Vol. 162, No. 3

Defective Secretion of Maltose- and Ribose-Binding Proteins Causedby a Truncated Periplasmic Protein in Escherichia coli

REGINE HENGGE AND WINFRIED BOOS*

Department ofBiology, University of Konstanz, D-7750 Konstanz, Federal Republic of Germany

Received 18 December 1984/Accepted 11 March 1985

The secretion in Escherichia coli of a C-terminally truncated periplasmic enzyme from Salmonellatyphimurium, the glpQ-encoded glycerolphosphate phosphodiesterase, was studied. Plasmid pRH100, carryingthe truncated glpQ gene, directs the synthesis of a 30,000-molecular-weight (30 K) protein that is processed toa mature 27.5 K protein. (The mature wild-type protein is a 38 K protein.) The truncated protein is not releasedinto the periplasm but remains membrane associated, although it becomes protease sensitive after conversionof cells to spheroplasts. The presence of pRH100 strongly reduces the amount of some other proteins in theperiplasm, including the maltose- and ribose-binding proteins. The reduction does not occur at the level oftranscription or early translation, as shown by lacZ fusions to the gene coding for the structural gene of themaltose-binding protein. Outer membrane proteins are not affected. A hydroxylamine-induced mutation in thesequence of glpQ corresponding to the mature polypeptide overcomes the inhibitory effect of pRH100. Themutated gene no longer directs the synthesis of the 30/27.5 K protein but directs that of a new 19 K proteinwhich is not membrane bound. We propose that sorting signals in the mature GlpQ protein are necessary foreffective translocation to the periplasm and that the C-terminal third of the protein is essential for release intothe perip!asm.

Studies of protein export in gram-negative bacteria havefocused on the early steps of the secretion process. The roleof the signal sequence in the initiation of export has beenelucidated, and components of the secretory machinerywhich are likely to interact with the signal sequence havebeen identified above all by genetic techniques which aresimilar for outer membrane and periplasmic proteins (forreview see reference 33). However, very little is knownabout the sorting of exported proteins into the outer mem-brane or the periplasm. Sorting has to be a late event duringthe secretion process, since signal sequences of proteinswith different final locations are not significantly different(33). They are even exchangeable, as was demonstrated byfusing the signal sequence and part of the mature polypep-tide of the periplasmic ,-lactamase to signal-sequence defi-cient PhoE, an outer membrane pore protein. The hybridprotein is correctly exported to the outer membrane (34).Therefore, sorting signals are likely to be located in thesequence of the mature exported protein. For another outermembrane protein, the X receptor (lamB gene product), suchouter membrane signals have been identified recently bystudying the gene products of various lamB-lacZ proteinfusions (3). Two distinct signals, one for initiating export andthe other for defining outer membrane location, have beenfound, both located before amino acid 50. Also, there isevidence for a consensus sequence of outer membraneproteins (27). Nothing is known about comparable signals inperiplasmic proteins or about the protein components rec-ognizing the sorting signals.

Blockage of the export machinery by late-fusion proteinshas been reported for lacZ fusions to malE (which codes forthe periplasmic maltose-binding protein [MBP]) or lamB(33). The jamming is caused by an abortive attempt of thecells to export the unphysiological fusion proteins, whichremain membrane associated. The effect can even be lethalwhen large amounts of fusion protein are produced as for

* Corresponding author.

some malE-lacZ fusions that confer a maltose-sensitivephenotype.Here we describe a jamming phenomenon affecting only

periplasmic proteins. The effect is exerted by the incompletesecretion of a truncated form of the periplasmic glycerol-phosphate phosphodiesterase, the glpQ gene product, and isespecially pronounced in an overproducing strain.glpQ belongs to the glpT operon, which is located near 48

min on the chromosome of Escherichia coli. It codes for aphosphodiesterase that hydrolyzes glycerol-phosphoryl es-ters (20); the promoter proximal glpT is the structural genefor a tightly inner membrane-bound permease for sn-glycerol-3-phosphate (21). The chromosomal location of the operon,its structural organization and regulation, and its gene prod-ucts and their activities are very similar in E. coli andSalmonella typhimurium (12). The gIpA operon is locatednext to glpT; its first gene codes for the large subunit of theanaerobic sn-glycerol-3-phosphate-dehydrogenase (18, 31).The glpT region of both organisms was cloned previously onmulticopy plasmids (see Fig. 1).

