THE .IOURNAL OF BIOLOGICAL CHEMISTRY Val. 258, No. 17, Issue of September 10. pp. 10761-10767, 1983 Prmted in U. S A .

Mannitol-specific Enzyme I1 of the Bacterial Phosphotransferase System 111. THE NUCLEOTIDE SEQUENCE OF THE PERMEASE GENE*

(Received for publication, January 12, 1983)

Catherine A. Lee and Milton H. Saier, Jr. From the Department of Biology, The John Muir College, Uniuersity of California, Sun Diego, La Jolla, California 92093

The nucleotide sequence of the mtlA gene, which codes for the mannitol-specific Enzyme I1 of the Esch- erichia coli phosphotransferase system, is presented. From the gene sequence, the primary translation prod- uct is predicted to consist of 637 amino acids (Mr = 67,893). This result is compared to the amino acid composition and molecular weight of the purified man- nitol Enzyme I1 protein. The hydrophobic and hydro- philic properties of the enzyme were evaluated along its amino acid sequence using a computer program (Kyte, J., and Doolittle, R. F. (1982) J. Mol. Biol. 157, 105-132). The computer analysis predicts that the NHz-terminal half of the enzyme resides within the membrane, whereas the COOH-terminal half of the enzyme has the properties of a soluble protein. The possible functions of such a protein structure are dis- cussed. RNA mapping has identified the promoter and mRNA start point for the mtl operon.

The mannitol-specific Enzyme I1 of Escherichia coli is a transmembrane component of the phosphoenolpyruvate-de- pendent sugar phosphotransferase system (1). The enzyme catalyzes the concomitant transport and phosphorylation of mannitol. This reaction requires phosphoenolpyruvate and two phosphoryl carrier proteins of the phosphotransferase system, Enzyme I and HPr. The phosphoryl group from P- enolpyruvate is first transferred to HPr by the Enzyme I and then it is transferred from phospho-HPr to mannitol by the Enzyme IImt" (Fig. 1A) ( 2 ) . This process is unlike the uptake of glucose by the phosphotransferase system, which requires a third phosphoryl carrier protein, the glucose-specific En- zyme 111, to transfer the phosphoryl group from phospho-HPr to the Enzyme I1 and the sugar (Fig. 1B) (3). Phospho-HPr and Enzyme IImtl are sufficient to transport and phosphorylate mannitol. The internalized mannitol 1-phosphate is then oxidized to fructose 6-phosphate by the cytoplasmic protein, mannitol-1-phosphate dehydrogenase (4).

In order to understand the role of the Enzyme 11"" in the transport and phosphorylation of mannitol, various investi- gative approaches have been taken. The protein components required for mannitol uptake have been purified and recon- stituted (1, 5 , 6). The Enzyme IImt' can be purified from membranes and is catalytically active in detergent micelles or in phospholipid vesicles (1, 7). This has allowed study of the

* The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "aduertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

11. ' The abbreviation used is: Enzyme II"", mannitol-specific Enzyme

catalytic and physicochemical properties of the Enzyme 11"'". In addition, mutagenesis of the structural gene for the Enzyme IImti, mtlA, has resulted in the genetic dissection of the cata- lytic activities of the enzyme (8).

We present here the amino acid sequence of the Enzyme IImtl, predicted from the nucleotide sequence of its structural gene. A computer analysis of the primary structure of the enzyme distinguishes those portions of the protein which have transmembrane or globular properties. This structural infor- mation gives new insight into the function of the Enzyme

We have also conducted RNA mapping on the sequenced DNA. The mtl operon contains mtlC, a cis-dominant regula- tory region, mtlA, the structural gene for the Enzyme IImtl, and rntlD, the structural gene for the mannitol-1-phosphate

rrmt l .

