Vol. 170, No. 6JOURNAL OF BACTERIOLOGY, June 1988, p. 2639-26450021-9193/88/062639-07$02.00/0Copyright C) 1988, American Society for Microbiology

Truncated Forms of Escherichia coli Lactose Permease: Models forStudy of Biosynthesis and Membrane Insertion

URSULA STOCHAJ,'t HANS-JOACHIM FRITZ,2 CARIN HEIBACH,' MARTINA MARKGRAF,tANTJE VON SCHAEWEN,1§ UWE SONNEWALD,1§ AND RUTH EHRING1*

Institut fur Genetik der Universitat zu Koln, D-5000 Cologne 41,1 and Max-Planck-Institut fur Biochemie, AbteilungZellbiologie, D-8033 Martinsried bei Munchen,2 Federal Republic of Germany

Received 2 March 1987/Accepted 1 March 1988

Using in vitro DNA manipulations, we constructed different lacY alleles encoding mutant proteins of theEscherichia coli lactose carrier. With respect to structural models developed for lactose permease, thetruncated polypeptides represent model systems containing approximately one, two, four, and five of theN-terminal membrane-spanning a-helices. In addition, a protein carrying a deletion of predicted helices 3 and4 was obtained. The different proteins were radiolabeled in plasmid-bearing E. coil minicells and were foundto be stably integrated into the lipid bilayer. The truncated polypeptides of 50, 71, 143, and 174 N-terminalamino acid residues resembled the wild-type protein in their solubilization characteristics, whereas the mutantprotein carrying an internal deletion of amino acid residues 72 to 142 of the lactose carrier behaved differently.Minicell membrane vesicles containing truncated proteins comprising amino acid residues 1 to 143 or 1 to 174were subjected to limited proteolysis. Upon digestion with proteases of different specificities, the samecharacteristic fragment that was also produced from the membrane-associated wild-type protein was found toaccumulate under these conditions. It has previously been shown to contain the intact N terminus of lactosepermease. This supports the idea of an independent folding and membrane insertion of this segment even in theabsence of the C-terminal part of the molecule. The results suggest that the N-terminal region of the lactosepermease represents a well-defined structural domain.

Lactose permease of Escherichia coli represents a well-studied example of a protein mediating proton:substratesymport (reviewed in references 17 and 46). Numerousmutational exchanges have been identified which affect thefunction of the carrier in different ways (3, 6, 14, 22, 24, 31,32, and references cited therein). However, despite thesedetailed studies, it was only recently that data began toaccumulate concerning the structure of the protein. Struc-tural models based on physicochemical investigations (11,43) predict the protein to be mostly embedded in the mem-brane without any larger domains protruding from either sideof the lipid bilayer and to consist predominantly of mem-brane-spanning a-helices. Both the N-terminus and theC-terminus of the protein are exposed towards the cyto-plasm (1, 26, 35). Different proteases have been used to testthe accessibility of the membrane-associated lactose carrier,and several regions of preferred proteolytic attack have beendefined (1, 13, 36, 38). A characteristic N-terminal fragmentextending into the region around amino acid residue 140 wasfound to accumulate and to be exceptionally resistant tofurther proteolytic degradation even in the presence ofnonionic detergents (38).So far, there is only little information available about the

biosynthesis of such polytopic membrane proteins that re-tain both termini on the cytoplasmic face of the membrane(30, 45). In view of the detailed studies on genetics and

* Corresponding author.t Present address: Institut fur Anatomie und Zellbiologie, Phi-

lipps-Universitat Marburg, D-3055 Marburg, Federal Republic ofGermany.

t Present address: Henkel KGaA Dusseldorf, Biotechnologie,D-4000 Dusseldorf, Federal Republic of Germany.

