Vol. 170, No. 10

Two Regions of Mature Periplasmic Maltose-Binding Protein ofEscherichia coli Involved in Secretion

PASCALE DUPLAY AND MAURICE HOFNUNG*

Unite de Programmation Moleculaire et Toxicologie Genetique, Centre National de la Recherche Scientifique UA271,Institut National de la Santg et de la Recherche Medicale U163, Institut Pasteur, 25 rue du Dr Roux, 75015 Paris, France

Received 9 February 1988/Accepted 18 June 1988

Six mutations in malE, the structural gene for the periplasmic maltose-binding protein (MBP) fromEscherichia coli, prevent growth on maltose as a carbon source, as well as release of the mutant proteins by thecold osmotic-shock procedure. These mutations correspond to insertion of an oligonucleotide linker, concom-itant with a deletion. One of the mutations (malE127) affects the N-terminal extension (the signal peptide),whereas the five others lie within the mature protein. As expected, the export of protein MalE127 is blockedat an early stage. This protein is neither processed to maturity nor sensitive to proteinase K in spheroplasts.In contrast, in the five other mutants, the signal peptide is cleaved and the protein is accessible to proteinaseK added to spheroplasts. This indicates that the five mutant proteins are, at least in part, exported through theinner membrane. We propose that the corresponding mutations define two regions of the mature protein(between residues 18 and 42 and between residues 280 and 306), which are important for release of the proteinfrom the inner membrane into the periplasm. We discuss the results in terms of possible conformationalchanges at this late step of export to the periplasm.

Instructions for the cellular localization of proteins arecontained within their structural genes. It is already wellestablished that bacterial proteins, which are exportedthrough the cytoplasmic membrane, are synthesized with anN-terminal extension, the signal peptide, which is cleavedupon export (21, 26, 28). For periplasmic proteins, such asthe maltose-binding protein (MBP) of Escherichia coli, ex-port requires at least three steps: initiation of transfer,translocation through the inner membrane, and release intothe periplasm. In a normal situation, these steps are parts ofa continuous process; some of them can be detected withradioactive labeling. Under some conditions (mutations,inhibitors, low temperatures, etc.), intermediate forms areobserved, which may, in favorable cases, represent trueintermediates of the export pathway (for recent reviews, seereferences 5 and 34).The best-defined part of the structural gene involved in

export corresponds to the signal peptide (for recent reviews,see references 4, 31, and 34). The signal peptide of theperiplasmic MBP, the product of the malE gene, presentscharacteristics which are common to all signal peptides fromthis bacterium (6, 7, 37). Three regions can be distinguished:the extreme amino-terminal sequence is composed of eightresidues, three of which are basic; it is followed by a stretchof hydrophobic and neutral residues (the hydrophobic core);the last six residues before the cleavage site are believed tocontain residues from the recognition sequence of the proc-essing enzyme. In particular, the sequence Ala-Leu-Ala, atpositions -3 to -1 relative to the site of proteolytic cleav-age, is similar to the consensus (Ala-X-Ala) for recognitionby signal peptidase 1 (16, 17, 35). Despite intense efforts, theexact roles played by the signal peptide in export are still notclear. The signal peptide appears to be involved in maintain-ing the precursor in an export competent conformation andto promote attachment of the precursor to the inner mem-brane (34). These effects may require additional factors.Later in the process, cleavage of the signal peptide is

* Corresponding author.

required to release the translocated protein from the innermembrane; cleavage is not required for translocation itself(11, 17).Much less information is available on the regions in the

mature protein important for export and on the role of theseregions in the export process. A priori, the sequence of thetransfered protein has to satisfy at least two conditions. Onthe one hand, it has to be export compatible: for example, itshould not include highly hydrophobic membrane anchorsequences (12). On the other hand, it may also play an activerole in export. For periplasmic proteins such as MBP, it wasalready shown that the C-terminal 25 to 35 residues areneeded for release into the periplasm but are not required fortranslocation (22). It was also shown that mutations in themature part of the protein could reverse, at least to someextent, the effect of mutations of the signal peptide affectinginitiation of export (for reviews, see references 10 and 36):three amino acids in the mature portion of the protein(Lys-1, Gly-19 and Tyr-283) have been identified by thiscriterion. On the basis of in vivo competition experimentsbetween wild-type and export-defective proteins, it has alsobeen proposed that amino acid residues 89 to 189 contain aninternal export sequence (1, 2). In addition, it has beensuggested that MBP is competent for export only before itfolds into its final stable tertiary conformation (33). All thispoints to a role for the mature part of the protein in export,but the exact functions and the nature of the sequencesinvolved remain to be elucidated.

