T H E JOURNAL 0 1988 by The American Society for Biochemistry and Molecular Biology. Inc.

OF BIOLOGICAL C H E M I S T R Y Vol. 263, No. 32, Iesue of November 15, pp. 16696-16703.1988 Printed in U.S.A.

Primary Structure of the Lactose Permease Gene from the Yeast Kluyveromyces lactis PRESENCE OF AN UNUSUAL TRANSCRIPT STRUCTURE*

(Received for publication, August 11, 1987)

Yuan-Di Chang and Robert C. Dickson From the Department of Biochemistry and the Lucille Parker Markey Cancer Center, University of Kentucky, Lexington, Kentucky 40536

The LAC12 gene of Kluyveromyces lactis codes for an inducible lactose permease. We have determined the nucleotide sequence of a DNA fragment which includes the complete LAC12 gene. The 4.7-kilobase (kb) mRNA carrying LAC12 contained two open reading frames, ORFI (1761 bases) and ORFII (1266 bases), separated by a 673-base pair noncoding region. Mung bean and exonuclease VI1 mapping showed that there was no splicing of the 4.7-kb transcript and thus no intron between the two open reading frames. Chro- mosomal disruption of ORFI with the URA3 gene de- stroyed lactose transport activity, suggesting that ORFI codes for a component of the permease. Disrup- tion of ORFII and the noncoding region between the two open reading frames did not affect the lactose permease function, indicating that they do not com- prise a part of the permease. We do not know if ORFII is translated, but in either case, the structure of the 4.7-kb mRNA is unusual. We discuss possible origins for it. The peptide predicted from ORFI is hydrophobic as would be expected for a membrane-bound protein. Compared with other membrane proteins, LAC12 (ORFI) protein showed sequence similarity to the hu- man glucose and the Escherichia coli xylose-H+ and arabinose-€I+ transporters. No obvious amino acid se- quence similarity was found with the lactose permease of E. coli.

Many procaryotic and eucaryotic organisms are able to selectively internalize sugars from their environment using membrane-bound transport proteins or permeases. Kluyver- omyces luctis is one of the few yeasts that can transport lactose across its cell membrane and subsequently use the sugar as a carbon and energy source. Lactose is transported by the product of LAC12 (1,2). Cells grown in the absence of lactose or galactose transport lactose very poorly; however, transport

* This work was supported by National Institutes of Health Grant GM22749 and by Biomedical Research Support Grant RR05374 from the Biomedical Research Support Branch, Division of Research Fa- cilities and Resources, National Institutes of Health. Computer re- sources used to carry out these studies were provided by the BIONET National Computer Resource for Molecular Biology, whose funding is provided by the Biochemical Research Technology Program, Divi- sion of Research Resources, National Institutes of Health, Grant 1U41RR-01685-02. The costs of publication of this article were de- frayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The nucleotide sequence(s) reported in this paper has been submitted

X06997. to the GenBankTM/EMBL Data Bank with accession number0

activity is induced to a high level by either sugar (3). Induction of transport activity is governed at the level of transcription (2), although other forms of regulation including activation- inactivation of the permease have not been ruled out. The LAC12 permease requires energy for activity and transports lactose against a concentration gradient (3). Since transport is inhibited by the proton ionophore 2,4-dinitrophenol, it would appear that the permease works in part by a proton symport mechanism, but this remains to be substantiated directly. Direct phosphorylation of lactose is not involved in the transport process (3). Lactose is hydrolyzed to glucose and galactose by an intracellular @-galactosidase, the LAC4 protein (4).

One of the most well characterized sugar transporters is the lactose permease of Escherichia coli, the product of the lacy gene. This intrinsic membrane protein belongs to a group of transporters that use a cation electrochemical gradient to cotransport a substrate across the membrane (5). The cation in this case is H+, and the substrates are @-galactosides. Recent studies using site-directed mutagenesis techniques to change specific amino acids have implicated several residues that are essential for activity; these include: Ala-177, Tyr-236 (6); Cys-154 (7,8); His-205 and His-322 (9,lO); Glu 325 (11); Gly-24 (12); Arg-302 (13). Some of these residues appear to form the substrate recognition site while others may couple H+ and substrate translocation (14).

To provide a basis for a detailed mechanistic study of the K. luctis lactose permease, we have determined the nucleotide sequence of LAC12. The predicted protein product of the gene shows similarity to several other sugar transporters, but sur- prisingly, shows no apparent similarity to the E. coli lactose permease.