A ) Mtl E Mtl-P

I P

~ H P r - P P E I - P - PEP

J II1?ZP

# l lG 'C

E) Glc G1c.P

FIG. 1. The E. coli phosphoenolpyruvate-dependent phos- photransferase system. The diagrams show the enzyme constitu- ents responsible for the transport and phosphorylation of ( A ) man- nitol (Mt l ) and ( B ) glucose (Glc). The Enzymes I1 (ZZM", Z P ) are integral membrane proteins which function as the sugar permeases. The other enzymes, Enzyme I (EZ), HPr, and Enzyme IIIgl' ( Z Z P ) are soluble, or peripherally associated with the membrane. The phos- phoryl moiety of phosphoenolpyruvate ( P E P ) is transferred down the phospho-carrier sequence to form the sugar phosphate (Mtl-P, Glc-P).

" ------- - - - - "

- - T Z A S H S S Z T S Z S 2 B B 8 5 8 2 2 2 S B T 2 5 S 2 T

f IIIU I I I I > I I I I I'D l%3,1 !3 1 I 3 1 D 3 1 1 1 - " ""- - - " a -

- FIG. 2. Restriction map and sequencing strategy for the

2162-base pair DNA fragment containing the mtlA gene. Restriction sites are AuaI ( A ) , BstNI ( B ) , DdeI (D), Hind111 ( H ) , Sau3AI (SI, TaqI (T) , AluI (I), HpaII ( 2 ) , and HaeIII ( 3 ) . The arrows above and below the restriction map indicate the beginning, direction, and extent of the nucleotide sequence determination of the noncoding and coding strands of the DNA, respectively.

1- -

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10762 Sequence of Mannitol Permease Gene

endonuclease sites, mapped in Fig. 2, are shown.

dehydrogenase. The gene order is mtl C-A-D (9,lO). We have identified the mtl operon promoter, the mRNA start site, and two RNA 3'-ends, on less than 5% of mtl transcripts, that map in the intercistronic region between the mtlA and mtlD genes.

EXPERIMENTAL PROCEDURES

Bacterial Strains and Plasmids-Plasmid DNA from E. coli strain L163sr/pCL2.0 was utilized for the nucleotide sequence determina- tion (11). RNA from E. coli strain C600 was utilized for the nuclease S1 mapping experiments (12).

DNA Preparation-Plasmid DNA was purified from E. coli lysates by equilibrium centrifugation in cesium chloride/ethidium bromide gradients (13). DNA was digested with restriction endonucleases using the conditions specified by the manufacturers. Restriction fragments were purified from agarose gels by electroelution (13) or from polyacrylamide gels by diffusion into elution buffer (14). Sepa- ration of DNA strands by electrophoresis through polyacrylamide gels was conducted at 4 "C (13). DNA was end-labeled with T, polynucleotide kinase or E. coli DNA polymerase Klenow fragment using 32P-labeled ATP or deoxynucleotide triphosphates, respectively (14).

DNA Sequencing-Nucleotide sequencing was conducted using the Maxam-Gilbert procedures (14) with the exception of the G-specific modification reaction. DNA was modified at G residues using meth- ylene blue and sunlight (15).

Nuclease SI Mapping-The hybridization reactions for RNA map- ping were conducted at 47 "C for 6 h (13). The nuclease S1 digestions were conducted a t 37 "C for 30 min (13). The RNA was isolated from log phase E. coli cells grown in L broth containing 0.5% mannitol using a guanidinium thiocyanate lysis procedure followed by centrif- ugation through a cesium chloride cushion as described for isolation of RNA from pancreatic cells (16).

Hydropathy Analysis-The hydropathy analysis of the amino acid sequence of the Enzyme IImt' was conducted by Dr. R. Doolittle using the SOAP computer program (17).

Enzymes and Radionucleotides-DNA restriction enzymes were obtained from Bethesda Research Laboratories and New England

Biolabs, Inc. T, polynucleotide kinase was from P-L Biochemicals and the Klenow fragment of E. coli DNA polymerase was from Bethesda Research Laboratories. 32P-labeled nucleotide triphosphates were products of Amersham Corp. or New England Nuclear.