MATERIALS AND METHODS

Bacterial strains and growth conditions. Strain DL291 is aderivative of E. coli K-12 MC4100 which is araDJ39 A(argF-lac)U169 relAl rpsL150flbB5301 deoCi ptsF25 (32); in ad-dition to these markers, DL291 is glpR A(glpT-gIpA)593 gyrArecA (23); strain RH62 is a malr derivative of DL291. Theminicell strain DS410-T is a A(glpT-glpA)593 derivative (21)ofDS410, which is minA minB ara lacYmalA mtl xyl rpsL thitonA azi (9).For cold osmotic shock, membrane preparation, and ,3-

galactosidase tests, the cells were grown in minimal mediumA (MMA; described in reference 24) supplemented with0.01% thiamine. Unless otherwise indicated, the carbonsource was sn-glycerol-3-phosphate for strains harboringplasmids carrying glpT+ and glycerol for the others. For allplasmid-harboring strains, tetracycline (10 ,ug/ml) was in-cluded in the medium. For preparation of minicells or

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SECRETION OF PERIPLASMIC PROTEINS 973

P

(pBR322J

HB E

(pACYC184)

p

(pBR322')

E P(BB)E H B EI I I III

,_ (pACYC184)glpQ Pmgl

FIG. 1. Plasmids carrying the glpT,Q region from E. coli(pGS31)and S. typhimurium(pRH100, pRH310, and pRH123). The restric-tion sites relevant for this study are: B, BamHI; Bg, BglII; E,EcoRI; H, HindIII; P, PstI.

plasmids, cells were grown in LB medium (24) containing 10jig of tetracycline per ml. All cells were grown in 37°C.

Minicell techniques. Minicells were prepared from station-ary-phase cultures on two subsequent continuous sucrosegradients as described by Reeve (29). They were stored inMMA-30% glycerol at -70°C. For labeling, the minicellswere washed in MMA and suspended in 0.5 ml of MMA-0.4% lactate (optical density, 0.5). After a 45-min preincuba-tion at 37°C, 25 ,ul of methionine assay medium (DifcoLaboratories, Detroit, Mich.) and 5 ,uCi of L-[35S]methionine(Amersham International, England) per sample were added.After 60 min of incubation at 37°C, unlabeled methionine(0.25 mM) was added for a 10-min chase. The minicells werewashed in 1 ml of 50 mM Tris hydrochloride (pH 7.5)-100mM NaCl-1 mM EDTA and suspended in 20 pul of elec-trophoresis sample buffer (12). For fractionation of theminicells by NaOH treatment (30) or conversion of theminicells to spheroplasts (28), they were washed in 50 mMTris hydrochloride (pH 7.5), and all further steps werecarried out at 4°C. Proteinase K treatment was performed bythe method of Randall (28).

Fractionation of unlabeled cells. Periplasmic proteins were

isolated by the cold osmotic shock procedure of Neu andHeppel (26). If not otherwise indicated, total membraneswere prepared from 10 ml of stationary-phase cells whichwere washed once and resuspended to an optical density (at578 nm) of 0.5 in MMA. The cells were passed three timesthrough a French press at 16,000 lb/in2; unbroken cells werespun down, and the membranes were collected from thesupematant by a 1-h centrifugation at 130,000 x g. Thepellet was suspended in 30 pu1 of electrophoresis samplebuffer. Protein concentrations were determined by themethod of Lowry et al. (22).SDS-PAGE. Sodium dodecyl sulfate-polyacrylamide gel

electrophoresis (SDS-PAGE) on 15% gels was done by themethod of Laemmli (19). Electrophoresis and sample buffersare described elsewhere (12). The samples were pretreatedfor 1 h at 37°C, then for 3 min at 95°C.

13-Galactosidase assay. P-Galactosidase activity in washed,toluenized, exponential-phase cells was assayed as de-scribed by Miller (24). A405 was measured (e = 4.86 mM-1cm-1) after clearing by centrifugation.

Maltose sensitivity tests. For quantification of sensitivity tomaltose, the procedure of Benson et al. (3) was followed.