EXPERIMENTAL PROCEDURES AND RESULTS’

DISCUSSION

Previous data demonstrated that the region between nucle- otides -1191 and +5512 (Fig. 2) would confer on Saccharo- myces cerevisiae the ability to transport lactose (1). Other data demonstrated that this region of DNA encoded a 4.7-kb’ mRNA transcript (2). Also, disruption of the chromosomal LAC12 region at the XbaI site shown in Fig. 1 with the URA3 gene caused a loss of lactose transport activity, suggesting

Portions of this paper (including “Experimental Procedures,” “Results,” and Figs. 1-8) are presented in miniprint at the end of this paper. Miniprint is easily read with the aid of a standard magnifying glass. Full size photocopies are included in the microfilm edition of the Journal that is available from Waverly Press.

The abbreviations used are: kb, kilobase(s); ORF, open reading frame; UAS, upstream activating sequence(s).

16696

Lactose Permease of Kluyveromyces lactis 16697

that the XbaI site is in the coding region of the lactose permease (2). Based upon these results, we expected that there would be one large open reading frame that would be slightly smaller than the 4.7-kb mRNA. However, analysis of the DNA sequence showed that there was no large ORF that would correspond to the 4.7-kb mRNA (Fig. 1C). Instead, there were two smaller ORFs that could code for proteins of 587 amino acids (ORFI) and 422 amino acids (ORFII). The presence of two small ORFs is unlikely to be an artifact of DNA sequencing since both strands of DNA were sequenced and many sequences were verified using both the Klenow fragment of DNA polymerase I and avian myeloblastosis virus reverse transcriptase.

To determine if ORFI and ORFII were exons separated by an intron, we looked for splicing of mRNA using a mung bean nuclease and an exonuclease VI1 assay. As the data in Fig. 5 demonstrated, the same sized fragment of the radiolabeled probe was protected from digestion by either nuclease. Thus, the 4.7-kb transcript is contiguous to the probe, and there are no introns present in this region of the genome. This conclu- sion was verified using Northern blot analysis. DNA frag- ments corresponding to part of ORFI, ORFII, and the region between them were used as hybridization probes against poly(A+) RNA isolated from K. lactis. All three probes hybrid- ized to the same sized mRNA, 4.7 kb (Fig. 6). Thus, the region of DNA between the two ORFs is not spliced out of the 4.7- kb transcript.

Even though both ORFI and ORFII are present on the same mRNA, our gene disruption experiments indicate that only ORFI is coding for a component of the lactose permease. For example, disruption of ORFI at the XbaI site with URA3 creates a Lac-Gal- phenotype, while a deletion-disruption of ORFII at the BstXI sites has no effect on the lactose and galactose phenotypes. An alternative explanation would argue that there is another copy of ORFII elsewhere in the genome so that disruption of the copy in the LAC12 region has no phenotypic affect. Consequently, disruption of only one copy of ORFII would not allow one to determine if it was necessary for lactose transport. By Southern blot analysis (Fig. 4 and data not shown) there appears to be only one copy of ORFII in K. lactis, the one downstream of ORFI.

If ORFII is translated, then the presence of two nonover- lapping coding regions on a single mRNA (a polycistronic mRNA) is unique in eucaryotes, although there are cases where a mRNA codes for more than one protein. For example, the yeast Ty element Ty-912 contains two open reading frames in one mRNA (30). The two ORFs of Ty-912 overlap and are translated by a frame-shifting mechanism. The prod- uct of the second ORF is a fusion protein consisting of the product of the first and second ORFs. Since ORFI and ORFII of LAC12 do not overlap and are separated by 573 base pairs, it is unlikely that the two ORFs are translated by a frame- shifting mechanism. The yeast tRNA ligase gene also has an unusual structure (31). There are three ORFs in the gene other than the coding region. Although the ORFs are ex- tremely close together, evidence suggests that they are tran- scribed as separate mRNAs. Many eucaryotic viruses and mammalian neurotransmitter receptor genes have bifunc- tional mRNAs (32, 33). In most of these mRNAs, the 5’- ORFs are very short and either code for a leader protein or they have noncoding regulatory functions. The LAC12 tran- script does not seem to fall into this category since ORFI and ORFII are both long and the 5’-ORF is ORFI which codes for a large protein.