§ Present address: Institut fur Genbiologische Forschung BerlinGmbH, D-1000 Berlin 33, Federal Republic of Germany.

structure-function relationships, the lactose carrier can beconsidered a prototype of this class of proteins in E. coli. Itis synthesized without a cleavable N-terminal signal peptide(9), as has also been found for other integral membraneproteins (see references 30 and 45 for review). Recently,lactose permease was shown to be synthesized on mem-brane-bound polysomes, and its N-terminal region wasshown to mediate contact with the lipid bilayer during chainelongation (39). To test the ability of different portions of thelactose carrier to interact with the lipid bilayer, we con-structed the plasmids described below. They encode trun-cated proteins of lactose permease as well as a mutantprotein carrying an internal deletion. These polypeptideswere compared with the wild-type carrier by investigatingtheir membrane association and their susceptibility to pro-teolytic attack.

MATERIALS AND METHODSAbbreviations. PAGE, Polyacrylamide gel electrophore-

sis; SDS, sodium dodecyl sulfate; kb, kilobase; bp, basepair.Enzymes. Restriction endonucleases and enzymes used in

the construction and analysis of plasmids described belowwere purchased from Boehringer, Mannheim Federal Re-public of Germany (FRG), New England Biolabs, Schwal-bach, FRG, and Bethesda Research Laboratories "GIBCO-BRL," Freiburg, FRG. Pancreatic DNase I (EC 3.1.21.1)was from Worthington Diagnostics.

Deoxyoligonucleotide linkers. EcoRI deoxyoligonucleotidelinkers (8-mer) were from New England Biolabs; Schwal-bach, FRG. The completely symmetrical deoxyoligonucleo-tide linker 5'-TAGCTAGGTAGATCTACCTAGCTA-3' wassynthesized on a DNA synthesizer (Applied Biosystems380A) tuned to phosphoamidite chemistry (7). After depro-tection, it was purified by electrophoresis through a dena-

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2640 STOCHAJ ET AL.

turing 20% polyacrylamide gel. When introduced into eitherplasmid pGM21 or pBR322, the linker provides a uniqueBglII recognition site as well as at least one translationalterminator within each reading frame. Sources for othermaterials have been indicated previously (19, 38).

Bacteria and plasmids. The starting plasmid pGM21 and E.coli T184 (41), used as the bacterial host for all plasmidconstructions and for DNA preparations, were kindly pro-vided by P. Overath. Expression of plasmid-encoded lacYalleles was studied in a derivative of the minicell-producingstrain DS410 (33). It contains an F' lacIq plasmid to ensureoverproduction of lactose repressor (38).

Construction of plasmids carrying mutant lacY alleles.Plasmids directing the synthesis of different truncated lac-tose carrier molecules were constructed by in vitro DNAmanipulations (Fig. 1). In addition, a plasmid containing aninternal deletion within the lacY gene is described. Standardmethods (21) were used for the cloning procedures and foranalysis of the newly constructed plasmids.Transformation was done as described elsewhere (15). A

simplified nomenclature is used for those plasmids express-ing lacY-related polypeptides: their designation indicates thesegment of the lactose carrier that they encode (see Fig. 2).pY50 was obtained by random insertion of the 24-mer

BglII linker described above into plasmid pGM21 (Fig. 1),essentially as described (16): linearized full-length plasmidDNA was produced by mild treatment with pancreaticDNase I in the presence of Mn2" ions and purified byagarose gel electrophoresis. It was incubated with E. coliDNA polymerase (large fragment) in the presence of the fourdeoxyribonucleoside triphosphates and was subsequentlyused for insertion of the double-stranded DNA linker. Se-quence analysis by the chemical degradation method (23)showed the linker to be inserted adjacent to the codonspecifying amino acid residue 50, with the first terminatorcodon of the linker in the position of codon 51. On the distalside of the DNA linker, a 22-bp deletion was found to beadjacent to the linker. The occurrence of similar deletionsupon insertion of linkers by the method described has beenreported previously (16).pVI-1 corresponds to the parent plasmid pGM21 except