RESULTS

Nucleotide Sequence of the mtlA Gene-A 2-kilobase pair fragment of DNA has been cloned which contains the mtlA gene (11). Fig. 2 shows a detailed map of the cloned DNA and indicates the restriction sites utilized for the nucleotide se- quencing method of Maxam and Gilbert (14). The ends of the DNA fragment were Hue111 restriction sites destroyed by ligation to EcoRI linkers during construction of the clone (11). The arrows above and below the restriction map indicate the beginning, direction, and extent of the nucleotide sequence determination of the noncoding and the coding' strands of the DNA, respectively (Fig. 2). Sequence determination of both complementary strands and across all restriction frag- ment junctures established the correct nucleotide sequence of the 2162 base pairs containing the mtlA gene (Fig. 3).

RNA Mapping with Nuclease SI-RNA mapping was con- ducted to localize the mtl mRNA on the sequenced DNA. The 5'-end of the mtl mRNA was mapped using the coding strand of the TuqI (position 6) to Hind111 (position 169) restriction fragment as a hybridization probe. The 164-base, single- stranded DNA probe was labeled at the HindIII-cut 5'-end, allowed to hybridize with RNA isolated from E. coli cells grown in the presence of mannitol, and subsequently digested with nuclease SI. The size of the fragment of the DNA probe which was protected from nuclease S1 digestion by hybridi- zation with RNA was determined by running the sample into

'The coding strand of DNA is that strand which serves as the template for RNA polymerase.

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4 5 6 7 0 -q T -2125

C

c

FIG. 4. RNA mapping with nuclease S1. The nucleotide sequence of the coding strand of the DNA probes is shown on the side of the autoradiographs. The nucleotides are numbered as in Fig. 3. The sequence of the noncoding strand of DNA is also shown at the sites where the RNA ends map. A, the 5‘-end of the mtl mRNA was mapped as descibed in the text. Lune 1,5’-end labeled single-stranded DNA probe; lane 2, DNA probe after nuclease S1 treatment; lanes 3-6, DNA probe after hybridization to 1, 2.5, 5, and 10 pg of E. coli RNA and subsequent nuclease S1 treatment; lane 7, C-specific Maxam-Gilbert sequencing ladder of the DNA probe; lane 8, C + T-specific Maxam-Gilbert sequencing ladder. B, two 3’-ends of the mtl mRNA are mapped within the intercistronic region as described in the text. Lanes 1-8 are identical to those in A except with a different single- stranded DNA probe labeled at the 3’-end.

a sequencing gel alongside the C- and C + T-specific Maxam and Gilbert sequencing ladders of the same labeled DNA (Fig. 4A). Gel autoradiography localized the rntl mRNA start-site near the T-A-T at positions 45-47. Upstream from the mRNA start site, sequences homologous to RNA polymerase recog- nition sites are identified. Fig. 5 compares these rntl promoter sequences to the consensus promoter sequences compiled by Rosenberg and Court (18). The putative rntl promoter region has reasonable spatial and nucleotide similarities to the con- sensus sequence. The mtl -35 homology (T-G-G-A-C-A) be- gins at position 10 and the mtl mRNA start site (T-A-T) is a t position 45.

The first initiation codon on such a transcript would be the A-U-G a t positions 138-140. Nine bases upstream from the A-U-G codon is an A-A-G-G at positions 125-128. This se- quence might be the “Shine-Dalgarno” ribosomal recognition site for mtlA (19). The initiation codon begins a continuous reading frame of 637 triplet codons which terminates a t a nonsense codon, U-A-A, a t positions 2049-2051. Alternatively another AUG codon in this reading frame could be the mtlA initiation codon, such as that at positions 193 or 199. How- ever, the corresponding ribosomal recognition regions for

MfL: 5’ -gt-acTGGACA-tto-7bp-cag!gcCAGATT t -5bp-!at 3’ IO 20 30 45

FIG. 5. Comparison of the mtl promoter sequences to the consensus sequences compiled by Rosenberg and Court (18). The consensus sequence-highly conserved bases are underlined, well conserved bases are indicated by upper case letters, and weakly conserved bases are indicated by lower case letters. The mtl se- quence-the bases are aligned with the positions in the consensus sequence. The highly conserved nucleotides in the -10 and -35 regions are capitalized. The positions are numbered as in Fig. 3.

these alternative initiation codons have little homology to the Shine-Dalgarno consensus sequence.