The unusual structure of the LAC12 gene could have arisen by fusion of two genes either by gene translocation or chro-

mosomal deletion. Such an event might have deleted the transcriptional termination region of the lactose permease gene so that a long mRNA containing both the permease coding region and ORFII gene would be made. The noncoding region between ORFI and ORFII contains an A-rich region (nucleotides 2266-2272) and a TATA box (nucleotides 2239- 2244). Those sequences are common cis-elements of yeast promoters, which suggest that ORFII had its own promoter at one time. There is a potential polyadenylation signal, AATAAA (position 1879), that may have been used at one time for ORFI. The region between the two ORFs also con- tains a nonperfect UAS sequence 5’-CGG(N7)CCG (nucleo- tides 2012-2024) which might have been used as a UAS by the LAC9 protein. It is not likely to be used that way now because the normal UAS has the sequence 5’-CGG(N11)CCG (34, 35). The ability of ORFII to be expressed is currently under investigation. If ORFII did originate from a once func- tional gene but is now not expressed, it may be retained because it gives a selective advantage to the LAC12 mRNA such as greater stability.

Another possible origin of ORFII is that the noncoding region is a nonfunctional intron resulting from mutation of a splicing junction. We consider this less likely because the consensus sequence of the 5”junction site was not found at the 5‘-end of the ORFI coding region and the size of the predicted lactose permease is comparable to other eucaryotic sugar transporters (400-500 amino acids long).

Many S. cerevisiue genes have an A-rich region on the 5’- side of the initiation codon (36-38). LAC12 has an A-rich region at nucleotides -21 to -34 and is thus similar in this regard to LAC4 (34, 35) and to genes of S. cereuisiue. It has been hypothesized that such A-rich regions govern basal level gene expression (39) and that they are devoid of nucleosomes (40, 41). Yeast genes typically have a TATAAA sequence between 60 and 120 nucleotides upstream of the transcription start site (42). These sequences are thought to be necessary for proper transcription initiation. LAC12 has such a se- quence, TATAAA, at nucleotides -217 to -212, which is 92 nucleotides upstream of the -120 transcript end point. Mu- tational analysis will be necessary to determine if the -217 to -212 sequence determines the transcription start sites.

LAC12 transcription (2,3), like other genes in the lactose- galactose regulon, is inducible by lactose. It is thought that induction is mediated by binding of the LAC9 positive regu- latory protein (15, 43) to 17-base pair UASc which are found at various distances in front of genes in the regulon (34, 35). A computer search of the LAC12 DNA sequence for similarity to a core UAS consisting of 5’-CGG(N11)CCG identified the two putative UAS shown upstream of LAC12 at -345 to -329 and -625 to -609 (Fig. 2).

The AUG initiation codon shown for ORFI (Fig. 2, nucle- otides +1 to +3) is the first AUG codon in the mRNA, and for this reason we believe that it is the true initiation codon. However, there is a second in-frame ATG codon at nucleotides +253 to +255 which is 85 codons downstream from the first methionine codon. Although it is less likely, the possibility that the second ATG of ORFI is the initiation codon cannot be excluded based upon current information.

Taking the first ATG codon to be the initiation site, the product of ORFI is a 587-amino acid protein with a predicted molecular weight of 65,384. The hydropathicity plot of this protein (Fig. 8) shows several hydrophobic regions as would be expected for a membrane-bound protein. There is not, however, a hydrophobic region at the N terminus to function as a signal peptide. The first 45 amino acid residues at the N terminus form the most hydrophilic region of the protein.

16698 Lactose Permease of Kluyveromyces luctis

Instead of having the signal peptide at the N terminus, the lactose permease may have an internal signal peptide. There are several membrane proteins that do not have a signal sequence at the N terminus but are still able to be inserted into a membrane. For example, the arginine permease se- quence deduced from the CANl gene of S. cereuisiae lacks an N-terminal hydrophobic sequence (44). It was shown that the secretory pathway of the CANl gene product is blocked in secretory mutants of S. cereuisiue, which suggested that trans- port proteins with or without signal sequence may share a common secretory mechanism (45). A similar result was re- ported for the GAL2 gene which codes for a galactose permease in S. cereuisiae (46) and for the E. coli melibiose carrier (47). Alternatively, if translation of LAC12 actually started at the second in-frame ATG codon, the gene product would be 84 amino acids shorter and have a hydrophobic domain at the N terminus that could serve as a signal sequence.

We compared the protein sequence deduced from ORFI with other membrane proteins of eucaryotes and bacteria. The three proteins with the highest similarity were the human glucose transporter (48), and the E. coli xylose-H+ and ara- binose-H+ transport proteins (49). There was significantly less similarity between the LAC12 permease and other mem- brane proteins including the lactose permease of E. coli (50) and Klebsiella pneumoniae (51). Little similarity was found between the LAC12 protein and amino acid permeases such as the histidine permease in Salmonella typhimurium (52,53), histidine permease of S. cereuisiae (54), the arginine permease of S. cereuisiae (44), and the leucine binding protein of E, coli (55).