In the present paper, we provide information pertinent tothe role of the mature part of the periplasmic maltose-binding protein of E. coli in the last step of the export:release into the periplasm. We show that instructions for thisstep are encoded in two distinct regions internal to themature protein and discuss possible implications of thisresult.

MATERIALS AND METHODS

Media, bacterial strains, and growth conditions. Minimalmedium M63 and indicator media have been described (29).

4445

JOURNAL OF BACTERIOLOGY, Oct. 1988, p. 4445-44500021-9193/88/104445-06$02.00/0Copyright C 1988, American Society for Microbiology

on January 21, 2019 by guesthttp://jb.asm

.org/D

ownloaded from

4446 DUPLAY AND HOFNUNG

All strains used in this study are isogenic derivatives of E.coli K-12 strain pop3325 (F- A/acU169 araDl39 rpsLIS0 thiflbBS301 deoC7 ptsF25 relAl malT(Con)l. The ma/T(Con)lallele confers constitutive expression on the maltose oper-ons. PMPD92 was constructed by introducing the metAallele into pop3325 by transduction to rifampin resistanceand screening for a Met- phenotype by using bacteriophageP1 vir grown on MM131 (9). The malE mutations werecrossed by P1 vir transduction into strain PMPD92. Met'-transductants were selected and screened for a Mal- pheno-type on MacConkey maltose plates.

All strains were grown overnight at 37°C in M63B1 me-dium containing 0.2% glycerol and diluted 1:50 into the samemedium containing 0.2% glycerol and 0.2% maltose. Cul-tures growing exponentially were labeled at a cell density of3 x 108 cells per ml.

Pulse-chase experiments. The cells were labeled with 50,uCi of [35S]methionine per ml for 15 s. A chase withunlabeled methionine was performed by addition of anexcess of nonradioactive methionine (final concentration,0.05%) for 45 s. The pulse-labeling and pulse-chase-labelingexperiments were terminated by mixing the sample with anequal volume of ice-cold 10% trichloroacetic acid. In exper-iments in which export was blocked, carbonyl cyanidem-chlorophenylhydrazone (CCCP) was added to a finalconcentration of 8 ,uM 5 min before the cells were labeledwith 50 ,Ci of [35S]methionine per ml for 1 min. CCCP is anuncoupler that dissipates proton motive force and blocksexport (32).

Localization of proteins. Cultures (1.5 ml) were labeledwith 40 ,uCi of [35S]methionine for 40 s. The samples werechilled by being mixed with 1.5 ml of a crushed-ice solutioncontaining 200 Rg of chloramphenicol per ml and 200 mMsodium azide. The cells were washed with 0.5 ml of 50 mMTris hydrochloride (pH 8)-S15 mM TAME (tosyl argininemethyl ester) and suspended in 0.3 ml of 0.1 M Tris hydro-chloride (pH 8)-0.5 M sucrose-5 mM EDTA-15 mM TAME.Lysozyme was added to a final concentration of 160 p.g/ml,and then 0.3 ml of ice-cold water was added. After incuba-tion for 30 min on ice, the spheroplasts were divided intothree portions of 0.2 ml and subjected to three paralleltreatments.

(i) Proteinase K treatment in lysed cells. The spheroplastswere centrifuged, and the supernatant was kept as theperiplasmic fraction. The spheroplasts were lysed by sus-pension in 0.8 ml of 50 mM Tris hydrochloride (pH 8)-2.5mM EDTA. After the spheroplasts were lysed, the periplas-mic fraction was added back. The samples were then treatedwith proteinase K at a final concentration of 50 jig/ml afteraddition of MgSO4 to a final concentration of 20 mM. Thesuspensions were incubated on ice for 20 min, and phenyl-methylsulfonyl fluoride was added to a final concentration of1 mM.