P GS31 DNA manipulations. Plasmid DNA was isolated by the....pGS technique of Birnboim and Doly (4). The digestion of DNA

with restriction nucleases (Boehringer GmbH, Mannheim,Federal Republic of Germany), religation of digested DNA,transformation, agarose gel electrophoresis with Tris-acetatebuffer, and elution of DNA fragments from agarose gels

pRHlOO were performed as described by Davis et al. (8).pRH100~~~~~~~~~

RESULTS

The gene products of pRH100. Plasmids pGS31, whichcarries the E. coli glpT-glpQ region, and pRH100, whichcarries an incomplete glpT region from S. typhimurium, areshown in Fig. 1 and Table 1. The gene products of theseplasmids were identified in a minicell system (Fig. 2). Thewild-type GlpQ protein coded by pGS31 appeared as aprecursor of 41,000 daltons and as mature form of 39,000daltons. The mature Salmonella protein has a molecularweight of 38,000 and the same activity as the E. coli enzyme(12). Plasmid pRH100 produced two bands with molecularweights of 30,000 and 27,500, the latter as a doublet. Neitherthe 30,000-molecular-weight (30 K) nor the 27.5 K polypep-tide were expressed by pRH310, which lacks an additional0.5 kilobase (kb) of the glpQ gene. These bands represent atruncated form of the GlpQ protein, most likely a precursorand a mature polypeptide (this truncated GlpQ protein willhereafter be referred to as GlpQ'). All three plasmids di-rected the synthesis of the sn-glycerol-3-phosphate-permease, which runs as a diffuse 40 K band in the gelsystem used here (15% polyacrylamide).

All plasmids produced a polypeptide of about 70,000daltons, the large subunit of the anaerobic sn-glycerol-3-phosphate-dehydrogenase, synthesized in its wild-type formby pGS31. In the case of pRH100 and pRH310, however, theEcoRI site at the end of the insert cuts off the C-terminal endof the GlpA protein. This could be demonstrated by invert-ing the EcoRI insert with respect to the vector plasmid. Thiscreated an in-frame fusion of glpA' to the C-terminal se-quence of the chloramphenicol transacetylase (CAT) gene,which is read from right to left in Fig. 1. The resulting fusionprotein has a molecular weight of about 85,000 (data notshown). Its formation proves the diverging transcription ofthe glpT and the glpA operons.

Cellular location of GlpQ'. To see unlabeled GlpQ' in cellfractionation experiments, the glpT promoter on pRH100

TABLE 1. Plasmids used in this study'Plasmid Description Source

pRH100 tet gipT glpQ' (S. typhimur- (12)ium)

pRH103 tet glpT (Am) glpQ' (S. typhi- (12)murium)

pRH310 tet gipT (S. thyphimurium) This study (Fig. 1)pRH110 tet gipT glpQ' (nonsense) (S. This study

typhimurium)pRH123 tet (Pmgl) glpQ' (S. typhimur- This study (Fig. 1)

ium)pRH124 tet (Pmgl) glpQ' (nonsense?) (S. This study

typhimurium)pGS31 tet glpT glpQ (E. coli) (20)pACYC184 tet cat (6)pBR322 tet bla (5)

a The gene symbols stand for wild-type alleles; a prime indicates a C-terminally incomplete gene.

P

glpQ gipT gipA

lkb

E P EBsB EI rs L-

I - igipa' glpT glpA'

PBoBTI

E

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974 HENGGE AND BOOS

was exchanged by the very strong Salmonella-mgl promoterwhich, on the other hand, is almost totally repressible byglucose. A BgIII-EcoRI fragment (2.5 kb) of the insert ofpRH100 was replaced by a BamHI-EcoRI fragment (0.88 kb)from pNM515 which carries the mgl promoter (N. Muller,personal communication; Fig. 1). The new plasmid, pRH123,produced GlpQ' bands in minicells that were identical tothose expressed by pRH100 (data not shown). Total mem-branes were prepared from strain DL291(pRH123). By SDS-PAGE, a 30 K band could be identified exclusively in themembrane fraction (Fig. 3). This band corresponds to GlpQ'since it is not present with plasmid-free DL291 or withDL291 carrying pRH124, a plasmid analogous to pRH123that in addition has a mutation in glpQ' described below.Only the 30 K precursor of GlpQ' could be seen on theCoomassie-stained gel. This could be due to a greaterinstability of the mature protein, or the cellular exportmachinery might be unable to process enough GlpQ' fordetection. By NaOH fractionation (30) of minicells, themembrane association of GlpQ' was confirmed (data notshown).