We noticed that the region immediately downstream from the mtlA gene translational termination codon codes for an RNA molecule which has the potential to form a hairpin secondary structure (Fig. 6A). The free energy of formation (AGO a t 25 “C) of this possible RNA secondary structure is estimated to be -23 kcal (20). This hairpin structure and the U-rich region following it show similarities to p-independent

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10764 Sequence of Mannitol Permease Gene

termination sites (18). In addition, the T at position 2092, which corresponds to the end of this U-rich region, begins a nucleotide sequence homologous to a procaryotic intercis- tronic regulatory element proposed by Higgins et at. (21). Fig. 6B shows the homologous sequences of the putative mtl intercistronic element. Notice that the entire regulatory ele- ment is not included on this mtlA subclone. These sequences are speculated to be involved in transcriptional termination or RNase processing (21). Therefore, RNA 3'-ends were sought in this region. The coding strand of the TaqI (position 1988) to HaeIII (position 2162) (EcoRI-linker end) DNA restriction fragment was labeled at the TuqI-cut 3'-end. DNA- RNA hybridizations, nuclease S1 digestions, and gel electro- phoresis were conducted as before. The autoradiographs show that most of the DNA probe (>95%) was protected along its entire length. However, a small percentage of RNA transcripts terminate in the U-rich region near position 2090, down-

A U C A

C G C G

U A C G U A

C A C G

A U C G

C G C G -

! i ' - U A A U C C A A U C G G U U A A U U - 3 ' 2050 2090

B)

5' TGCCTGATGCGCTACGCT;ATCAGGCCTACA-XW-GTAGG(CQ 3' " L

2092 2120 2 160

FIG. 6. Nucleotide sequences in the intercistronic region. A, possible secondary structure of the RNA transcribed from the inter- cistronic region. The hairpin structure and the downstream U-rich region show similarities to p-independent termination sites (18). B, The mtl intercistronic sequences homologous to the procaryotic in- tercistronic regulatory element proposed by Higgins et al. (21). The conserved palindromic units are indicated by arrows. The Had11 site at position 2162 is reconstructed here to demonstrate further homol- ogy. The base positions are numbered as in Fig. 3.

TABLE I Amino acid composition of the Enzyme II""

Amino acid Of purified From gene In first 336 protein" sequence amino acids

Alanine 59 64 33 Arginine 23 5 Asparagine 12 Aspartic acid 5 Cysteine 5 4 2 Glutamic acid 8

Glycine 63 67 47 Histidine 15 13 6 Isoleucine 34 54 37 Leucine 52 67 39 Lysine 28 31 13 Methionine 15 25 19 Phenylalanine 23 29 22 Proline 28 26 18 Serine 42 40 19 Threonine 29 30 12 Tryptophan 4 4 Tyrosine 8 11 6 Valine 42 50 25

Totals 553 637 336 The amino acid analysis of purified Enzyme IImt' was performed

by Drs. R. A. Laursen and G. R. Jacobson, Boston University, as described in Ref. 23. Values represent averages of two determinations except for cysteine which was an average of five determinations and methionine which was an average of eight determinations.

} :: ;73} 50

Glutamine I 51 49 4

stream from the predicted hairpin structure. In addition, another RNA 3'-end was mapped near position 2119, imme- diately downstream from the small hairpin structure of the proposed regulatory element (Fig 4B). Both of these 3'-ends appear in less than 5% of the mtl transcripts and could be due to minor transcriptional termination or RNase process- ing.

Amino Acid Sequence of the mtlA Translation Product- The primary translation product of the mtlA gene can be predicted from the nucleotide sequence of the gene. The amino acid sequence corresponding to the translation of the 637 triplet codons identified above is shown in Fig. 7. The molec- ular weight of such a protein is 67,893. The purified mannitol Enzyme I1 protein migrates, in sodium dodecyl sulfate-poly- acrylamide gels, with an apparent molecular weight of 60,000 (+5%) (2). This minor discrepancy between the predicted and

FIG. 7. The amino acid sequence of the primary translation product of the mtlA gene. The 637 amino acids are listed from the N R to the COOH terminus.