(ii) Proteinase K treatment in intact spheroplasts. MgSO4was added to a final concentration of 20 mM, and thespheroplasts were treated with proteinase K as describedabove for lysed spheroplasts.

(iii) Cell fractionation and control experiment in the absenceof proteinase K. The spheroplasts were either precipitatedwith 5% trichloroacetic acid or centrifuged to separateperiplasmic proteins from spheroplast-associated proteins.In the latter case, the spheroplasts were stabilized with 20mM MgSO4 (final concentration) and centrifuged for 10 min.The supernatant was kept as the periplasmic fraction. Thespheroplasts were washed once with 50 mM Tris hydrochlo-

ride (pH 8)-0.25 M sucrose and lysed in 50 mM Trishydrochloride (pH 8)-2.5 mM EDTA.

Immunoprecipitation and polyacrylamide gel electrophore-sis. All the samples were precipitated with 5% trichloroaceticacid (final concentration) and solubilized in 50 mM Trishydrochloride (pH 8)-1% sodium dodecyl sulfate (SDS)-1mM EDTA-1 mM phenylmethylsulfonyl fluoride. MBP wasimmunoprecipitated with rabbit anti-MBP serum and proteinA (21). During the immunoprecipitation procedure, all thesolutions contained 1 mM phenylmethylsulfonyl fluoride.Immune precipitates were analyzed by SDS-polyacrylamidegel electrophoresis (11% acrylamide) and autoradiography.

RESULTS

We described previously a set of MBP mutant proteinsgenerated by random insertion of an oligonucleotide linkerinto malE and selection for stable mutant proteins whichcould be immunoprecipitated with an anti-MBP serum. Incontrast to wild-type MBP and to the other 13 mutantproteins isolated, 6 mutant proteins could not be releasedfrom intact bacteria by cold osmotic shock (14). This proce-dure is taken as an operational criterion that the proteins arelocated in the periplasm (23, 24). Interestingly, the sixmutant proteins were unable to function in the transport ofmaltose (14).The nature of the six mutations is shown in Fig. 1. In all

cases, a deletion of several base pairs took place in additionto the insertion of the linker. One insertion (ma/E300)changed the malE reading frame, resulting in an earlytermination of MBP.

Processing. To obtain more information on the cellularlocation of the six mutant proteins, we examined whethertheir signal peptides were processed. Since the processingenzyme, signal peptidase I, is located at the external face ofthe cytoplasmic membrane (38), processing points to at leasta partial export of MBP for these mutants. Conversely, adefect in processing is a strong indication of a defect inexport. There are, however, some exceptions, since muta-tions have been described which prevent signal peptideprocessing but not export (8, 17).For the five mutants affected in the mature part of the

protein, as well as for the wild type, immunoprecipitationafter a 15-s pulse of radioactive [35S]methionine revealedtwo species of MBP on SDS-polyacrylamide gel electropho-resis (MaIE302, MalE345, MalE312, and MalE364 [Fig. 2];MalE300 [data not shown]). The slower-migrating bandsdisappeared after a chase of 45 s with an excess of nonra-dioactive methionine. These results suggest that the minorband corresponds to the precursor form (pMBP) and themajor band corresponds to the mature form (mMBP) of theprotein. They indicate that the signal peptides of these fivemutant proteins are processed. In contrast, mutant MalE127presented only one band, in which radioactivity continued toaccumulate during the chase period. This band migrated withthe same apparent molecular weight as pMBP, suggestingthat no processing occurs (Fig. 2). The proposal that thesignal peptide of mutant protein MalE127 is not processed isin good agreement with the fact that the malE mutationresponsible for this mutant deletes amino acids 19 to 24 ofpMBP, within the N-terminal extension and near the cleav-age site (see Discussion).

