Proc. Nati. Acad. Sci. USAVol. 91, pp. 4539-4543, May 1994Cell Biology

A cytoplasmic domain is important for the formation of aSecY-SecE translocator complex

(protein export/Escherichia col/doinant negative mutatlon/suppressor/protein interacdon)

TADASHI BABA, TETSUYA TAURA, TAKASHI SHIMOIKE, YOSHINORI AKIYAMA, TOHRU YOSHIHISA,AND KoREAKI ITODepartment of Cell Biology, Institute for Virus Research, Kyoto University, Sakyo-ku, Kyoto 606-01, Japan

Communicated by Jon Beckwith, January 24, 1994 (received for review December 6, 1993)

ABSTRACT An approach to identifying the interactionsite ofmulticomponent protein assembly has been applied to themembrane-bound SecY-SecE complex, which mediates pro-tein export across the Escherichia coli cytoplasmic membrane.A dominant negative secY allele, secY-dl, inactivates SecY butpreserves its ability to interact with SecE. Thus, the mutantprotein sequesters SecE in an inactive complex. Second sitemutations that disrupt the SecE binding site will suppress theexport interference. We introduced insertion/deletion muta-tions that intragenically suppressed secY d1. After eliminatingknock-out mutations by virtue of the expression of a LacZasequence that had been attached to theC terminus, we obtaineda striking clustering of mutations in cytoplasmic domain 4. Onthe basis of this result, the secY24 (Ts) substitution mutation inthis domain was examined for its effects on interaction withSecE. It indeed suppressed secY-dl. Although the instabilityassociated with excess SecY can be alleviated by overproduc-tion of SecE, the secY24 mutant protein was not stabilized bySecE. The basal-level SecY24 protein was also destabilized at4MC. SecE was coimmunoprecipitated with SecY+ but not withthe SecY24 protein. These results indicate that the secY24mutation weakens SecY's interaction with SecE. Taken to-gether, we propose that cytoplasmic domain 4 is important forthe association between SecY and SecE.

Little information exists on the sites of interaction betweenproteins that form complexes in a membrane. One approachto this problem is to obtain mutants that disrupt such inter-actions, which could then be used to define the sites ofinteraction. A class of mutations in a membrane protein,those that disrupt the function of a protein but do not affectits interaction with its partner(s), could be useful for thispurpose. These mutants should be dominant negatives, andadditional mutations in the altered proteins that eliminatetheir interaction with their partners should relieve the dom-inant negative effect. These mutations, appropriately char-acterized, could define the interaction site of the protein.A possibility for doing such an analysis exists for the

SecE-SecY membrane protein complex of Escherichia coli.SecY is a multispanning membrane protein whose homologsplay major roles in protein translocation in various organisms(1, 2). SecY and SecE function in close mutual interaction (3,4). The SecY/E complex can be isolated after membranesolubilization and chromatographic purification (4). Over-synthesized SecY, which by itself is very unstable in the cell,can be stabilized by simultaneous overproduction of SecE (5,6). A decreased cellular content of SecE results in destabi-lization of SecY (6, 7). Particular combinations of a priAmutation in sec Y and a priG mutation in secE are syntheti-cally lethal (8, 9). It was proposed that SecE and SecY cycle

between association and dissociation (8, 10), although recentstudies argue against this notion (6, 37).We previously showed that a SecY variant with an internal

deletion (secY-dl) of three residues at the interface betweencytoplasmic domain 5 (CS) and transmembrane segment 9(TM9) interfered with protein export when it was overex-pressed from a plasmid (11). The dominant export interfer-ence was suppressed by overexpression of secE. We pro-posed that the secY-dl mutation inactivates an essentialtranslocation activity of SecY but not its ability to bind SecE.Thus, the mutated SecY molecules sequester SecE in inac-tive complexes. We therefore attempted to identify a regionof SecY that is important for the SecE binding by isolatingintragenic suppressor mutations of secY-d]. Results of in-sertion/deletion mutagenesis as well as characterization of atemperature-sensitive missense mutation, secY24 (12), indi-cate that the C4 domain is important for SecY-SecE binding.This contrasts with a recent proposal (9) that a specific pairof transmembrane segments mediates the SecY-SecE asso-ciation.

MATERIALS AND METHODSBacterial Strains. E. coli K-12 strain MV1184 (13) was used

as a host in plasmid constructions as well as a LacZw donor.All other strains were derived from MC4100 (14). K1437carried the secA-4acZgene fusion as well as APR9 (secA+; ref.15) and was constructed as described (11). AD202 (MC4100,ompT::kan; ref. 16), TW130 (AD202, zhd33::TnJO; ref. 17),and BB030 (KI437, ompT::kan) were used as secY+ strains.AD206 (AD202, secY24 zhd33::TnJO) and AD208 (AD202,secY39 zhd33: TnlO) carried secY mutations that had been P1cotransduced with the TnlO marker.

