented here offer additional strong myces lactis lactose permease encoded by the LAC12 evidence that the MAL61 gene encodes the maltose gene, and the Escherichia coli xylose and arabinose permease. Transcription of the MAL61 gene is malt- transporters (MUECKLERet al. 1985; MAIDEN et al. ose induced and glucose repressed (NEEDLEMANet al. Sequence of Maltose Permease 48 1

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-0.50 c, -4 U -0.75 -rl A 1.00 2 I I I I I 1 I I a 100 200 300 400 600 500 700 800 0 k 0.75 a h SN F3 0.50

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-0.75.

-1.00, I I I I I I I I 100 200 300 400 500 600 700 800 Residue number FIGURE4,"IIydrophobicity profile of the predicted MAL61 protein and SNF3 proteins. The profiles were determined as described in MATERIALS AND METHODS and in Figure 2.

1984). Genetic evidence indicates that MAL61 is re- residues in length andsuch structure is consistent with quired for maltose transport in MAL6 strains (Y. S. that of anintegral membrane protein (KYTE and CHANG,R. A. DUBIN, E. PERKINS,C. A. MICHELSand DOOLITTLE1982; EISENBERC1984). In anotherstudy R. B. NEEDLEMAN,unpublished results). Our results (tobe reported elsewhere), analysis ofa series of clearly demonstrate that the MAL61 protein is ho- MAL61-PhoA fusions selected in E. coli by transposition mologous both in sequence andin secondary structure of a Tn5-derivative carrying a truncated copy of the to other sugar transporters, particularly the Saccha- E. coli phoA gene into a plasmid carrying the MAL61 romyces high affinity glucose transporter encoded by gene also supports the localization of the MAL61 the SNF3 gene. While the results presented here do protein to theplasma membrane (MANOIL,BOYD and not demonstrate that the MAL61 protein is a plasma BECKWITH1988; C. A. MICHELSand L. SEECCOOMER, membrane protein, they support thisconclusion. The unpublished results). E. coli strains carrying these fu- primarysequence of the MAL61 protein exhibits sion plasmids express alkaline phosphatase activity in twelve highly hydrophobic regions approximately2 1- whole cells and preliminarymapping localizes the 482 Q. Cheng and C. A. Michels fusion junction sites to theregion of the second group (reviewed in HOLZER1984; JONES 1984; ACHSTETTER of six transmembrane domains. and WOLF 1985). These include enzymes of the glu- The homology demonstrated here among the Sac- coneogenic pathway (fructose-l,6-bisphosphatase,cy- charomyces maltose permease, the Saccharomyces toplasmic malate dehydrogenase and phosphoenolpy- high affinity glucose transporter, the human HepG2 ruvate carboxykinase), aminopeptidase I, uridine nu- glucose transporter, the K. lactis lactose permease and cleosidase, the high affinity glucose transporter (SNFS the E. coli xylose and arabinosetransporters is re- protein) and galactose permease (WITT, KRONAUand markable. These proteins appear to be members of a HOLYER 1966;FERGUSON, BOLL and HOLYER1967; family ofrelated sugar transporters,even though their GANCEW 197 1;HAARASILTA and OURA 1975; MA- mechanisms of transport differ.Some are active trans- TERN and HOLZER 1977;MACNI et al. 1977; FREY and porters utilizing proton symport and others function ROHM 1979; BISSONand FRAENKEL1984; RAMOS, by facilitated diffusion. The Saccharomyces galactose SZKUTNICKAand CIRILLO1988). Maltose uptake is transporter, which transports by facilitated diffusion, almost completely inactivated within 90 minutes fol- also is reported to be a memberof this family of sugar lowing the addition of glucose to the culture medium transporters (SZKUTNICKAet al. 1989). An evolution- (GORTS 1969; BUSTURIAand LAGUNAS1985). The ary relationship is strongly implied among all of these most recent results indicate that this deadaptation proteins. However, homology resulting from a com- inactivates both the high and low affinity uptake sys- mon ancestry is difficult to