In conclusion, the five mutations located within the ma-ture part of the protein do not prevent in vivo cleavage of thesignal peptide, whereas the one mutation located within thesignal peptide prevents cleavage. The rate of maturation of

J. BACTERIOL.

on January 21, 2019 by guesthttp://jb.asm

.org/D

ownloaded from

SECRETION OF MALTOSE-BINDING PROTEIN 4447

MalE127 metl8 arg ile arg leu25 ala26 lyslmetl9 phe ser ala ser ala24ku25ala26hs1

MalE302 glyl6 tyr17 gly ser glu leu43asnl8 S1lleu ala glu val gly lys lys phe glu lys asp thr gly ile lys val tir val glu his pro asp lys42

MaIE345 phe28 asp gly ser asp pro4Oglu28 lys asp thr gly ile lys val dir val glu his39

MalE364gly32 ile33

asp3O thr3l asp pro lys34

MaIE312 glu278 phe279 gly ser ala tyr3O7leu280 glu asnI3t leu leu dtr asp glu gly leu glu ala val asn lys asp lys pro leu gly ala val ala leu lys ser3O6

MalE300 gly252 his gly ser val pro ***

FIG. 1. Amino acid sequence changes in the six mutants studied. The mutations correspond to insertion of a BamHI linker, concomitantwith a small deletion. The amino acid changes, as well as the locations of the six mutations, are indicated. In each case the upper line givesthe sequence in the mutant and the lower line (in small characters) gives the wild-type sequence in the corresponding region. Residues inbold-face type correspond to residues not found in the wild-type sequence (insertions, or frame shift for MalE300). Residues are numberedaccording to their positions in the mature wild-type protein, except for residues in the signal peptide (italic), which are numbered accordingto their position with respect to the N terminus of the wild-type signal sequence. The signal peptide is 26 residues long (6), and the matureprotein is 370 residues long (14). Residues that are both underlined and in bold-face type in the wild-type sequence correspond to the threeknown sites where mutations have been found to suppress mutations in the signal peptide (3, 10, 36). The net changes in the number ofresidues of the mutant proteins are as follows: MalE127, 3 additions and 7 deletions (net loss of 4 residues; mutation in signal peptide);MalE302: 3 additions and 16 deletions (net loss of 13 residues); MaIE345; 4 additions and 12 deletions (net loss of 8 residues); MaIE364: 2additions and 2 deletions (no change); MalE312: 3 additions and 28 deletions (net loss of 25 residues); MalE300: 5 additions and 118 deletions(net loss of 113 residues).

these mutant proteins seems comparable to that of wild-typeMBP (16): only small amounts of mutant pMBP were de-tected after 15 s of labeling, and only the mature form wasdetected after a short chase period of 45 s.

Accessibility to proteinase K. To obtain independent evi-dence for localization, we examined the effect of the additionof proteinase K to the mutant proteins in spheroplasts.Sensitivity to protease shows that the protein, or at leastpart of it, has been exported through the cytoplasmic mem-

364 345 312 302 127

45 0 45 0 45 0 45 0 45 0

WT* 0

FIG. 2. Pulse-chase experiment. Strains were labeled for 15 s

with [35S]methionine (lanes 0), and an excess of nonradioactivemethionine was added for 45 s (lanes 45). The corresponding malEalleles are indicated above each lane. For the wild-type strain (WT),8 ,uM CCCP was added for 5 min prior to labeling (lane *). MBP was

immunoprecipitated and analyzed by SDS-polyacrylamide gel elec-trophoresis and autoradiography.

brane. For each of the six mutants, cells were labeled for 40s with [35S]methionine and spheroplasts were prepared andsubjected to three parallel treatments before immunoprecip-itation with an anti-MBP serum, SDS-polyacrylamide gelelectrophoresis, and autoradiography: (i) treatment withproteinase K, (ii) no addition of proteinase K as a control forlabeling and protein stability, and (iii) treatment with pro-teinase K after spheroplasts lysis. This last condition servedas a control for the sensitivity of the proteins to proteinaseK.At this stage it is worth recalling that wild-type mMBP is

resistant to proteases. For wild-type pMBP, treatment withproteases removes an amino-terminal domain correspondingapproximately to the signal peptide but does not degrade themature portion of the protein (13). Similarly, pMBP withsignal sequence mutations usually yield the mature protease-resistant portion on protease treatment (33). The data shownin Fig. 3 confirm these results. Protease sensitivity wasdetected by the decrease or absence of the total amount ofmutant protein immunoprecipitated. As expected, wild-typemMBP was resistant to proteinase K added to the celllysates (Fig. 3, panel WT, lane K1), to the spheroplasts(panel WT, lane S), or to the periplasmic fluid (panel WT,lanes P and Sp). After treatment with proteinase K, mutantprotein MalE127 was degraded to a form migrating like thewild-type MBP in lysed spheroplasts (Fig. 3, panel 127, lane51). This is in agreement with the idea that MalE127 is aprecursor species, which is processed to a form identical orsimilar to the mature wild type. MalE127 remained unde-graded in intact spheroplasts (panel 127, lane Sp). In addi-tion, in contrast to wild-type MBP, most of the MalE127protein remained associated with spheroplasts (panel 127,