Plasmids. pKY259 carried the secY-dl-lacZa fusion gene(secY-dla). A 1.9-kb Eco52I fragment of pKY234 (6) wasfirst replaced by the corresponding fragment from pKY241(11), yielding pKY249, whose HindIII secY fragment wasthen cloned into a pACYC184-based lac promoter vector,pKY238 (11). pKY248, pKY247, and pKY258 were similarpKY238-derived plasmids with secY+, secY-dl, and secY-lacZa, respectively. pKY132 (pBR322 replicon) and pKY316(pACYC184 replicon) carried the secY24 mutation; the for-mer was a derivative of pKY6 with its Sma I-Ban IIIfragment replaced by the corresponding region from pKY12(12), and the latter was a pKY248 derivative with its HindIII-Eag I secYfragment replaced by the corresponding fragmentfrom pKY132. pKY324 (pBR322 replicon) carried secY withboth the secY24 and the secY-dl mutations and was con-structed by replacing the 0.3-kb EcoRI (within secY)-EcoRI(multicloning site) fragment ofpKY132 by the correspondingfragment from pKY241.

Abbreviations: C, cytoplasmic domain(s); P, periplasmic domain(s);TM, transmembrane segment(s); MBP, maltose-binding protein.

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pKY250 (11) and pKY271 carried secE under the control ofthe lac promoter on pACYC184- and pBR322-based vectors,respectively. The latter contained the BamHI secE ragmentof pKY250 cloned into a lac promoter vector, pKY225 (17).DNA manipulations were according to the standard proce-dures (18). The secYregion was sequenced as described (19).

Linker Insertion Mutagenesis. Plasmid pKY259 was linear-ized either by treatment with pancreatic DNase I in thepresence of 1 mM Mn2+ (18) or by partial digestion with AccII. After agarose gel electrophoresis, molecules of almostintact sizes (4.4 kb) were isolated, blunted by T4 DNApolymerase (for the DNase I products), and then ligated in thepresence of a 500-fold molar excess of nonphosphorylatedoligonucleotides, 5'-CCTCGAGG-3' (for the DNase I sam-ple) or 5'-CTCGAG-3' (for theAcc II sample). Transformantswere pooled for extraction of a mixture of plasmids, whichwere then treated with Xho I. After electrophoretic separa-tion, linear molecules were again isolated, recircularized, andallowed to transform KI437 (secA-lacZ) on peptone agarplates containing 5-bromo-4-chloro-3-indolyl (-D-galactopy-ranoside and phenylethyl t3-D-thiogalactopyranoside (15).White colonies were picked up, restreaked on the samemedium, and pooled for plasmid extraction. The plasmidmixture was then introduced into MV1184 carrying lacZw.Blue transformants with unimpaired expression of the lacZaregion of the secY-dla gene were saved.

Topology Analysis of SecY with the X4 Deletion/Insertion.TnphoA was allowed to transpose onto the target plasmid (20,21). A plasmid with a P4 PhoA fusion in which PhoA followsGly-299 was identified and its HindIII-Pst I secY-phoAfragment was cloned into pTWV229, a vector identical topTWV228 (17) but-with an inverted multicloning site to yieldpYXP299. A site-directed deletion (22) was then introducedinto pYXP299 using an appropriate synthetic primer designedsuch that the resulting plasmid (named pYXP268) encoded afusion protein with a junction that follows Lys-268 of secY.

RESULTSInsertion/Deletion Suppressors of secY-dl Cluster in the C4

Region. The approach to defining the interaction site of aprotein was applied to the SecY-SecE system, since thedominant negative mutation, secY-dl, appeared to fit thisstrategy (11). Expression of the mutated gene from a plasmidinterferes with the protein translocation system. This domi-nant negative effect is moderate in that it does not lead togrowth inhibition (11). We isolated intragenic suppressormutations to define regions of SecY that are important forSecY's interaction with SecE. The target of our mutagenesiswas plasmid pKY259, which carried secY-dl to which lacZahad been attached in frame to the 3' end (termed secY-dl a).An attachment of the LacZa sequence to the SecY+ Cterminus, which is localized in the cytoplasm, did not impairthe activities of SecY or of LacZa (6). The LacZa tail did notinterfere with the dominant negative phenotype of SecY-dleither. We used the secA-lacZ gene fusion as an indicator ofprotein export proficiency; the expression of secA (secA-lacZ) elevates when the translocation system is compro-mised, leading to an increase in ,B-galactosidase activity (15).To monitor the production of the SecY derivative proteinsthemselves, we used the ability of the LacZa portion of thefusion protein to complement the lacZc gene product for,B3galactosidase activity.pKY259 was linearized by a limited and random DNase I

reaction, and an Xho I linker was introduced. Mutant plas-mids that lost the dominant export interference but retainedthe LacZa activity were screened for as described (Materialsand Methods). The LacZa screening should have excludedchain terminators and other undesired mutations that non-specifically knocked out the sec Y region of pKY259.