distinguish from conver- tems butearlier studies reportthat only the high gentevolution. Sequence convergence could result affinity transport is affected. Glucose specifically ini- fromthe fact that all are sugartransporters with tiates the inactivation since the transfer of a maltose similar functional constraints placed upon their struc- fermenting culture to a noninducing medium contain- tures. ing ethanol does notlead to the rapid loss of maltose Transmembrane domains 1, 2, 4, 5, 7, 8 and 1 1of transport activity. Recovery from glucose inhibition MAL61 protein contain several polar and negatively requires thatthe cells be returned to inducing medium charged residues (serine, threonine, asparagine, glu- and recovery does not occurif de novo protein synthe- tamine, aspartate and glutamate). Particularly note- sisis inhibited. These results imply that glucose-in- worthy is domain 1, which contains seven polar resi- duced inactivation irreversibly destroys the maltose dues (4 threonine, 2 serine,1 glutamine) and two transport protein. charged residues (1 aspartate, 1 glutamate) and has The mechanism of this irreversible inactivation is an average hydrophobicity value that justexceeds the unknown. Studies on aminopeptidase I and the glu- 0.42 minimum proposed by EISENBERG(1984). coneogenic enzymes fructose- 1,6-bisphosphatase, cy- Graphic analysis of the membrane-spanning domains toplasmic malate dehydrogenase and phosphoenolpy- indicates that the polar and charged residues con- ruvate carboxykinase from Saccharomyces indicate tained in these domains would largely be localized to that the glucose-induced inactivation of these enzyme the same face of the proposed alpha-helical structure activities is paralleled by a decrease in the amount of in the case of domains 2, 4, 5, 8 and 1 1. MUECKLERet cross-reacting material suggesting that inactivation re- al. (1985) suggest that the hydroxyl and amide side sults fromtheir selective proteolysis (NEEFF et al. chains in an amphipathic a-helix could line a trans- 1978; FREY and ROHM 1979;FUNA-YAMA, GAN- membrane channel and function in the transport of CEDO and GANCEDO1980; MULLER,MULLER and the sugar. A similar structural organization may exist HOLZER 1981;TORTORA et al. 1981). While maltose in the maltose permease. Additionally, since the trans- permease is an integral membrane protein and these port of maltose in Saccharomyces is a proton symport enzymes are cytosolic proteins, it is nevertheless system, it is possible that the chargedresidues located tempting to propose that the irreversibleinactivation in the transmembrane domainsof the MAL61 protein of maltose permease is the result of proteolytic deg- function in proton transport in a fashion similar to radation. In surveya of several proteins from different that seen in the lactose permease of E. coli (HERZLIN- eukaryotic organisms, RECHSTEINERand coworkers GER, CARRASCOand KABACK 1985; CARRASCOet al. have found a correlation between short half-life and 1986; SERRANO 1977). the presence of sequences rich in proline, glutamate, The addition of glucose to maltose-induced fer- serine and threonine (so-called PEST-regions) which menting cultures not only leads to the cessation of they propose target proteins for proteolysis (ROGERS, synthesis of maltose permease but also leads to the WELLand RECHSTEINER1986; RECHSTEINER1987; loss of any existing maltose permease activity by an as RECHSTEINER,ROGERS and ROTE 1987). A search of yet undefined process referred to as glucose-induced the MAL61 and SNFS proteins reveals potential inactivation (GORTS 1969; BUSTURIAand LAGUNAS PEST sequences located in the N-terminal cytoplasmic 1985). Glucose-induced inactivation also affects the regions. These are found residues at 49-78 of MAL61 activity of several other enzymes in Saccharomyces protein (score of 0.64) and at residues 1-13 (score of Sequence of Maltose Permease 483