Since GlpQ' was apparently processed, it was of interestto us whether it reached the outer side of the inner mem-brane or remained on the cytoplasmic side. Therefore, theprotease sensitivity of GlpQ' was tested in minicells con-verted to spheroplasts. The 27.5 K bands of GlpQ', presum-ably representing the mature polypeptide, were sensitive toproteinase K, whereas the 30 K precursor was resistant (Fig.4). The fact that the 27.5 K protein was shortened but nottotally degraded might indicate that most of the polypeptideis still protected by the membrane. Membrane proteins suchas the sn-glycerol-3-phosphate-permease or the TetA pro-tein, as well as the GlpA protein, which is attached to thecytoplasmic side of the inner membrane, were proteinase Kresistant (Fig. 4).

oo C- oo,- C') v

-_. -

U, -

26b-4-M26-

12-

FIG. 2. SDS-PAGE followed by autoradiography of proteinssynthesized in minicells harboring pRH100, pRH310, pGS31, andpRH110. Size standards are given in kilodaltons. Arrowheadsindicate GlpQ' in its precursor and mature forms. The arrowindicates the glpT gene product that appears as a diffuse band. WithpGS31 as template this protein is hardly visible since the completeGlpQ protein runs at the same position and since the other proteinsare preferentially labeled; however, in a 12% gel or in unlabeledinner membrane preparations it can be clearly identified (21).Plasmid pRH110 is described in the text below.

-9255*:F- 66.2_

- 4~~5.0

t- ~~31,0-_

-21.5 _

-14.41 2 3 :

FIG. 3. SDS-PAGE of membrane and soluble fractions of strainDL291 harboring various plasmids shown with Coomassie bluestaining. Cells were grown overnight on MMA and 0.4% glucose,washed once in MMA, suspended in MMA and 0.4% glycerol to anoptical density of 0.6, and incubated for an additional 3 h. Totalmembrane fractions (lanes 1 through 3) and soluble fractions (lanes4 through 6) are from strain DL291 harboring no plasmid (lanes 1and 4), pRH123 (lanes 2 and 5), or pRH124 (lanes 3 and 6).Membrane samples were treated at 37'C for 1 h, and soluble fractionsamples were boiled for 3 min before electrophoresis. (GlpQ' is heatstable and appears also in boiled membrane samples; data notshown.) The arrowhead indicates GlpQ'.

The protease sensitivity of mature GlpQ' demonstratesthat some portion of the polypeptide chain is translocatedacross the membrane and that this translocation is tightlycoupled to processing. Yet, GlpQ' is not released into theperiplasm. It did not appear among the proteins of the coldosmotic shock fluid of strain DL291 transformed with pRH100(Fig. 5). This absence was not due to proteolytic degradationas is frequently found for unphysiological proteins (7), sinceGlpQ' remained intact in minicells converted to spheroplastsand incubated with cold osmotic shock fluid for 30 min atroom temperature (data not shown).

Effects of GlpQ' on other exported proteins. The amount ofseveral other periplasmic proteins was strikingly reduced incold osmotic shock fluids of strain DL291(pRH100). Ribose-binding protein (RBP), which is constitutively expressed inDL291 (Fig. 5), MBP, and the binding proteins for galactose(GBP) were released in much smaller amounts (Fig. 6) thanfrom strains carrying pGS31 or pRH103, which carries anamber mutation in glpT that is polar on glpQ. Some peri-plasmic proteins (indicated by the arrowheads in Fig. 5)were not affected. Also, the profile of the major outermembrane proteins OmpF, OmpC, and OmpA (Fig. 7) aswell as that of LamB (data not shown) was not changed incells harboring pRH100.