We obtained 23 plasmids with a structural alteration withinsec Y. Pulse-labeling experiments addressed whether eachplasmid synthesized a SecYa derivative protein (Fig. 1A,lanes 6-11) and whether each plasmid lost the ability tointerfere with export of MBP and OmpA (Fig. 1B, lanes6-11). Eleven clones passed these tests. Sequence analysesidentified six different mutations, X4, X17, X54, X101, X34,and X63, with Xho I linker insertions and accompanieddeletions (Fig. 2). The X4 alteration was scored six times.Strikingly, the alterations were all located in the C4-TM7interval, mostly within C4 (Fig. 2). In contrast, linker inser-tions in another set of clones that were obtained without theLacZa positive screening were scattered along the wholesecY sequence (data not shown). We also obtained severalmutations that were LacZa-positive but suppression-negative and mapped outside the C4-TM7 region (notshown).Another intragenic suppressor (designated X213) with a

less extensive sequence alteration was isolated after partialAcc II digestion followed by similar ligation and screeningprocedures. Five Acc II sites exist within secY (Fig. 2). Weobtained eight clones with the same X213 insertion (Arg-Ala)in C4 (Fig. 2). The product of the X213 derivative ofSecY-dla and its effect on protein export are shown in Fig.1 (lane 12). We also obtained a deletion removing the TM6-C4-TM7 segment (not shown) by the Acc II approach.Transformation without the screenings allowed us to isolatein-frame insertions at the four remaining Acc II sites (Fig. 2),which did not suppress secY-dl Thus, of the five Acc II sitesin secY, only the X213 insertion in the C4 domain suppressedintragenically the secY-dl mutation.

Effects of the Suppressor Mutations on Stability and Mem-brane Integration of SecY. A population of SecY moleculesthat are synthesized in excess over SecE are rapidly (half-life,2-3 min) degraded in vivo, whereas the other populationrapidly associates with SecE and remains stable (6). In thepresent experimental system, newly synthesized moleculesof SecY+ and SecY-dl derivatives, both of which are intrin-sically unstable, compete with one another for the stableassociation with the limited partner molecules, SecE. If thesuppressor mutations further shorten the half-life of newlysynthesized molecules, they will decrease the chance ofSecY-dla's competition with SecY+. We compared in vivostability of SecY-dla with its X4, X17, and X213 derivatives.The initial phase of their degradation did not differ signifi-cantly from one another (Fig. 3). The stable population wasobserved for SecY-dla, but not clearly for the X4, X17, and

A 2 3 4 5 6 ~ 8 9-424 o6SecYu[SecY - M _

BMBP EP _

OmpAEP _. ....

FIG. 1. Synthesis of various SecY derivatives from plasmids andtheir effects on export of maltose-binding protein (MBP) and OmpA.Cells of K1437 that carried a plasmid with a variant of secY weregrown at 370C and pulse-labeled with [35S]methionine for 0.5 min.Whole cell proteins were solubilized with SDS and immunoprecip-itated with anti-SecY serum (A) or a mixture of anti-MBP andanti-OmpA sera (B). After SDS/PAGE, radioactive SecY species (A)as well as precursor (p) and mature (m) forms ofMBP and OmpA (B)were visualized. Experimental procedures were essentially as de-scribed (6, 19, 23). Plasmids carried were as follows: lane 1, pKY238(vector); lane 2, pKY248 (secY+); lane 3, pKY247 (secY-dl); lane 4,pKY258 (secY+ a); lane 5, pKY259 (secY-dl a); lane 6, pKY259-X4;lane 7, pKY259-X17; lane 8, pKY259-X34; lane 9, pKY259-X54; lane10, pKY259-X63; lane 11, pKY259-X101; lane 12, pKY259-X213;lane 13, pKY324 (secY-d1-secY24 double mutant).