that nonessential DNA sequences have been deleted fromthe vector. It was derived from an intermediate, plasmidpAS142. The latter is similar to plasmid pYS0 except that itwas found to carry the newly introduced BglII linker (seeabove) within the DNA sequence of the vector (Fig. 1). Theshortened vector plasmid pV142 was constructed frompAS142 by attaching EcoRI linkers to both ends of theAvaI-BglII fragment after the recessed ends had been filled.The resulting 2.5-kb plasmid contains the origin of replica-tion and the tetracycline resistance gene of pACYC184 (21).The original 2.3-kb insert from pGM21 containing the lacYgene was introduced into the EcoRI site to yield plasmidpVI-1. It served as the starting material for the constructionof plasmids pY71/1, pY143, and pAY72-142.For pY71/1, the unique AvaI restriction site within the

lacY gene of plasmid pVI-1 was cleaved, the recessed endswere filled in, and the BglII linker (24-mer) described abovewas introduced into this site. DNA sequence analysis re-vealed the linker to be inserted on the carboxy-terminal sideof codon 71 so that amino acid residue 71 was followed firstby a serine residue and then by a UAG terminator codonspecified by the linker.The construction of plasmid pY143 is analogous to that of

pY71/1 except that a recognition site for BssHII locatedwithin the lacI gene was first destroyed by linker insertion.

210bp f ragment

FIG. 1. Construction of plasmids specifying mutant forms oflactose permease. In vitro manipulations used in the derivation ofthe plasmids are described in the text. The newly constructedplasmids used in this study are indicated by larger letters.

The remaining unique BssHII site in the lacY gene was usedto insert the 24-mer BglII linker. DNA sequence analysisascertained that codon 143 of the lacY structural gene wasfollowed immediately by an amber terminator codon.The same BssHII site within the lacY gene (now unique on

the plasmid) was used in conjunction with the unique AvaIsite to construct a plasmid (pAY72-142) carrying a deletion ofcodons 72 to 142 of the lacY sequence. As expected from theknown DNA sequence, filling in the recessed ends andreligation resulted in concomitant loss of the AvaI and

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TRUNCATED FORMS OF E. COLI LACTOSE PERMEASE

restoration of the BssHII recognition sites. As the mutantprotein carrying this internal deletion reacts with an anti-body preparation specifically elicited against a syntheticpeptide identical to the C-terminus of lactose permease (35),the accidental introduction of a frameshift mutation can beruled out (unpublished experiments by U. Sonnewald and U.Stochaj).

It should be pointed out that these last two plasmids,pY143 and pAY72-142, are very similar to plasmidspUR2YR1 and pUR2YA1, respectively, described previ-ously (M. Markgraf, diploma thesis, University of Cologne,1983). The lacY-related polypeptides specified were found tobe practically identical to those described in this study asproducts of pY143 and pYA72-142, except that plasmidpUR2YR1 encodes a truncated protein comprising 143 resi-dues of the lactose carrier followed by four additional aminoacids unrelated to the lacY sequence. As a consequence ofthe particular cloning procedure and of the vector employed,those previous plasmid constructions were less advanta-geous, as they were found to specify the synthesis ofadditional proteins unrelated to the lactose carrier (39;unpublished experiments by U. Stochaj).

Plasmid pY174 encodes the N-terminal 174 amino acidresidues of the lactose carrier followed by the UGA termi-nator codon at position 492 of the pBR322 sequence (40).Using the BanII restriction site at position 485 of the pBR322sequence, we replaced the EcoRI-BanII fragment of pBR322by the 1,225-bp EcoRI-BanII fragment from the insert ofpGM21 (Fig. 1).

Analysis of plasmid-encoded proteins. Biosynthetic radio-labeling of proteins in plasmid-bearing E. coli minicells with[35S]methionine and preparation of a crude membrane frac-tion have been described (38, 39). Extraction of membranevesicles with urea followed by treatment with sodiumcholate was carried out as described (29). Solubilization ofthe lactose carrier by octylglucoside in the presence of E.colj phospholipids followed published procedures (28, 39).Electrophoretic separation on polyacrylamide gradient gels(11 to 20% acrylamide in the presence of SDS [20]) was usedto detect wild-type lactose permease and proteins AY72-142,Y174, and Y143. Fractions containing the truncated poly-peptides Y71/1 and Y50 were subjected to electrophoresis inSDS and urea (39).