Pulse-labeling experiments with [3S]methionine followedby precipitation with antibodies against MBP revealed thatthe presence of GlpQ' did not result in the accumulation ofprecursor MBP. It did, however, strongly reduce the amountof mature MBP (data not yet published).The reduction of RBP is so strong that strain DL291

containing pRH100 grows very poorly with ribose as solecarbon source at 13.3 mM (0.2%) and not at all below 3.3

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SECRETION OF PERIPLASMIC PROTEINS

FIG. 4. SDS-PAGE and autoradiography of proteins synthesizedin minicells harboring pRH100. After labeling, the minicells wereconverted to spheroplasts by the method of Randall (28). ProteinaseK was added (lane 1, 0 ,ug/ml; lane 2, 10 ,ug/ml; lane 3, 30 ,ug/ml),and after a 30-min incubation at 0°C the samples were precipitatedwith trichloroacetic acid, washed with acetone, and suspended in 30,ul of electrophoresis sample buffer. The arrow indicates the precur-sor of GlpQ', and the arrowheads indicate the mature protein thatappears to be protease sensitive.

mM. This ribose-negative phenotype was not dependent onthe orientation of the insert in pRH100, indicating that GlpQ'is not an in-frame fusion between the N-terminal part ofGlpQ and the C-terminus of the CAT.pRH123, where glpQ' is under the control of the mgl

promoter, even confers a conditional lethal phenotype.DL291(pRH123) does not grow at all under fully inducedconditions, i.e., in minimal medium containing carbonsources that are not active as catabolite repressors.

o c,

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Effect of GlpQ' on malE-LacZ fusions. As shown above,MBP, the malE gene product, is affected by GlpQ'. Todetermine whether GlpQ' acts at the level of transcription,translation, or secretion, we used malE-lacZ fusions. Withan operon (Xp72-12) or a protein (Xp4-81) fusion to the signalsequence of MBP (2), P-galactosidase activity was notreduced by GlpQ' [RH62(Xp72-12), 28.6 nmol/min per ml ofcells of optical density 0.5 at 578 nm; RH62(X72-12, pRH100),38.5 nmol/min per ml of cells; RH62(Ap4-81) and RH62(Ap4-81, pRH100), 3.1 nmollmin per ml of cells]. This indicatedthat transcription and at least the initiation of translation ofMBP are normal.A different picture was obtained with a late fusion in malE

(Xp72-47), known to exert inducible sensitivity to maltose(2).When strain MC4100 lysogenized with this Xp72-47 was

transformed with pRH100, maltose sensitivity was greatlyincreased (data not shown). Thus, the effects of the fusionprotein and of GlpQ' were additive, both preventing exportof other proteins. Plasmid pGS31 had no effect on maltosesensitivity.Thejamming effect can be overcome by a mutation affecting

the mature part of GlpQ as well as by chromosomal muta-tions. In vitro mutagenesis by hydroxylamine was carriedout with pRH100. After transformation into DL291 andtetracycline selection, colonies were screened for growth onribose. A plasmid (pRH11O) was isolated from a ribose+colony and retransformed into DL291. The resulting strain

0 .0o oz ._cc a:

I Ql I ffi

67 -

40-i

26

00

ar.

WI -MBP

":- -RBP

w . _= .4

_-A- -

_ 4m -_RB P26-1 am --

I'domm I.:: Amwmw~~~~~~~~

FIG. 5. SDS-PAGE of cold osmotic shock fluids of strain DL291harboring various plasmids. Cells were grown on MMA and 0.4%sn-glycerol-3-phosphate (if harboring pRH100, pGS31, or pRH110)or 0.2% glycerol (if harboring no plasmid or pRH103). Arrowheadsindicate unidentified proteins which are not dramatically affected bypRH100.

FIG. 6. SDS-PAGE of cold osmotic shock fluids of the strainsDL291 and DL291(pRH100) grown on MMA and carbon sources asindicated (0.2% glycerol, maltose, or galactose). Fucose (2 mM) wasadded as an inducer of the GBP. Since another protein runs atalmost the same position as MBP, the reduction of the amount ofMBP was confirmed by binding tests (data not shown). Glyc,Glycerol; Mal, maltose; Gal/Fuc, galactose and fucose.

G4yc:: Mat Gal/Fuc

.M-

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976 HENGGE AND BOOS

no longer exhibited reduced levels of periplasmic proteins(see Fig. 5) or increased maltose sensitivity in MC4100carrying Xp72-47. When the mutation was under the controlof the mgl promoter (pRH124), the conditional lethal pheno-type was relieved.The mutation on pRH110 could be located by a technique