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apparent molecular weights of the Enzyme 11"'" could be due to atypical mobility in sodium dodecyl sulfate gels or post- translational processing of the protein. NH,-terminal proc- essing of secreted proteins occurs during their export into the bacterial outer membrane or the periplasmic space. Thus far, integral membrane proteins of the bacterial inner membrane, with the exception of the M13 coat protein, and the membrane penicillinase of Bacillus licheniformis, do not appear to have such processed "signal" peptides. The predicted NHZ-terminal sequence of the Enzyme IImt', an inner membrane protein, does not have the features found in bacterial signal peptides (22). However, preliminary sequence analysis of the NHp terminus of the purified protein has failed to reveal a free amino acyl residue, suggesting that the NH, terminus is b l ~ c k e d . ~ Therefore, the possibility that the NH2 terminus of the Enzyme I P ' is processed cannot be eliminated.

The amino acid composition of the predicted mtlA trans- lation product is compared to the amino acid analysis of the purified protein (23) in Table I. The number of residues from the amino acid analysis, 553 total, was derived assuming a protein molecular weight of 60,000. Regardless, there is a general correspondence between the two amino acid compo- sitions with the exceptions of methionine and the hydrophobic amino acids valine, isoleucine, leucine, and phenylalanine. These discrepancies are probably due to inaccuracies in the amino acid analysis. The underestimation of the hydrophobic amino acid residues is probably due to their large number in the Enzyme IImt' and the hindered hydrolysis of peptide bonds between such amino acids (see "Discussion").

Hydropathic Analysis of the Enzyme IImt' Amino Acid Se- quence-A computer program has been developed to evaluate the hydrophobic and hydrophilic properties of a protein. Hy- dropathy values are assigned to each amino acid side chain which reflect its inclination to seek an aqueous or a hydro- phobic environment. The hydropathy scale ranges from +4.5 for isoleucine to -4.5 for arginine (17). Soluble proteins were shown to have an average hydropathy of -0.4, while Halobac- terium halobium bacteriorhodopsin, an integral membrane protein which traverses the membrane seven times, has an average hydropathy of +0.70. The average hydropathy of the Enzyme II"", +0.33, is intermediate between these values. However, closer examination of the distribution of the hy- dropathy values along the Enzyme IImt' reveals that the av- erage hydropathy of the first 336 amino acids is +0.90 whereas the last 301 amino acids average -0.32. This uneven distri- bution of hydrophobic and hydrophilic amino acids is also observed by comparing the composition of the first 336 amino acids to that of the entire 637 (Table I). The NHp-terminal half of the Enzyme 11"'" has more tryptophan, phenylalanine, glycine, and isoleucine residues and fewer arginine, glutamine, glutamic acid, and aspartic acid residues than would be ex- pected for a random distribution of the amino acids.

This split nature of the Enzyme 11"" is visualized in greater detail by plotting a progressive evaluation of its hydropathy (Fig. 8). The average of the hydropathy values of seven consecutive amino acids is plotted at the position of the central amino acid residue. The seven residue segments over- lap and are displaced from one another by one residue. In Fig. 8, the first value at residue 4 is the average of the hydropathies of residues 1-7, and the next value a t residue 5 is the average for residues 2-8. The midline is -0.4, the mean average value for soluble proteins. Long stretches above the midline are observed in the plot of the NHp-terminal half of the Enzyme

M. J. Novotny, F. Eshe, and M. H. Saier, Jr., unpublished exper- iments.

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10766 Sequence of Mannitol Permease Gene

IImt'. At residue 336, the plot changes character and fluctuates about the midline.

Membrane-spanning portions of proteins were shown to have an average hydropathy of +1.22 to +2.65 over a 19- residue segment (17). The average hydropathies of the long hydrophobic regions of the Enzyme IImt' were calculated (Fig. 8). At least seven regions in the NHp-terminal half of the Enzyme IImt' are predicted to have sufficient hydophobic character to be able to traverse the membrane.

The region between residues 190 and 260 has sharp peaks of hydrophobicity interspersed with segments below the mid- line. This might indicate that this part of the polypeptide chain is only partially embedded in the membrane and inter- acts with the aqueous environment on the membrane surface or that it traverses the membrane having protein-water inter- actions within a transmembrane aqueous channel.