Conditions for indirect immunoprecipitation have beendescribed recently (39). Immunoglobulin G preparations ofpeptide-specific antibodies (36) directed against syntheticpeptides comprising either residues 125 to 135 (anti-P125_135)or residues 408 to 417 (anti-CT) were kindly provided by R.Seckler and P. Overath. The N-terminal proteolytic frag-ment of lactose permease extending approximately to resi-due 140 as well as the truncated proteins Y143 and Y174have been detected in indirect immunoprecipitation experi-ments with absorption of the immune complexes to Staphy-lococcus aureus cells (38, 39). So far, neither of thesepolypeptides has been observed in immunoblotting proce-dures (36; unpublished experiments by U. Stochaj).

Proteolytic digestion of wild-type or mutant lactose per-mease in inside-out membrane vesicles derived from radio-labeled minicells was done in 38.5 mM potassium phosphate(pH 7.5)-0.8 mM dithiothreitol-1.5 mM MgSO4-385 ,ug ofbovine serum albumin per ml (38). Treatment with thermoly-sin was done in this buffer with the addition of 3.1 mMCaCl2; cleavage was done with clostripain in the presence of3.1 mM CaCl2 and 6.0 mM dithiothreitol. The final concen-tration of thermolysin, trypsin, and chymotrypsin was 7.7,ug/ml and of clostripain was 135 ,ug/ml.

a) L 100 200 300

143

71 Ser pY71/150 pY50

-17 pGM21pY 174pY 143

cytoplaslTh side of membran

FIG. 2. Diagrammatic representation of polypeptides encodedby mutant lacY alleles described in this study. The relative length ofdifferent mutant proteins of the lactose carrier is compared with thatof the wild-type carrier. Codon numbers indicate positions of theC-termini of truncated polypeptides or, for the internal deletionprotein AY72-142, the deleted amino acids. The additional serineresidue at position 72 of protein Y71/1 in the diagram is not presentin the wild-type sequence of lactose permease; it resulted from thein vitro DNA manipulations. The different mutant proteins arealigned to a simplified form of the model proposed by Vogel et al.(43) for the structure of lactose permease. According to theseauthors, the four more hydrophilic a-helices, 7, 8, 9, and 12, are notpredicted to span the membrane with certainty (43). The figure isfrom reference 39, with minor modification. Plasmids are referred toby names based on the mutant lac Y protein they encode, as detailedin Materials and Methods.

After 30 min of incubation at 20°C, proteolysis wasstopped by the addition of inhibitors to yield final concen-trations of 10 mM phenylmethylsulfonyl fluoride and 35 mMEDTA (pH 8.0), and a mixture of protease inhibitors (anti-pain, aprotinin, chymostatin, leupeptin, and pepstatin [1[Lg/ml each]) was added.Accumulation of [14C]lactose by cells of strain T184 (41)

harboring plasmid pAY72-142, pY143, or pY71/1 was testedas described (2). No significant transport has been detectedin cells grown for several generations in the presence ofinducer. Residual levels of less than 5% of the transportactivity obtained from the same strain carrying the parentplasmid pGM21 would not have been detected by ourassays.

Preliminary tests for substrate protection against modifi-cation by N-ethylmaleimide were performed essentially aspreviously described (38). So far, results have been negativewith crude membrane preparations obtained from cultures ofstrain T184 (41) harboring plasmids pAY72-142 and pY174grown in the presence of inducer. Other mutant derivativeshave not been tested.

RESULTS

Construction of plasmids carrying different mutant lacYalleles. The mutant lacY alleles described in this study havebeen derived from the wild-type allele carried on the 2.3-kbinsert of the widely used plasmid pGM21 (41). Their tran-scription from the wild-type control region of the lactoseoperon is subject to regulation by lactose repressor. Detailsof their construction are described in Materials and Methods(Fig. 1). The fractional length of each truncated lacY proteinis shown diagrammatically in Fig. 2 in relation to the lengthof the complete polypeptide as derived from its DNA se-quence (4). A simplified nomenclature is used for the plas-mids based on the mutant lacY polypeptide they encode.