VOL. 170, 1988

on January 21, 2019 by guesthttp://jb.asm

.org/D

ownloaded from

4448 DUPLAY AND HOFNUNG

., I t ' " 9 x > , El5 e~

MON_ _mim~~d

FIG. 3. Localization and proteinase K sensitivity experiments. Strains were labeled for 40 s with [35S]methionine. Spheroplast-associatedproteins (lanes S) and periplasmic proteins (lanes P) or proteins released after the spheroplasts were washed (lanes P') (see Materials andMethods) were separated for MalE127 and wild-type MBP. Unfractionated spheroplasts (lanes Sp) or lysed unfractionated spheroplasts (lanesS1) were subjected to treatment with proteinase K (+) or not treated.

lanes P and S). These results are in agreement with the ideathat MalE127 is not exported.The five MBP carrying a mutation in the mature part of

MBP were degraded by proteinase K in spheroplasts in theabsence of lysis (Fig. 3, lanes Sp+): this confirmed theirextracytoplasmic location. The extent of degradation variedamong the five mutants: in particular, MalE364 was clearlyless sensitive than the others. In all cases, this indicated thatthe conformation of the five mutant proteins was differentfrom that of the wild type.The integrity of the spheroplasts in the experiment was

confirmed by the fact that neither the precursor form ofwild-type MBP nor that of MalE127 was degraded by theaddition of proteinase K to intact spheroplasts (Fig. 3, panelsWT and 127, lane Sp).

DISCUSSION

We have examined six stable mutant MBP, which aredeficient for maltose transport and are not released byosmotic shock. The mutations were due to insertion of anoligonucleotide linker concomitant with various deletions(Fig. 1). Five of the mutations lay in the part of the malEsequence corresponding to mMBP. One is internal to thesignal peptide. We will discuss successively the resultsobtained with each of these two categories of mutants.

Mutations in the mature protein. The five mutant proteinsaffected in the mature part of the sequence are at leastpartially exported through the cytoplasmic membrane, sincetheir signal peptide is processed and since they are sensitiveto proteinase K in intact spheroplasts. The sensitivity toproteinase K also indicates that these proteins are not foldedlike wild-type mMBP, which is resistant to proteases.What is the status of these mutant proteins with respect to

export? Several arguments suggest that these five mutantscould be affected in a late step of export, whereby theprotein is released from the inner membrane into the peri-plasm. Despite their sensitivity to proteases, these proteinsmust be quite stable in vivo, since they can be detected inunlabeled form on a Coomassie blue-stained gel. This iscompatible with the idea that they could be protected from

degradation by being partially inserted in the membrane. Inaddition, cells harboring the five different mutated alleles ona multicopy plasmid lysed under inducing conditions, i.e., inthe presence of maltose. Thus, the five mutant proteins,when overproduced, were toxic to the cell. Toxicity isusually observed for poorly exported hybrid proteins. Mu-tants blocked in a late stage of the secretion process havealready been reported. Truncated forms of several periplas-mic proteins (1-lactamase, MBP, and glycerol-phosphatephosphodiesterase) were processed but stayed associatedwith membranes and were sensitive to externally addedproteases (6, 19, 22). For glycerol-phosphate phosphodies-terase, the accumulation of the truncated form appears toblock a step common to export of different proteins. Re-cently, it has been shown that substitution at either of thetwo cysteines in 1-lactamase prevented release from themembrane during secretion (18).These five mutants can be organized into three groups.