The amino acid sequence comparison of ORFI (LAC12) with the glucose, xylose, arabinose, and lactose transporters is shown in a dot matrix form in Fig. 9. A more detailed

analysis of the regions of similarity is described below. A dot matrix of ORFI against itself showed no extensive repeat sequences (data not shown). Due to the similar function of the LAC12 protein and the lacy protein, homology was ex- pected to be found between them. We did not, however, see any strong homology in either the DNA or protein sequences by computer analysis. In Fig. 9D, the 40 amino acids at the N terminus of the lacy protein showed some similarity to amino acids 50-90 and amino acids 480-530 of the LAC12 protein. These similarities are due to similar amino acids rather than to identical amino acid residues, since the dot matrix com- parison scored similar amino acids as identities (56).

The hydrophobicity profile of ORFI was very similar to those for the glucose, xylose, and arabinose transporters (49) with five to six hydrophobic domains being located on both sides of a central hydrophilic domain. Maiden et al. (49) identified a motif RXGRR (where R may be replaced by K) that is duplicated in the glucose, xylose, and arabinose trans- porters as well as in the E. coli citrate, lactose, and melibiose transporters. Each of the duplicated motifs occurs in a hydro- philic region that is bordered on each side by a hydrophobic region. This motif, marked X in Figs. 7 and 9, is also present in the LAC12 permease, although the copy at amino acid residues 140-143, K-GRK, is not a perfect match. Since the RXGRR motif is highly conserved in sugar transporters, Maiden et al. (49) suggest that it reflects functionally impor- tant parts of an ancient sugar transporter. The function of this motif remains to be determined.

A second region of similarity between sugar transporters and the LAC12 protein occurs in the central hydrophilic domain, region Y (Figs. 8 and 10). Using the Chou and Fasman algorithm (57), we found that this region of LAC12 protein and the glucose transporter is predicted to have an a-helical

FIG. 9. Amino acid sequence sim- ilarities. The protein sequence deduced from LAC12 (ORFI) was compared to sugar transporters using the dot matrix method of Staden (56). A window length of 20 amino acids and a stringency of 225 were chosen. The axes are labeled in amino acid residues with LAC12 protein on the x axis. Proteins on the y axis are: A , glucose transporter from human hep- atoma cells (48); B, xylose-H+ transport protein from E. coli (49); C, arabinose- H+ transport protein from E. coli (49); and D, lactose permease from E. coli (50).

Lactose Permease of Kluyveromyces lactis 16699 REGION X

GLUCOSE 66 SLSVA-IFSVG6nI-GSFSVGLFVNRFGRNSHMNLLAFVSAVL 109

LAC12 ORFI 117 GL-VFSIFNVGQ-ICGAFFVPlMOW-6RKPAILIGCLGVVI~II 159

XYLOSE 58 GFCiAkL-ikIiGML6-SYCSNRF&RDSiKiAAiLFFiSG\jG 101

ARABINOSE 63 - Y V ~ S ~ ~ & A i ~ F f f i W L S F R L G R K Y S L r V \ G A I L F V L G S I G 105

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

REGION Y

GLUCOSE 141 PMYVGEVSPTAFRGALGTLHQLGIVVGILIAQVFGLOSIffi-NKDLUPL

ORFI 188 PTYCAEVAPAHLRWVAGLYNTLYSV6SIVAAF-STYGTNKNF-PN . . . . . . . . . ..... . . . . . . . . . . . . . . ..... . . . . . ............. . . ............. . .

XYLOSE 148 PMYIAELAPAHIRW-LVSFNQFAIIFGQLLVYCV-NYFI-AR . . . . . . . . . . . . . . . . ARABINOSE 133 PLYLSEIV\SEN~RGKMIS~IYQLMVTLG-IVLAFL-SDTAF

GLUCOSE 189 LLSIIFI---PALLQCIVLPFCPESPRFLLIN-RNEEN 222

LAC12 ORFI 232

XYLOSE 188

ARABINOSE 172

-

GLUCOSE 262

LAC12 ORFI 319

XYLOSE 278

ARABINOSE 252

GLUCOSE 303

LAC12 ORFI 363

XYLOSE 313

ARABINOSE 295

-

GLUCOSE 432

ORFI 493

XYLOSE 445

ARABINOSE 428

. . . . . . . . . . . . . . . . . . . . . . ....... SSKA~Ki-"-PLYLQIIQPGLV-CIFGYLIPESPRULVGVGRE~EA 273 . . . . . . . . . . . . . . . . . ~GDASW~NTDGYRYMFASECIP~~~FLM~LYTVPESPRULMSRGKQ~QA 236