of crosswise religation of restriction fragments as illustratedin Fig. 8. The ribose+ character was contained in the smallerPstI-HindIII fragment of pRH110 left of the PstI site ofpRH110 and in the EcoRI insert of pRH110. Therefore, themutation must be located in the 0.5-kb region where thesetwo fragments overlap. Since the PstI site is within glpQ(12), the pRH110 mutation is located within DNA sequencescoding for GlpQ'. This was confirmed by minicell analysis ofthe gene products of pRH110 (Fig. 2). Instead of the 30/27.5K polypeptide coded by pRH100, pRH110 directed thesynthesis of two smaller polypeptides with apparent molec-ular weights of 19,500 and 15,500. Both polypeptides were inthe soluble fraction after NaOH treatment of minicells (datanot shown). Also, when expressed under the control of themgl promoter (pRH124), these polypeptides could not bedetected either in the membranes or in the soluble fraction.Both were proteinase K resistant in spheroplasted minicellsbut became sensitive when the labeled spheroplasts wereperturbed by addition of 1% Triton X-100 before incubationwith proteinase K (Fig. 9). These observations argue for acytoplasmic location of these two polypeptides.The jamming effect of GlpQ' could also be overcome by

chromosomal suppressor mutations, isolated by selectingribose+ colonies in tetracycline-containing plates. After isola-tion of plasmid DNA from these 'strains and retransformationof DL291, the jamming effect could again be demonstrated.The nature of these chromosomal suppressor mutations hasnot yet been elucidated.

00

I-I =L

Cl_

C,,

IL

0

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w'-w 67

-_'umd_~ mak41i

WANWO, :.

40

26

FIG. 7. SDS-PAGE of total membrane preparations of strainDL291 harboring no plasmid (-), pRH100, pGS31, or pRH110. Noapparent changes in the protein pattern were observed.

Crosswise religation of restriction fragments

Rib Rib-

RiV

Restriction fragments of pRH110 carrying the mutation

FIG. 8. Localization of the mutation on pRH110. Both pRH100and pRH110 were digested with PstI (P) and HindIII (H); therestriction fragments were separated and eluted from an agarose geland religated in a crosswise fashion. In a second analogous experi-ment, the EcoRI (E) insert of pRH110 was isolated and religated intothe vector. After transformation of the various religated plasmidsinto DL291, the ribose phenotype was tested.

DISCUSSION

Here we describe a jamming effect on export of someperiplasmic proteins in E. coli due to the synthesis of aC-terminally truncated protein, the glpQ' product.

It is clear that not all periplasmic proteins were subjectedto the effect of the incompletely exported GlpQ' protein.This may indicate the existence of more than one specificexport machinery for periplasmic proteins. In the presentcase, periplasmic proteins belonging to catabolite-repressi-ble operons such as the MBP-, RBP-, and GBP-dependenttransport systems were clearly affected.

In its intact form, the glpQ+ gene product itself is aperiplasmic glycerolphosphate phosphodiesterase. The glpQ'product is not the result of a protein fusion of GlpQ and CATcreated by the cloning. The gene product of GlpQ', the30/27.5 K protein, is too small to be a fusion protein, whichshould have at least 20,000 daltons from GlpQ' and 15,000daltons from CAT, and the ribose-negative phenotype causedby GlpQ' is not dependent on the orientation of the glpT-glpQ' insert. Therefore, the jamming of secretion by GlpQ'does not seem to be caused by the same mechanism as thejamming by certain lamB-lacZ (10) and malE-lacZ (2) fu-sions.From the nucleotide sequence of the CAT gene (1) into

which glpT,Q' was cloned with the EcoRI site, two possiblereadthrough peptides at the C-terminal end of GlpQ' can be

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SECRETION OF PERIPLASMIC PROTEINS 977

FIG. 9. SDS-PAGE and autoradiography of proteins synthesizedin minicells harboring pRH110. Spheroplasted minicells were incu-

bated with proteinase K (lane 1,0 pg/ml; lane 2, 10 ~Lg/ml; lane 3, 30

~Lg/ml) for 30 min at O0C. As a control, Triton X-100 was added to a

final concentration of 1% 1 min before the addition of protease (30

~Lg/ml) (lane 4). Further treatment was as described in the legend to

Fig. 3. Arrowheads indicate the position of the GlpQ' protein bands

coded by pRH110.

derived: -Asn-Ser-Val-Trp-Gln(op) and -Ile-Pro-Tyr-Gly-

Asn-Glu-Arg-Arg(op) in the +1 and +2 reading frames,

respectively. In the case of ompA fusions, a special C-ter-

minal readthrough peptide was shown to cause a block in

secretion and had a lethal effect on the cells (13). This

peptide, however, was much longer (31 amino acids) and

contained a series of highly hydrophobic amino acids. There-

fore, we believe that the effect of GlpQ' on secretion is not

due to one of the two possible short readthrough peptides.It is unlikely that the Salmonella-glpQ on pRH100 is not

compatible with the E. coli secretory apparatus. Other

Salmonella periplasmic proteins, for instance GBP, are

normally exported in E. coli (25).