Three mutations (malE302, malE345, and malE364) areclustered between residues 18 and 42 of the mature MBPsequence. These three deletions overlap at residues 32 and33. Mutation malE312 defines a second region betweenresidues 280 and 306. Finally, malE300 encodes a truncatedform of the protein missing the last 20 C-terminal residues. Itis striking that two regions described here include two of thethree sites identified so far by mutations suppressing theeffects of signal peptide mutation: site 19 (Gly to Val) (3) andsite 283 (Tyr to Asp) (10). This strongly suggests that theseregions play a critical role in the export. However, atpresent, it is not possible to exclude other explanations suchas complete export of an insoluble protein which staysadsorbed on the outer face of the inner membrane.How could an alteration in the mature portion of the MBP

prevent the release of the protein into the periplasm? Con-formational changes have been suggested to play a specificrole during the release of the protein into the target compart-ment. Studies of 1-lactamase export suggested that an inter-mediate, which would be protease sensitive and bound to theouter face of the inner membrane, may exist in the normalexport of this periplasmic protein (30). The two cysteine

J. BACTERIOL.

klw

AMVNmmdm-. WillII"

11,411111111111011W

on January 21, 2019 by guesthttp://jb.asm

.org/D

ownloaded from

SECRETION OF MALTOSE-BINDING PROTEIN 4449

residues of P-lactamase were proposed to be involved in theconformational change during the release of the protein intothe periplasm (18). For the carboxyterminal nonsense mu-tants (19, 22, 25), the lack of the C-terminal sequence mightprevent the protein from achieving the conformation re-quired for its release into the periplasm. The two regions ofthe mature protein described here appear to correspond tosites important for conformational changes of the proteinduring export. In this respect, it would appear logical that ifa conformational change was involved in the insertion of aprotein from the cytoplasm into the membrane, anotherchange would be required for the protein to emerge on theother side (34). Finally, it is relevant to indicate that foldinginto the active form and protease resistance may not berequired for release into the periplasm. Three linker inser-tion mutant proteins described previously (MalE120,MalE183, and MalE220) are released by osmotic shock butare inactive for transport; one of them (MalE220) is partiallydegraded in vivo (14). Further studies of the relation be-tween sequence, conformation, and export will certainlybenefit from the elucidation of the structure of the proteinand of its mode of folding.Mutation in the signal peptide. Mutant MalE127 is affected

in the signal peptide. The protein is neither exported norprocessed. The strain is completely unable to grow onmaltose, which is not the case for most MalE signal peptidemutants (36). This may be due to two additive effects. On theone hand, the mutation corresponds to the introduction of anArg residue (instead of Met) at position 19 of the signalpeptide. It was already shown that such a mutation results ina tight block in export (36). This is enough to prevent in vivoprocessing of the signal peptide. In addition, there is also anArg at position 21, which is -3 with respect to the normalcleavage site of the signal peptide. Introduction of a chargedresidue at this position is also known to be sufficient, byitself, to prevent processing by signal peptidase, but notexport (16). It is also worth noting that the mature portion ofMalE127 is resistant to proteinase K, as is wild-type MBP(13, 33). MalE127 is also able to bind amylose (6a, 14). Thisconfirms that export and processing of the signal peptide arenot required for pMBP to fold in a conformation similar tothat of wild-type mMBP, i.e., to be able to bind substrateand to be proteinase K resistant (15, 20, 33).

ACKNOWLEDGMENTSWe thank Pierre Martineau, Sevec Szmelcman, and Kalle Gehring

for comments on the manuscript. M.H. thanks Philip Bassford andhis group for stimulating discussions on maltose-binding proteinexport during a week in Chapel Hill.Our laboratory benefited from support by the Association pour la

Recherche contre le Cancer, the Fondation pour la RechercheMedicale, and the Ligue Nationale contre le Cancer.

LITERATURE CITED1. Bankaitis, V. A., and P. J. Bassford, Jr. 1984. The synthesis of

export-defective proteins can interfere with normal proteinexport in Escherichia coli. J. Biol. Chem. 259:12193-12200.

2. Bankaitis, V. A., and P. J. Bassford, Jr. 1985. Sequences withinthe mature maltose-binding protein of Escherichia coli may beactively involved in initiating the export process. Ann. Inst.Pasteur Microbiol. 136B:3-7.