SYSGNURAMLG--VLALPAVLLIILVVFLPNSPRYLAEKGRHIEA 214

........ , . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Y

REGION X

LFR--SPAYRQPILIAVVLOLSOOLSGINAVFYYS-TSIFEKAG---- . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . LFRTRSDRYRAMLVILMAY-FGQFSGNNVCSYYLPTMLRN-VGMKS--

IVIGV-"-"IILSI-- FQQFVGINVVLYYAPEVFKT-LGA-STD . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . .

NRNVRRAVFLGMLLQAn"QQFTGMN1IMYYAPRIFK~AGFTTTE

---VQQPVYATIGSGIVNTAFTVVSLFVVERAGRRTLHLIGLA"AG 346 . . .. . . . . . . . . . . . --VSLN~LMNGVYSi~TUISSiCGAFFIDKIGRREGF~-GSISG~L 406

IAL~QTI i~GVIN- -~~F-T~LAVMTVOKFGRKP--~QI I -GALG 351

. . . . . . . . . . . . . . . . . . . . ...... .. ....... . . . . . . . . . . . . . ... ........ . . . . . . . . . . . .

QQMIATLVVGLT----F~~-ATFIAVFTVDKAGRKPALKIGFSVU~~G 337 A

REGION Z

YVFIIFTVLLVLF-FIFTY-FKVPETKGRTFKEIASGFRQGG 471

533 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . YYFYVFYVFFOIFEFIVIYFFFV-ETKGRSLE~~EVV~EAPN ~UIYGCWVLAA-~FMFVPETKGKTLEELEALU~PET 482 . . . . . . . . . . . . . . . . . . ............. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

FYLYTALNIAF-VGITFY-LIPETKNVTLEHIERKLMAGE -z 465

FIG. 10. Similar regions of LAC12 protein and other sugar transporters. The protein sequence of LAC12 (ORFI), and the glucose, xylose-H+, and arabinose-H+ transport proteins were aligned to search for the similar regions using the procedures of Wilber and Lipman (62) as modified by BIONET. The relative positive of regions X, Y, and Z in the LAC12 protein is shown in Fig. 7. The underlined sequences are the regions of highest similarity (see "Discussion"). A cobn between two sequences indicates identical amino acids. Leucine, isoleucine, and valine were considered structurally similar, as were arginine and lysine.

configuration. Allowing for conservative amino acid changes, the sequence AEVAPAHLRGK in the Y region is conserved between the LAC12 protein (amino acids 192-202) and the xylose transporter (amino acids 152-162). Another sequence PESPRW(F)L, residues 215-221 of the glucose transporter, is also conserved in the other three proteins (Fig. 10).

A third region of similarity occurs at the C terminus of the proteins (region 2, Fig. 10). Within this region, the sequence V-(P)ETKGRS(T)LEELE is highly conserved in all four proteins. This sequence of 13 amino acids contains many charged residues and was shown to be at the cytoplasmic surface of the membrane in the glucose transporter (48,58).

Since the LAC12 protein is more similar to the three monosaccharide transporters described in Figs. 8 and 10 than to the lactose permease of bacteria, we suggest that one or more of the similar regions represent domains that are critical for H+ or Na' transport or coupling of the electrochemical gradient to substrate transport, rather than being critical for substrate recognition. We assume that the LAC12 protein uses H+ or Na+ ions for symport, but this remains to be established.

The lacy permease transports ,&galactosides using a H+ symport mechanism. His-322 and Glu-325 have been reported to have critical roles in H+ translocation and in charge-relay (11,12). Identical residues with a similar location and spacing were not found in the LAC12 protein. The thiol group of cysteine has been reported for a long time to be important in substrate protection against inactivation of the permease by N-ethylmaleimide (59). Of the 8 cysteine residues in the lacy protein, only Cys-148 and Cys-154 are involved in substrate protection and only Cys-154 seems to be essential for trans- port activity (7,s). There are 9 cysteine residues in the LAC12 protein. Cys-76 and Cys-82 are separated by 5 amino acid residues and Cys-82 is flanked by Leu and Ala. The spacing between these two residues is identical to that for Cys-148 and Cys-154 in the lacy protein. Also, the second Cys is flanked by the same two amino acids in both proteins. Thus, although the two proteins show little overall amino acid sequence similarity, they may still have similar functional domains and use similar mechanisms to recognize and to transport lactose. Further structural and functional studies using mutant molecules should help to establish if the two permeases function in a similar manner.