DISCUSSION

We have determined the nucleotide sequence of a 2162- base pair DNA fragment which contains the mtlA gene. We have identified on this sequence, using nuclease S1 mapping, the start point for the rntl mRNA. Upstream from the mRNA start, putative rntl promoter sequences have been identified.

I t is interesting to note that the rntl -35 homology begins only 10 base pairs from the end of the sequenced DNA fragment. Previous measurements of the expression of the mtlA gene from this 2-kilobase pair DNA fragment demon- strated that, unlike in the intact rntl operon, expression was not inducible by cyclic AMP and mannitol (11). Operons regulated by cyclic AMP and the catabolite gene-activator protein contain a binding site for the cyclic AMP-catabolite gene-activator protein complex 60 base pairs upstream from the mRNA start site (24). Obviously, the mtlA subclone does not include such cis-dominant regulatory sequences for cyclic AMP control and, therefore, cyclic AMP cannot affect its expression.

The mechanism of induction by mannitol is not known. The lack of effect of mannitol on the gene expression of this mtlA subclone is probably due to the absence of the sequences necessary for mannitol regulation. However, noninduction could also be due to low levels of the putative rntl regulatory protein and the presence of the rntl operator region on a high copy plasmid. Therefore, we cannot eliminate the possibility that sequences on this DNA fragment are required for man- nitol regulation. For example, the lac repressor binds to the lac operon in the region between the lac promoter and the lac2 gene translational initiation codon. In fact, there are 90 base pairs between the rntl mRNA start point and the mtlA gene initiation codon. However, it should be noted that pre- vious genetic studies suggest that the cyclic AMP-catabolite gene-activator protein binding site overlaps or is closely as- sociated with the mannitol regulatory region (25).

The observation of a possible p-independent termination site and of a regulatory element in the intercistronic region between the mtlA and mtlD genes prompted a search for 3'- ends of RNA transcripts within this region. A small percent- age of the RNA transcripts appear to terminate in the U-rich region immediately downstream from the first hairpin struc- ture (Fig. 6A) and at a site immediately downstream from the second hairpin structure (Fig. 6B). These short transcripts could be a result of transcriptional termination or RNase processing. The majority of the RNA transcripts continue downstream. None of the nucleotide sequences in this inter- cistronic region corresponds to the beginning of the mtlD structural gene which was deduced from the NHp-terminal amino acid sequence of the purified mannitol-1-phosphate

dehydrogenase protein.' We presume that the mtlD gene begins further downstream.

The amino acid sequence of the mtlA gene primary trans- lation product was predicted from the nucleotide sequence of the gene. The molecular weight and amino acid composition of the purified Enzyme 11"" protein are reflective of those of the predicted gene product. A discrepancy was observed be- tween the amino acid analysis of the purified protein and that predicted for the gene product, based on the nucleotide se- quence. Specifically, hydrophobic residues (particularly Ile, Leu, and Val) were underestimated in the amino acid analysis. This apparent anomaly has been observed for all of seven integral membrane proteins for which the amino acid com- positions and the complete gene sequences are available (sub- units I and I1 of cytochrome oxidase and the CY, p, y, and 6 subunits of the acetylcholine r e ~ e p t o r ) ~ a s well as the mannitol Enzyme 11. Thus, amino acid analyses of integral membrane proteins may generally be inaccurate. Only sequence analyses of these proteins can be considered to be fully trustworthy. It should be noted that while the analyses reported here provide the complete amino acid sequence of the mannitol Enzyme I1 in the unprocessed form, determination of the NHp-terminal sequence of the purified protein will be necessary to demon- strate whether the NH, terminus of the Enzyme IImt' is post- translationally processed.

An analysis of the hydrophobicity and hydrophilicity of the Enzyme 11"'" amino acid sequence suggests that the NHp- terminal half of the enzyme resides in the membrane, and that the COOH-terminal half resembles a globular, soluble protein. Previous studies on the asymmetric binding proper- ties, protease sensitivity, and antigenic determinants of the Enzyme 11"" suggested that a large portion of the protein was exposed on the cytoplasmic surface of the inner membrane (23, 26, 27). Therefore, we propose that the COOH-terminal half of the enzyme is a large globular structure on the cyto- plasmic side of the membrane (Fig. 9).