3. Bankaitis, V. A., B. A. Rasmussen, and P. J. Bassford, Jr. 1984.Intragenic suppressor mutations that restore export of maltosebinding protein with a truncated signal peptide. Cell 37:243-252.

4. Bankaitis, V. A., J. P. Ryan, A. B. Rasmussen, and P. J.Bassford, Jr. 1985. The use of genetic techniques to analyseprotein export in Escherichia coli. Curr. Top. Membr. Transp.

24:105-150.5. Beckwith, J., and S. Ferro-Novick. 1986. Genetic studies on

protein export in bacteria. Curr. Top. Microbiol. Immunol. 125:5-27.

6. Bedouelle, H., P. J. Bassford, Jr., A. V. Fowler, I. Zabin, J.Beckwith, and M. Hofnung. 1980. Mutations which alter thefunction of the signal sequence of the maltose-binding proteinfrom Escherichia coli. Nature (London) 285:78-81.

6a.Bedouelle, H., and P. Duplay. 1988. Production in Escherichiacoli and one-step purification of bifunctional hybrid proteinswhich bind maltose. Eur. J. Biochem. 171:541-549.

7. Bedouelle, H., and M. Hofnung. 1981. On the role of the signalpeptide in the initiation of protein exportation, p. 361-372. In B.Pullman (ed.), Intramolecular forces. The Jerusalem Sympo-sium on Quantum Chemistry and Biochemistry, n° 14. ReidelPublishing Co., Dordrecht.

8. Boyd, D., C. D. Guan, S. Willard, W. Wright, K. Strauch, andJ. Beckwith. 1987. Enzymatic activity of alkaline phosphataseprecursor depends on its cellular location, p. 89-93. In A.Torriani-Gorini, F. G. Rothman, S. Silver, A. Wright, and E.Yagil (ed.), Phosphate metabolism and cellular regulation inmicroorganisms. American Society for Microbiology, Washing-ton, D.C.

9. Brass, J. M., and M. D. Manson. 1984. Reconstitution ofmaltose chemotaxis in Escherichia coli by addition of maltose-binding protein to calcium-treated cells of maltose regulonmutants. J. Bacteriol. 157:881-890.

10. Cover, W. H., J. P. Ryan, P. J. Bassford, Jr., K. A. Walsh, J.Bollinger, and L. L. Randall. 1987. Suppression of a signalsequence mutation by an amino acid substitution in the matureportion of the maltose-binding protein. J. Bacteriol. 169:1794-1800.

11. Dalbey, R. E., and W. Wickner. 1985. Leader peptidase cat-alyzes the release of exported proteins from the outer surface ofthe Escherichia coli plasma membrane. J. Biol. Chem. 260:15925-15931.

12. Davis, N. G., and P. Model. 1985. An artificial anchor domain:hydrophobicity suffices to stop transfer. Cell 41:607-614.

13. Dierstein, R., and W. Wickner. 1985. The leader region ofpre-maltose binding protein binds amphiphiles. A model forself-assembly in protein export. J. Biol. Chem. 260:15919-15924.

14. Duplay, P., S. Szmelkman, H. Bedouelle, and M. Hofnung. 1987.Silent and functional changes in the periplasmic maltose bindingprotein of Escherichia coli K12: transport of maltose. J. Mol.Biol. 194:663-673.

15. Ferenci, T., and L. L. Randall. 1979. Precursor maltose-bindingprotein is active in binding substrate. J. Biol. Chem. 254:9979-9981.

16. Fikes, J. D., V. A. Bankaitis, J. P. Ryan, and P. J. Bassford, Jr.1987. Mutational alterations affecting the export competence ofa truncated but fully functional maltose-binding protein signalpeptide. J. Bacteriol. 169:2345-2351.

17. Fikes, J. D., and P. J. Bassford, Jr. 1987. Export of unprocessedprecursor maltose-binding protein to the periplasm of Esche-richia coli cells. J. Bacteriol. 169:2352-2359.

18. Fitts, R., Z. Reuveny, J. van Amsterdam, J. Mulholiand, and D.Botstein. 1987. Substitution of tyrosine for either cysteine inbeta-lactamase prevents release from the membrane duringsecretion. Proc. Natl. Acad. Sci. USA 84:8540-8543.