Using different malk.-lacZ fusion strains, we could demon-

strate that G1pQ' does not interfere with transcription or

translation of the amino-terminal portion of the MBP. How-

ever, very few mature MBPs were found in the periplasm.

First, pulse-labeling experiments indicated that the concom-

itant expression of GlpQ' does not result in an accumulation

of MBP precursor but that the complete synthesis of MBP is

reduced. This is reminiscent of the situation in a secA (17) or

secC (11) mutant and again suggests a tight coupling between

secretion and synthesis of exported proteins. The membrane-

bound state of GIpQ' also indicates a blocking of secretion.

The protease sensitivity test for the proteins produced in

spheroplasted minicells allowed more precise localization of

GlpQ'. At least some part of the processed polypeptidechain reaches the outer side of the inner membrane and

becomes protease sensitive. However, the truncated proteinis not released into the periplasm.A mutation in glpQ' alleviating the inhibiting effects of

GlpQ' was isolated (plasmid pRH110) and localized within

the 0.5-kb PstI-EcoRI fragment of pRH110. It is clear that

this cannot be a signal sequence mutation since the start

point of glpQ', including the signal sequence, is located

outside the EcoRI-Pstl fragment (12). Instead of the 30/27.5

K GlpQ' polypeptides produced by pRH100, pRH11O syn-

thesized two new bands with apparent molecular weights of

19,500 and 15,500. These polypeptides, one of which mightbe a degradation product of the other, are most likely locatedin the cytoplasm. They seem to be quite unstable, since theycannot be detected in unlabeled form, even under conditionsfor very strong expression. pRH110 might carry a nonsensemutation in glpQ', resulting in a further truncated geneproduct that no longer contains the sequence(s) necessaryfor translocation through the membrane.These results indicate that there are two distinct signals in

the GlpQ protein necessary for its complete secretion intothe periplasm, a releasing signal in the last third of theprotein and a signal located somewhat earlier but still in thesecond half of the mature polypeptide necessary for effectivetranslocation.The first observation has also been made for amber

fragments of P-lactamase (16) and of MBP (14). It wasconcluded that the C-terminal part of these proteins is notrequired for their translocation across the inner membranebut is required for their release into the periplasm.The location of GlpQ' at the external side of the mem-

brane and its processing to the mature form suggest thatGlpQ', in contrast to fusion proteins, blocks the secretion ofother periplasmic proteins at a late stage of the exportprocess. Several previous observations indicate that exportinto the periplasm might even be a more complex processthan protein export to the outer membrane. For instance,there is a longer delay in the appearance of newly synthe-sized protein in the periplasm than in that of outer membraneproteins (15). In addition, no ,-galactosidase fusions toperiplasmic proteins have been found in the periplasm,whereas fusions to the LamB protein have been identified intheir proper location in the outer membrane (3).

In line with this view is the observation of Randall (28),who reported that the synthesis of RBP and MBP has toproceed far beyond the signal peptide before the proteinreaches the outer surface of the membrane. The formation ofa translocation-competent conformation specific for peri-plasmic proteins at the amino-terminal part of the protein israther likely. Our GlpQ' protein can achieve this conforma-tion and thus can be translocated through the membrane, butdue to the lack of its carboxy-terminal sequence it is unableto be released into the periplasm. At this level, it interfereswith the secretion of other periplasmic proteins but not withthe proper localization of outer membrane proteins, whoseroute diverged at an earlier stage.

ACKNOWLEDGMENTSWe thank P. J. Bassford and T. J. Silhavy for malE-lacZ fusion

phages.This work was supported by grants from the Deutsche Forschungs-

gemeinschaft (SFB 156), the Fonds der Chemischen Industrie, andthe North Atlantic Treaty Organization. R. Hengge was supportedby a fellowship from the Studienstiftung des Deutschen Volkes.

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