The lack of homology between the lacy and LAC12 per.. mease is surprising because other proteins coded by the lac- tose-galactose regulon of K. lactis, /3-galactosidase, and the Leloir enzymes are homologous to the cognate proteins of E. coli (35): The homologous proteins probably have a common ancestor, while the nonhomologous permeases probably evolved convergently from separate ancestors to transport lactose across a membrane.

Acknowledgments-We thank Dr. Suradej Wicheanvonagoon and Janet Tamaren for assistance with initial DNA sequencing experi- ments, and Dr. Michael Riley for assistance with computer analysis of data.

REFERENCES

1. Sreekrishna, K., and Dickson, R. C. (1985) Proc. Natl. Acad. Sci. U. S. A. 82, 7909-7913

2. Riley, M. I., Sreekrishna, K., Bhairi, S., andDickson, R. C . (1987)

3. Dickson, R. C., and Barr, K. (1983) J. Bacteriol. 164,1245-1251 4. Sheetz, R. M., and Dickson, R. C . (1981) Genetics 9 8 , 729-745 5. Wright, J. K., Seckler, R., and Overath, P. (1986) Annu. Rev.

Mol. Gen. Genet. 208,145-151

Biochem. 65,225-248 6. Brooker, R. J., and Wilson, T. A. (1986) J. Biol. Chem. 2 6 1 ,

11765-11769 7. Menick, D. R., Sarkar, H. K., Poonian, M. S., and Kaback, H. R.

(1985) Bwchem. Biophys. Res. Commun. 132 , 162-170 8. Menick, D. R., Lee, J. A., Brooker, R. J., Wilson, T. H., and

Kaback, H. R. (1987) Biochemistry 2 6 , 1132-1136 9. Paden, E., Sarkar, H. K., Viitanen, P. V., Poonian, M. S., and

Kaback, H. R. (1985) Proc. Natl. Acad. Sci. U. S. A. 82,6765- 6768

10. Puttner, I. B., Sarkar, H. K., Poonian, M. S., and Kaback, H. R. (1986) Biochemistry 25,4483-4485

11. Carrasco, N., Antes, L. M., Poonian, M. S., and Kaback, H. R.

12. Overath, P., Weigel, U., Neuhaus, J., Soppa, J., Seckler, R., Riede, (1986) Biochemistry 25,4486-4488

T. D. Webster and R. C. Dickson, unpublished results.

16700 Lactose Permease of Kluyveromyces h t i s I., Bocklage, H., Muller-Hill, B., Aichele, G., and Wright, J. K. (1987) Proc. Nutl. Acad. Sci. U. S. A. 84,5535-5539

13. Menick, D. R., Carrasco, N., Antes, L., Patel, L., and Kaback, H. R. (1987) Biochemistry 26,6638-6644

14. Kaback, H. R. (1987) Biochemistry 26,2071-2076 15. Wray, L. V., Witte, M. M., Dickson, R. C., and Riley, M. I. (1987)

Mol. Cell. Bwl. 7 , 1111-1121 16. Miller, J. H. (1972) Experiments in Molecular Genetics, p. 433,

Cold Spring Harbor Laboratory, Cold Spring Harbor, NY 17. Riley, M. I., and Dickson, R. C. (1984) J. Bacteriol. 158 , 705-

712 18. Dale, R. M. K., McClure, B. A., and Houchins, J. P. (1985)

Plasmid 13,31-40 19. Sanger, F., Nicklen, S., and Coulson, A. R. (1977) Proc. Nutl.

Acad. Sci. U. S. A. 74, 5463-5467 20. Mills, D. R., and Kramer, F. R. (1979) Proc. Nutl. Acad. Sei. U.

21. Bina-Stein, M., Thoren, M., Salzman, N., and Thompson, J. A.

22. Weaver, R. F., and Weissman, C. (1979) Nucleic Acids Res. 7 ,

23. Maniatis, T., Fritsch, E. F., and Sambrook, J. (1982) Molecular Cloning: A Laboratory Manual, pp. 171-172, Cold Spring Har- bor Laboratory, Cold Spring Harbor, NY

24. Maniatis, T., Fritsch, E. F., and Sambrook, J. (1982) Molecular Cloning: A Laboratory Manual, pp. 382-389, Cold Spring Har- bor Laboratory, Cold Spring Harbor, NY