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2642 STOCHAJ ET AL.

a)1 2 3 4

b)1 2

69-

46-

184 lrT12.3- a

69-

46-

30- _

184-.i1t

FIG. 3. Biosynthesis of wild-type and mutant lactose permeasein E. coli minicells. Wild-type and mutant proteins of the lactosecarrier were biosynthetically radiolabeled in plasmid-bearing mini-cells. The reaction was terminated by the addition of trichloroaceticacid. Total proteins were subjected to SDS-PAGE and autoradiog-raphy. Positions of marker proteins (Mr in thousands) are indicated.(a) Radiolabeled proteins of minicells bearing plasmids pGM21 (lane1), pAY72-142 (lane 2), pY174 (lane 3), and pY143 (lane 4) areshown. The labeled proteins visualized in this figure include solubleproteins not retained in the minicell membrane fractions preparedfor the experiments shown in Fig. 4 to 6. (b) Radiolabeled proteinsof minicells harboring pY71/1 (lane 1) and pY50 (lane 2) wereprepared as described above and separated by SDS-PAGE in 7 Murea. The positions of wild-type and mutant proteins of lactosepermease are marked by arrows.

Plasmids pY50, pY143, and pY174 (Fig. 1) specify exactlythe N-terminal 50, 143, and 174 amio acid residues of thelacY sequence, respectively. In pY71/1 an additional serinecodon introduced by the DNA manipulations follows thesequence of the 71 N-terminal lacY codons. In the deletionplasmid pAY72-142, codon 71 is followed immediately bycodon 143 of the wild-type sequence.The truncated proteins Y71/1 and Y143 are of particular

interest, as they contain those segments of the proteinpredicted to form two and four N-terminal membrane-spanning a-helices, respectively. This is evident from com-parison with the structural model (43) outlined in Fig. 2. Onthe other hand, the internal deletion in pAY72-142 is pre-dicted to result in a loss of the third and fourth membrane-spanning ao-helices. It may be noted that models proposed byother authors (1, 11) for the structure of lactose permeasealso predict that the region between residues 10 and 125 ofthe lactose carrier forms four membrane-spanning a-helicesfollowed by a less tightly folded region extending approxi-mately to amino acid residue 140.

Identification of lactose permease-related mutant polypep-tides. The different mutant plasmids were expressed in E.coli minicells. As expected from the nature of the mutationalchanges introduced into the coding region of the lacY gene(see Materials and Methods), the intact lactose permeaseencoded by the parent plasmid (lane 1, Fig. 3a) is replaced bymutant polypeptides of increased electrophoretic mobilities:a protein migrating slightly faster than the Mr30,000 markerwas observed with minicells carrying the internal deletionplasmid pAY72-142 (lane 2); plasmids pY174 (lane 3) andpY143 (lane 4) specified products which migrated as Mr-16,000 and -13,000 proteins, respectively. The bands oflower electrophoretic mobilities marked by arrows in lanes 1and 2 of Fig. 3b were specific for minicells carrying plasmidspY71/1 and pY50, respectively. (Differences between thesetwo protein samples with respect to additional bands are

attributed to slight differences between the two vector plas-mids. As shown in Fig. 1, nonessential sequences present inpY50 have been removed in the construction of the vectorplasmid pV142.)The polypeptides indicated by arrows in Fig. 3 as related

to lactose permease were identified as follows. We haveshown previously (39) that synthesis of the truncated poly-peptides in E. coli minicells containing lactose repressor isstimulated by the inducer isopropyl-3-D-thiogalactopy-ranoside. This was also observed for the mutant polypeptideAY72-142 (data not shown).

Plasmids pGM21 and its derivatives, except pY174, en-coded the tetracycline resistance gene product in addition tothe lactose permease-related polypeptides. Under the con-ditions for electrophoretic separation used here (20), itcomigrated with the wild-type lactose carrier. pY174 is aderivative of pBR322 that did not express the tetracyclineresistance gene (Fig. 1). Instead, one recognized the matureform and residual precursor of the P-lactamase. Identifica-tion of the vector-specific gene products rests on comparisonwith the proteins expressed by E. coli minicells harboringthe corresponding vector plasmids and deletion derivativesthereof (data not shown).