7.85) and 63-91 (score of 1.66) ofSNFS protein. CHANG,Y. S., R. A. DUBIN,E. PERKINS,D. FORREST,C. A. MICHELS ROGERS,WELL and RECHSTEINER(1986) also suggest and R. B. NEEDLEMAN,1988 MAL63 codes fora positive regulator of maltose fermentation in Saccharomyces cerevisiae. that a protein containing a region with a low positive Curr. Genet. 14: 201-209. PEST score could besubject to degradation only CHARRON,M. J., R. A. DUBINand C. A. MICHELS,1986 Structural under certain physiological conditions, such as when and functional analysis of the MALI locus of Saccharomyaes that region was phosphorylated, thereby increasing cereuisiae. Mol. Cell. Biol. 6: 3891-3899. the negative charge of the region. In such cases, the CHARRON,M. J., and C. A. MICHELS,1987 The constitutive, glucose repression insensitive mutation present at the MAL4 degradation of a protein could be regulated, as is seen locus of Saccharomyces maps to MAL43. Genetics 116 23-3 1. in glucose-induced inactivation of the gluconeogenic CHARRON,M. J., and C. A. MICHELS, 1988 The naturally occur- enzymes. It is worth noting that the N-terminal pep- ring mutant alleles of MALI in Saccharomyces species evolved tide ofSaccharomyces fructose-1,6-bisphosphatase by various mutagenic processes including chromosomal re- contains a PEST-region (score of 1.7). Whether or arrangement. Genetics 120 83-93. CHARRON,M. J., E. READ, S. R. HAUT and C. A. MICHELS, not PEST regions are relevant in the Saccharomyces 1989 Molecular evolution of the telomere-associated MAL system remains to be determined, but the possibility loci of Saccharomyces. Genetics 122: 307-3 16. thata commonmechanism exists for the glucose- DE KROON,R. A., and V. V. KONINCSBERGER,1970 An inducible induced inactivationof all of these proteins even transport system for alpha-glucosides in protoplasts of Saccha- though they are located in different cellular compart- romyces carlsbergensis. Biochim. Biophys. Acta 204: 590-609. ments and lack any obvioussequence similarities is an DUBIN,R. A., R. B. NEEDLEMAN,D. GOSSETT and C. A. MICHELS, 1985 Identification of the structural gene encoding maltase interesting one to pursue. within the MAL6 locus of Saccharomyces carlsbergensis. J. Bac- teriol. 164 605-610. The authors wish to thank JOHN CELENZA,LINDA MARSHALL- EISENBERG,D., 1984 Three-dimensional structure of membrane CARLSONand CARLSONfor providing the hydropathy plots MARIAN and surface proteins. Annu. Rev. Biochem. 53: 595-623. of MAL61 and SNFS protein shownin Figure 4, and MARTIN FEDEROFF,H. J., J. D. COHEN,T. R. ECCLESHALL,R. B. NEEDLEMAN, RECHSTEINERfor providing the program used to perform the PEST- B. A.B. BUCHFERER,J. GIACALONEand J. MARMUR, search analysis. The authors areindebted to HOWARDSHUMAN for 1982 Isolation of a maltase structural gene from Saccharomy- many hours of enlightened discussion. They also wish to thank ces carlsbergensis. J. Bacteriol. 149 1064-1070. EVANREAD for help in the preparation of the figures. FERCUSON,J. J., M. BOLLand H. HOLYER,1967 Yeast malate This work was supported by a National Science Foundation dehydrogenase: enzyme inactivation in catabolite repression. Visiting Professorship for Women Award (RII-8700006) to Eur. J. Biochem. 1: 21-25. C. A. M., by a National Institute of Health Research Award FREY,J., and K. H. ROHM,1979 The glucose-induced inactivation (GM28216) to C. A. M. and by a Board of Higher Education- Professional Staff Congress Research Award. of aminopeptidase I in Saccharomyces cereuisiae. FEBS Lett. 100: 261-264. FUNAYAMA,S.,J. GANCEDOand C. GANCEDO,1980 Turnover of LITERATURE CITED yeast fructose bisphosphatase in different metabolic conditions. Eur. J. Biochem. 109: 61-66. ACHSTETTER,T., and D. H. WOLF,1985 Proteinases, proteolysis GANCEDO,C., 1971 Inactivation of fructose 1,6-diphosphatase by and biological control in the yeast Saccharomyces cerevisiae. 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KABACK, 1986 Lac permease of Escherichia coli: histidine-322 and glu- 1985 Functional and immunochemical characterization of a tamic acid -325 may be components of a charge relay system. mutant of Escherichia coli energy uncoupled for lactose trans- Biochemistry 250 4486-4488. port. Biochemistry 24 221-229. CELENZA,J. L., L. MARSHALL-CARLSONand M. CARLSON, HOLZER,H., 1984 Mechanism and function of reversible phos- 1988 The yeast SNF3 gene encodes a glucose transporter phorylation of fructose-l,6-bisphosphatasein yeast, pp. 143- homologous to the mammalian protein. Proc. Natl. Acad. Sci. 154 in Enzyme Regulation by Reversible Phosphorylation-Further USA 85: 2 130-2 134. Advances, edited by P. Cohen. Elsevier, Amsterdam. CHANG,Y. D., and R. C. DICKSON,1988 Primary structure of the JONES,E. W., 1984 The synthesis and function of proteases in lactose permease gene from the yeast Kluyueromyces lactis. J. Saccharomyces: genetic approaches. Annu. Rev. Genet. 18: 233- Biol. 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