The NH,-terminal half of the enzyme has seven hydropho- bic segments which probably traverse the membrane (Fig. 9). These long hydrophobic structures contain few uncharged polar amino acids, and only three of the seven regions contain a charged amino acid residue. The hydropathy plot of the protein segment between residues 190 and 260 (Fig. 8) shows several short hydrophobic stretches which may reside in the membrane. The hydrophilic regions of this protein segment could interact with the aqueous environment at the surface of the membrane (as shown in Fig. 9) or in the interior of the membrane, as would be expected for a hydrophilic channel (not shown in Fig. 9). In other words, since this region of the Enzyme IImt' is hydrophobic but has a number of hydrophilic amino acid residues, it might serve as a hydrophilic transmem- brane channel. The possibility that a portion of the protein would form a channel is intriguing since such a structure may serve to translocate the hydrophilic sugar substrate across the hydrophobic membrane bilayer. It is not known whether the enzyme functions as a monomer or as a multimer, but in either case, a hydrophilic channel could facilitate the trans- location process.

We suggest that the Enzyme I P ' might function in the transport and phosphorylation of mannitol in the following way, The membrane-spanning portions of the enzyme might form a hydrophilic opening to allow partial passage of the sugar across the bilayer. This channel might normally be blocked, but binding of the sugar to a site within the channel

M. J. Novotny, F. Eshe, and M. H. Saier, Jr., unpublished exper- iments. ' R. A. Nicholas, personal communication.

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IN I I OUT

FIG. 9. One possible disposition of the Enzyme 11"" across the membrane. The model shows the seven transmembrane seg- ments within the hydrophobic NH, terminus of the enzyme. The protein segment between residues 190 and 260 is represented as a small circle, partially embedded in the membrane interacting at one surface of the membrane. An alternative interpretation is that this region forms a membrane-embedded hydrophilic channel (see "Dis- cussion"). The hydrophilic COOH terminus, thought to he a globular structure on the cytoplasmic surface of the membrane, is depicted as the large circle. The NH, and COOH termini of the Enzyme 11"" are indicated by N a n d C, respectively.

might induce a conformational change in the enzyme which results in the exposure of the bound sugar to the cytoplasmic side of the membrane. The sugar might then be phosphoryl- ated and subsequently released inside the cell. The large globular COOH-terminal half of the enzyme might be involved in the phosphorylation of the sugar. This portion might bind phospho-HPr to allow for direct transfer of the phosphoryl group to the sugar, or it might be phosphorylated by phospho- HPr and then subsequently transfer the phosphoryl group to the sugar. It is interesting to note that the transport and phosphorylation of mannitol does not require an Enzyme I11 phospho carrier. I t is possible that the large globular portion of the Enzyme 11"'" serves the Enzyme I11 function in the mannitol phosphotransferase system.

Future genetic and biochemical studies will be required to gain support for these speculations on the structure and function of the Enzyme I P ' . T h e fact that this protein is multifunctional (l), catalyzing (a) both unidirectional and bidirectional transport (28); ( b ) both P-enolpyruvate-depend- ent and mannitol 1-phosphate-dependent sugar phosphoryl- ation (2); ( c ) chemoreception of mannitol (29-31); and ( d ) transcriptional regulation of the mtl operon (1) renders these studies of particular interest.

Acknowledgments-We are indebted to Dr. D. R. Helinski for allowing the nucleotide sequencing to be conducted in his laboratory. We thank Drs. M. Filutowicz, D. R. Corbin, and J. R. deWet for their instruction and advice. We also thank Dr. R. F. Doolittle for con-

ducting the computer analysis of the amino acid sequence and Drs. R. F. Doolittle and J. Kyte for helpful discussions.

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C A Lee and M H Saier, Jrnucleotide sequence of the permease gene.

Mannitol-specific enzyme II of the bacterial phosphotransferase system. III. The

1983, 258:10761-10767.J. Biol. Chem. 

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