In addition, the two larger truncated lactose permeasederivatives, Y143 and Y174, have been identified by indirectimmunoprecipitation (39). Whereas these polypeptides con-tain the sequence of residues 125 to 135 of lactose permeasethat is recognized by the peptide-specific antiserum anti-P125.135 (36), no suitable antiserum was available for indirectimmunoprecipitation of the short truncated polypeptidesY50 and Y71/1. In contrast, the internal deletion polypeptideAY72-142 (lane 2, Fig. 3a) has also been identified by indirectimmunoprecipitation (data not shown). It reacts with thepeptide-specific antibodies directed against the C-terminusof lactose permease described by Seckler et al. (35).When compared with marker proteins in the presence of

SDS, the mutant proteins had a higher electrophoretic mo-bility in relation to marker proteins than expected from theirrelative molecular masses. The values calculated from theiramino acid composition (as derived from the DNA se-quence) are 16,400 and 20,000 for the truncated proteinsY143 and Y174, respectively, and 38,600 for protein AY72-142. They migrated, however, like proteins of Mr 13,000,16,000, and 28,000, respectively (Fig. 3a). Such anomalouselectrophoretic mobility is well known for intact lactosepermease. In the gel system used here, it migrated like a32,000-dalton protein in SDS despite an Mr of 46,500 (46).The relative electrophoretic mobility of the shorter truncatedpolypeptides Y50 and Y71/1 has not as yet been criticallyevaluated, as they were separated in the presence of 7 Murea and SDS (5).Membrane association and solubilization behavior of dif-

ferent mutant lactose carrier proteins. Methods developedspecifically for the solubilization of wild-type lactose perme-ase were applied to study the membrane association of themutant polypeptides. The different truncated polypeptideswere found to sediment with the membrane fraction. Theyresembled the. wild-type protein in their solubilization char-acteristics. A large fraction of the E. coli membrane proteinscan be solubilized by treatment of membrane vesicles withurea and sodium cholate. In contrast, the intact lactosepermease remains insoluble under these conditions (29).Similarly, the different mutant proteins were recovered inthe lipid-containing sediments after extraction of membranevesicles with urea and sodium cholate. This indicates a tightinteraction of the different proteins with the lipid bilayer

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a)

1 2 3 415 6I7 8

wt Y174 Y143 s£Y72-142M UC M UC M UCim UC

.I * i

b)

2 3i 4

Y71/I1 Y150UCIM UC

-~~~~~~ ~ ~ ~~~~~~~~~~~~~~~~~~~..h

....mM... :-

FIG. 4. Extraction of membrane vesicles containing wild-type or

mutant lactose permease with urea and sodium cholate. (a) Mem-branes of minicells containing wild-type lactose permease (wt, lanes1 and 2), protein Y174 (lanes 3 and 4), protein Y143 (lanes 5 and 6),or protein AY72-142 (lanes 7 and 8) were subjected to extractionwith urea followed by treatment with sodium cholate. Comparableamounts of radioactivity of minicell membranes (M) and sedimentsafter treatment with urea-cholate (UC) were electrophoreticallyseparated in parallel. Positions of wild-type and mutant proteins ofthe lactose carrier are marked by arrows. (b) The same procedure as

described for panel a was applied to minicells containing proteinY71/1 (lanes 1 and 2) or protein Y50 (lanes 3 and 4). SDS-PAGE was

done in 7 M urea.

even for segments containing only 50 or 71 N-terminal aminoacid residues of the carrier (Fig. 4). On the other hand, thetruncated proteins could be efficiently solubilized with thenonionic detergent octylglucoside in the presence of phos-pholipids by the procedure described (28) (lanes 3, 7, and 9,Fig. 5a; lanes 3 and 5, Fig. Sb). Whereas these truncatedproteins clearly behaved similarly to the wild-type protein,polypeptide AY72-142, which carries an internal deletion of

09, S Og

6 7 8 9

S Og S Og

b)

1 2 3 4

S Og S...... .......69- 4

69-.;

46-:

184-

30i-w.12.3- -In0s~...