25. Feinberg, A. P., and Vogelstein, B. (1983) Anal. Biochm. 132 ,

26. Rudolph, H., Koenig-Rauseo, I., and Hinnen, A. (1985) Gene

27. Marsh, J. L., Erfle, M., and Wykes, E. J. (1984) Gene (Amst.)

28. Langford, C. J., Klinz, F. J., Donath, C., and Gallwitz, D. (1984)

29. Eisenberg, D., Schwarz, E., Kamaromy, M., and Wall, R. (1984)

30. Clare, J., and Farabaugh, P. (1985) Proc. Natl. Acad. Sci. U. S.

31. Westaway, S. K., Phizicky, E. M., and Abelson, J. (1988) J. Bwl.

32. Kozak, M. (1986) Cell 47,481-483 33. Green, S., Walter, P., Kumar, V., Krust, A., Bornert, J., Argos,

34. Ruzzi, M., Breunig, K. D., Ficca, A. G., and Hollenberg, C. P.

S. A. 76,2232-2235

(1979) Proc. Natl. Acad. Sci. U. S. A. 7 6 , 731-735

1175-1193

6-13

(Amst.) 36,87-95

32,481-485

Cell 36,645-653

J. Mol. Biol. 179 , 125-142

A. 82,2829-2833

Chem. 263,3171-3176

P., and Chambon, P. (1986) Nature 320 , 134-139

(1987) Mol. Cell. Biol. 7 , 991-997

35. Leonardo, J. M., Bhairi, S. M., and Dickson, R. C. (1987) Mol.

36. Zalkin, H., Paluh, J. L., van Cleemput, M., Moye, W. S., and

37. Taussig, R., and Carlson, M. (1983) Nucleic Acids Res. 11,1943-

38. Alber, T., and Kawasaki, G. (1982) J. Mol. Appl. Genet. 1 , 419-

39. Struhl, K. (1985) Proc. Natl. Acad. Sci. U. S. A. 82,8419-8423 40. Kunkel, G. R., and Martinson, H. G. (1981) Nucleic Acids Res.

41. Prunell, A. (1982) EMBO J. 1, 173-179 42. Hahn, S., Hoar, E. T., and Guarente, L. (1985) Proc. Nutl. Acad.

43. Salmeron, J. M., and Johnston, S. A. (1986) Nucleic Acids Res.

44. Hoffmann, W. (1985) J. Biol. Chem. 260,11831-11837 45. Tschopp, J. F., Eemon, P. D., and Schekman, R. (1984) J.

Bacteriol. 160,966-970 46. Tschopp, J. F., Emr, S. D., Field, C., and Schekman, R. (1986) J.

Bacteriol. 166,313-318 47. Yazyu, H., Shiota-Niiya, S., Shimamoto, T., Kanazawa, H., Futai,

M., and Tsuchiya, T. (1984) J. Bid. Chem. 259,4320-4326 48. Mueckler, M., Caruso, C., Baldwin, S. A., Panico, M., Blench, I.,

Morris, H. R., Allard, W. J., Lienhard, G. E., and Lodish, H. F. (1985) Science 229,941-945

49. Maiden, M. C. J., Davis, E. O., Baldwin, S. A., Moore, D. C. M., and Henderson, P. J. F. (1987) Nature 325,6414343

50. Biichel, D. E., Gronenborn, B., and Muller-Hill, B. (1980) Nature

51. Buvinger, W. E., and Riley, M. (1985) J. Bacteriol. 163,850-857 52. Hogg, R. W. (1981) J. Biol. Chem. 256,1935-1939 53. Higgins, C. F., Haag, P. D., Nikaido, K., Ardeshir, F., Garcia, G.,

and Ames, F. (1982) Nature 2 9 8 , 723-727 54. Tanaka, J., and Fink, G. R. (1985) Gene (Amst.) 38,205-214 55. Landick, R., and Oxender, D. L. (1985) J. Bwl. Chem. 260,8257-

56. Staden, R. (1982) Nucleic Acids Res. 10,2951-2961 57. Chou, P. Y., and Fasman, G. (1973) J. Mol. Bwl. 74,263-281 58. HasDel. H. C., Rosenfeld. M. G., and Rosen, 0. M. (1988) J. Bwl.

Cell. Biol. 7 , 4369-4376

Yanofsky, C. (1984) J. Biol. Chem. 259 , 3985-3992

1954

434

9,6869-6888

Sci. U. S. A. 82,8562-8566

14,7767-7781

283,541-545

8261

C h i . 263; 398-403 . 59. Fox. C. F.. and Kennedv. E. P. (1965) Proc. Natl. Acad. Sci. U.