19. Hengge, R., and W. Boos. 1985. Defective secretion of maltose-and ribose-binding proteins caused by a truncated periplasmicprotein in Escherichia coli. J. Bacteriol. 162:972-978.

20. Ito, K. 1982. Purification of the precursor form of maltose-binding protein, a periplasmic protein of Escherichia coli. J.Biol. Chem. 257:9895-9897.

21. Ito, K., P. J. Bassford, Jr., and J. Beckwith. 1981. Proteinlocalization in Escherichia coli: is there a common step in thesecretion of periplasmic and outer-membrane proteins? Cell 24:707-717.

22. Ito, K., and J. R. Beckwith. 1981. Role of the mature proteinsequence of maltose-binding protein in its secretion across theEscherichia coli cytoplasmic membrane. Cell 25:143-150.

VOL. 170, 1988

on January 21, 2019 by guesthttp://jb.asm

.org/D

ownloaded from

4450 DUPLAY AND HOFNUNG

23. Jacobson, G. R., and J. P. Rosenbusch. 1976. Abundance andmembrane association of elongation factor Tu in Escherichiacoli. Nature (London) 261:23-26.

24. Jacobson, G. R., and J. P. Rosenbusch. 1976. Properties of a

major protein released from Escherichia coli by osmotic shock.Biochemistry. 15(11):2297-2303.

25. Koshland, D., and D. Botstein. 1982. Evidence for post-transla-tional translocation of P-lactamase across the bacterial innermembrane. Cell 30:893-902.

26. Kuhn, A., and W. Wickner. 1985. Isolation of mutants in M13coat protein that affect its synthesis, processing, and assemblyinto phage. J. Biol. Chem. 260:15907-15913.

27. Laemmli, U. K. 1970. Cleavage of structural proteins during theassembly of the head of bacteriophage T4. Nature (London)227:680-685.

28. Lin, J. J. C., H. Kanazawa, and H. C. Wu. 1980. Assembly ofouter membrane lipoprotein in an Escherichia coli mutant witha single amino acid replacement within the signal sequence ofprolipoprotein. J. Bacteriol. 141:550-557.

29. Miller, J. H. 1972. Experiments in molecular genetics. ColdSpring Harbor Laboratory, Cold Spring Harbor, N.Y.

30. Minsky, A., R. G. Summers, and J. R. Knowles. 1986. Secretionof P-lactamase into the periplasm of Escherichia coli: evidencefor a distinct release step associated with a conformationalchange. Proc. Natl. Acad. Sci. USA 83:4180-4184.

31. Pollitt, S., and M. Inouye. 1987. Structure and functions of thesignal peptide, p. 117-137. In M. Inouye (ed.), Bacterial outermembranes as model systems. John Wiley & Sons, Inc., NewYork.

32. Randall, L. L. 1986. Function of proton motive force in trans-location of protein across membranes. Methods Enzymol. 125:129-138.

33. Randall, L. L., and S. J. S. Hardy. 1986. Correlation ofcompetence for export with lack of tertiary structure of themature species: a study in vivo of maltose-binding protein inEscherichia coli. Cell 46:921-928.

34. Randall, L. L., S. J. S. Hardy, and J. R. Thom. 1987. Export ofprotein: a biochemical view. Annu. Rev. Microbiol. 41:507-541.

35. Ray, P., I. Dev, C. MacGregor, and P. J. Bassford, Jr. 1986.Signal peptidases. Curr. Top. Microbiol. Immunol. 125:75-102.

36. Ryan, J. P., M. C. Duncan, V. A. Bankaitis, and P. J. Bassford,Jr. 1986. Intragenic reversion mutations that improve export ofmaltose-binding protein in Escherichia coli malE signal se-quence mutants. J. Biol. Chem. 261:3389-3395.

37. Von Heine, G. 1985. Signal sequences: the limits of variation. J.Mol. Biol. 184:99-105.

38. Wolfe, P. B., W. Wickner, and J. M. Goodman. 1983. Sequenceof the leader peptidase genes of Escherichia coli and theorientation of leader peptidase in the bacterial envelope. J. Biol.Chem. 258:12073-12080.

J BACTERIOL

on January 21, 2019 by guesthttp://jb.asm

.org/D

ownloaded from