FIG. 5. Solubilization of wild-type and mutant lactose carrierwith octylglucoside. Lactose permease was biosynthetically labeledin minicells, and samples of crude membranes were treated withoctylglucoside in the presence of E. coli phospholipids (28). Thesolubilized proteins (Og) were electrophoretically separated in par-

allel with equivalent amounts of the corresponding starting material(S), sediments of untreated membrane vesicles. Marker proteins (Mrin thousands) are indicated on the left. (a) Membranes were fromminicells bearing plasmids pGM21 (lanes 2 and 3), pAY72-142 (lanes4 and 5), pY174 (lanes 6 and 7), and pY143 (lanes 8 and 9). (b)Minicells harboring pY71/1 (lanes 2 and 3) and pY50 (lanes 4 and 5)were subjected to the procedure described above. Their proteinswere separated by SDS-PAGE in urea.

71 amino acid residues, was not solubilized under identicalconditions (lane 5, Fig. 5a).

Similarly, we have previously observed that the truncatedproteins can be recovered from the organic solvent when themembrane vesicles obtained after treatment with urea andsodium cholate are subsequently extracted with 1-butanol(39). This was expected from the known properties of theintact protein (18). In contrast, the same treatment provedineffective for solubilization of protein AY72-142, carryingthe internal deletion (data not shown).The N-terminal portion of lactose permease can fold inde-

pendently of the C-terminal half of the molecule. Previousexperiments with limited proteolysis used to study thetopology of lactose permease have revealed only a fewhigh-molecular-weight proteolytic fragments, indicating thatmany potential cleavage sites are not accessible to proteasesacting on the membrane-associated carrier (1, 13, 36, 38). Bythe use of lactose permease radiolabeled 'biosynthetically' inE. coli minicells, a characteristic cleavage product derivedfrom the N-terminal segment of the protein has been identi-fied (38). It was found to be exceptionally resistant to furtherproteolytic attack even in the presence of nonionic deter-gents. Most likely, it represents a tightly folded portion ofthe molecule. Under the conditions used here for electro-phoretic separation, it migrated like a protein of Mr 13,000.It was shown to contain the intact N-terminus of lactosepermease and to extend into the region around amino acid140. It reacted with antibodies directed against a syntheticpeptide comprising amino acids 125 to 135 of the lactosecarrier (38). No such fragment was produced when mem-brane vesicles derived from minicells harboring a vectorplasmid were exposed to proteolytic enzymes under thesame conditions (data not shown).To examine the properties of this N-terminal segment

within the truncated proteins Y174 and Y143, they weresubjected to limited proteolysis in parallel with intact lactosepermease. Membrane vesicles from minicells containingthese polypeptides were treated with proteases of differentspecificities for the cleavage site. The typical N-terminalfragment was produced from the truncated protein Y174 aswell as from the intact carrier (Fig. 6).As expected from the cleavage site previously deduced to

be near amino acid residue 140 (38), polypeptide Y143 wasnot significantly affected by treatment with proteases underthe conditions used here. It comigrated with the N-terminalproteolytic fragment produced from the intact protein as wellas from protein Y174. In the presence of SDS, however,protein Y143 could be degraded to low-molecular-weightfragments not retained in the gel. This reaction was en-hanced by prior boiling of the suspension of membranevesicles (data not shown).

DISCUSSION

Internal signal sequences have been identified within somemembrane proteins lacking a transient N-terminal signalpeptide (25, 37, 45). Bovine opsin, one of the model systemsfor complex integral membrane proteins, was shown tocontain more than one internal signal sequence (12). ThelacY plasmids described in this study were specificallyconstructed to test different segments of the E. coli lactosecarrier for their ability to interact with the membrane. Allmutant proteins examined so far were intimately associatedwith the lipid bilayer. Thus, they contain at least onesequence permitting stable integration into the membrane.This is true even for the small truncated polypeptides Y50

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112 3 4 5 6 7 8 9101112 13 141516Protease | Cl Th Tr

46-=w;

30 1" .