" I

S.'A. 54,891-899 . .

60. Meinkoth, J., and Wahl, G. (1984) Anal. Biochem. 138,267-284 61. Kyte, J., and Doolittle, R. F. (1982) J. Mol. Bwl. 1 5 7 , 105-132 62. Wilber, W. J., and Lipman, D. J. (1983) Proc. Natl. Acad. Sci. U.

S. A. 80, 726-739

Lactose Permease of Kluyveromyces lactis 16701

The nuc leo t ide semence o f the iplclp r e g i o n i s shown. NUCleotidFI a ~ e

numbered from the ATG i n i t l l t l o n codon of ORFI. which i s d e l i a n a t e d '1. The

deduced amino a c l d SeOuence o f O R f I , nuc leo t ides +1 t o *1161. and ORFII ,

nuc leo t ides '2334 t o '3599. a ~ e shown. The two major t ranscr ip t endpo in ts determined I s desc r ibed i n F ig . 3 dTe Ind icated by an aster isk under

nuc leo t ides -120 and -122. and t h e two minor t ranscr ip t endpo in ts are

reovence f rom nuc leot ides -329 t o -345 and from -609 t o -625 w e s i m i l a p t o

ind icated by an aster isk under nuc leot ides -127 and -131. The under l ined

the UPI tVelm Act ivator Sewencer. UAS, f o r t h e gene (34. 3 5 ) .

16702 Lactose Permease of Kluyveromyces &tis

"

- 2.3 kb - 20 kb

The Region 8 e t m n ORFl and ORFll i s Not an l n t ron To d e t a n i n r If the 573 bD nohcodinq region bet- DlFl and DRFll I s an Intron. R

cmsmsus SeownCe at the 5 ' end Of the intron i s 61AT6T and the s e o w ~ c TACTMC I s first searched the seoumcc for the dU spl ic ing consensus sequence of 5. cemls lae . The

located between 9 and 57 nuclwt fdes frm the 3' Intron-cam junction (28). rlthouqh a

d m r t r e r fvm the 5' end of MIFII. no 6 ~ ~ 1 6 ~ seoouc(ye (the 5' end of thc intron) .as

actin qene Of 5. I.Ct(I (Rossin. pcr%onal c o u n i c a t t m l . AgaIn. thnr was no s ign i f icant DM seouence s im i la r i t y svqqestIn9 that the region bet- ORFl and ORFlI I s mt an intmn.

32P-labelled Xbal to Hpal DIU fra-nt ( F l a r e 5) t o po Iy (A* )RU followed by digestion The presence of an Intron was ermine4 e a p c r l m t a l l y by hWid I l Inq a 3'

Of unhybridized. S I n g l c S t r a n M DNA with e i ther u n 9 bean nuclease or exmyclease VII. 8 u a u ~ c u n g bean nuclease i s an endonKlease it should y ie ld a 0.85 kb p m t u t e d rdk- active f r a w n t of the probe (fm Xb.1 t o the end of P F I ) If there I s an intron behtm ORFl and P F I I . I f no intron i s pretmt. a 3.4 kb fr1-t of thc pmbc shovld be

radioactive f r a g m t . The resu l ts shom in Figure 5 reveal that a 3.4 kb pmbc Ira-t

T E T M C " 4 6 seoycnce, rh ich Could be the 3' intron JunctIon Slte. 11s fwnd 69 bases

found near the 3' end of ORFI. Ye also coqarcd our data t o the Intron (-e of the

protected. I n elther Lase. the exonuclease V I 1 I S S I Y Should y i e l d I 3.4 kb PWtUted

.as pmtuted fm MCleaw dipcstim. These data rtronqly sqqcst that there I s 110 intron bet- ORFl and ORFll.

To l u r t h e r c m f i n t h a t thc wn-coding rqIm I s t r rnrcr lbed into dU aod I s mt In

RR used f o r Northern b lo t ma lys l s ( F i m 6). Thc r n u l t s Ihm that only one dM Of

intron. r a d I o Y t i v e wobcs coq lemnta ry t o QFl . t o the wn-codlnp rqion. and t o mll

4.7 kb hybridiles -4th a l l three wobcs. Ik c m l & that thc 4.7 kb dU CmtaInS ORFl.

the non-coding region bet- ORFl and CUF11. aod ORFll SCOYmCtS.

P 08765 4321

3.4KB- -4.3KB

- 1.9KB

4 3 2 1 I

". PI P2 PR

P4

Lactose Permease of Kluyveromyces lactis 16703