184-NE;

12 3- . 1 0" -

FIG. 6. Limited proteolysis of wild-type lactose permease andproteins Y174 and Y143. Wild-type and mutant proteins of thelactose carrier were biosynthetically labeled in minicells, and crudemembrane fractions were digested with clostripain (Cl, lanes S to 7),thermolysin (Th, lanes 8 to 10), trypsin (Tr, lanes 11 to 13), andchymotrypsin (Ch, lanes 14 to 16). Digestion with clostripain wasperformed in the presence of 3.1% (wt/wt) Triton X-100, as thisenzyme acts only weakly on lactose permease in the absence ofdetergents. Untreated controls are shown in lanes 2 to 4. Wild-typelactose permease was applied to lanes 2, 5, 8, 11, and 14; proteinY174 is in lanes 3, 6, 9, 12, and 15; and protein Y143 is in lanes 4, 7,10, 13, and 16. The position of the N-terminal proteolytic fragmentis marked by an arrow. Marker proteins (lane 1, Mr in thousands) areindicated on the left.

and Y71/1, comprising only 50 and 71 N-terminal amino acidresidues of lactose permease, respectively, as well as for thedeletion polypeptide AY72-142, which lacks the segmentpredicted to form the third and fourth membrane-spanninga-helices (Fig. 2).The truncated proteins resembled the wild-type carrier in

their solubilization characteristics. They were resistant toextraction of the membrane fraction by urea and sodiumcholate, whereas they were solubilized efficiently by proce-dures specifically developed for the intact protein. In con-trast, the deletion polypeptide AY72-142 was not obtained ina soluble form under these conditions. It remained tightlyassociated with the lipid bilayer. One might speculate thatthe loss of the segment comprising residues 72 to 142 and inparticular those in positions 125 to 142 could result in a lessflexible structure, preventing successful interaction withnonionic detergents and organic solvents. So far, we have no

evidence suggesting any functional difference between cellsharboring the internal deletion plasmid pAY72-142 and cellscarrying one of the plasmids encoding a truncated lactosecarrier. The differences observed in the solubilization prop-erties of the mutant proteins cannot at present be correlatedwith any differences in residual levels of transport activity or

substrate binding.As mentioned above, structural studies predict the lactose

carrier to be almost completely folded into up to 14 mem-brane-spanning a-helices (11, 43). The mutant polypeptideY71/1 contains the N-terminal segment predicted to fold intoa helical hairpin; the two larger truncated proteins Y143 andY174 could form in addition the second helical hairpin (Fig.2). Such a structure may indeed be formed by polypeptidesY143 and Y174, as suggested by their response to limitedproteolysis (see below). Although such helical hairpins may

be presumed to promote spontaneous partitioning of theseextremely hydrophobic truncated polypeptides into the lipidbilayer (10, 44), it remains to be tested whether binding toand/or integration into the membrane require additional

cellular factors, such as the products of genes secA and/orsecY (30).

Proteins Y174 and Y143 resembled the wild-type proteinnot only in their solubilization behavior but also in theirresponse to different proteases: exposure of these polypep-tides to different proteolytic enzymes resulted in accumula-tion of the characteristic N-terminal proteolytic fragment, atypical cleavage product of the wild-type carrier. The un-treated protein Y143 (which comprises 143 N-terminal aminoacid residues) comigrated with the wild type tryptic fragmentand was hardly affected by proteases in the absence ofnonionic detergents. These results suggest that the N-ter-minal region of lactose permease represents an autonomousdomain able to fold independently from the C-terminalportion of the molecule and to assume a structure resemblingthat of the corresponding segment within the intact mem-brane-bound protein.

ACKNOWLEDGMENTS

We thank Barbara Bieseler and Benno Muller-Hill for helpfuldiscussions and Robert Seckler and Peter Overath for generous giftsof antisera and immunoglobulin preparations.

This work was supported by the Deutsche Forschungsgemein-schaft through grant SFB 74-A2 to B. Muller-Hill and grants SFB74-Il and Eh 25/2-1 to R. Ehring.

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