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[IlI 1 i. Ii

C2 C304$00 D 1 05 06 LacZ(,

socY24 .il

TM6 C4

X17X54 -- LX101-4 -_ _xi o

.if, 11 SulcY '"I

TM7 P4

FIG. 2. Deletion/insertion mutations isolated as intragenic sup-pressors of secY-dl. The horizontal boxes show regions deleted,with concomitant insertion of amino acid residues as indicated byone-letter codes. The numbers to the left and to the right ofeach boxindicate the SecY residues just adjacent N-terminally and C-termi-nally to the deletion, respectively. Deletions at the nucleotide levelwere as follows: X4, residues 5025-5065; X17, 5013-5092; X54,5022-5035; X101, 5004-5086; X34, 5143-5193; X63, 5114-5134 (num-bering according to ref. 24). Inserted sequences were CCTCGAGGfor X4, X17, X54, and X101, (CCTCGAGG)3 for X34 and X63, andCTCGAG for X213. P, periplasmic domain.

X213 derivatives (Fig. 3). The unaltered initial half-livesindicate that the X mutations did not primarily destabilize theprotein, whereas the lack of the stable population supportsthe notion that the mutant proteins did not associate withSecE. It should be noted that SecY-SecE association andhence the competition in question will occur only amongnewly synthesized molecules (6, 37).To study effects of the X4 suppressor mutation on mem-

brane integration and topology of the SecY protein, the X4derivative of pKY259 was subjected to the TnphoA analysis(20, 21). Fusions with high alkaline phosphatase activity wereobtained at periplasmic domains of SecY, with distributionssimilar to those obtained previously for the wild-type secYgene (25). A P4 fusion after Gly-299 and a site-directedlyconstructed C4 fusion after Lys-268 were characterized byalkaline phosphatase assays as well as trypsin-resistance

20Chase (min)

FIG. 3. Stability of SecY-dla and its derivatives. Cells of K1437carrying aplasmid as indicated below were pulse-labeled at 37°C with[35S5methionine for 0.5 min and chased for the indicated periods.Labeled SecY species were immunoprecipitated (6) and only theSecYa species (see Fig. 2) were quantitated. The radioactivity at the0.5-min chase point was taken as unity for each experiment. Plasmidscarried were as follows: o, pKY259 (secY-dl a); *, pKY259-X17; *,pKY259-X54; o, pKY259-X213.

tests (26). The results (not shown) suggested that the X4mutation did not alter the topological configuration of SecY.The secY24 Mutation within the C4 Domain Weakens SecY-

SecE Interaction. The secY24 (Ts) mutation (Gly-240 Asp) islocated within C4 (12, 17). This mutation, when combinedwith secY-dl in cis, was found to suppress the dominantexport interference (Fig. 1, lane 13). Like the X mutations,the secY24 mutation itself did not affect the initial phase ofdegradation of overproduced SecY-dl (data not shown).We examined whether the SecY24 mutant protein can be

stabilized by SecE. Accumulation of SecY was examined forcells harboring two plasmids, one carrying secY+ (Fig. 4A) orsecY24 (Fig. 4B) and another carrying secE, ydr, or nochromosomal gene. The product of gene ydr, identified as amulticopy suppressor of secy-d) (1), stabilizes SecY al-though less efficiently than SecE (unpublished data). Accu-mulation of SecY24 was only slightly enhanced by the secEplasmid (Fig. 4B, lane 1), although it was enhanced markedlyby the ydr plasmid (lane 2). Pulse-chase experiments (Fig. 5)showed that oversynthesized SecY24 (open circles) wasdegraded as rapidly as the oversynthesized SecY+ (opensquares). Unlike the wild-type SecY protein (closed squares),the mutant protein was only negligibly stabilized by SecE(closed circles).SecE is the limiting factor that determines the stable SecY

pool (6). We examined stability of chromosomally encodedSecY protein. The SecY24 protein but not wild-type SecYstarted to break down slowly (half-life, about 10 min) whencells were shifted to 42°C (data not shown).To directly examine SecY-SecE complexes, we immuno-

precipitated them from solubilized cytoplasmic membranes(27) using an anti-SecY peptide serum. Those proteins thatwere competitively inhibited by the antigen peptide wereconsidered to be specifically (co)immunoprecipitated (Fig.6). As reported by Brundage et al. (4), SecE was coimmu-noprecipitated with wild-type SecY (Fig. 6A, lanes 1 and 5).The identity of the SecE band was confirmed by immuno-blotting (Fig. 6B, lane 1). In contrast, SecE was not coim-munoprecipitated with SecY24 (Fig. 6A, lanes 3 and 7; Fig.6B, lane 3). This was true for both the cells grown at 30°C(Fig. 6 A and B, lanes 1-4) and those grown at 37°C (Fig. 6A,lane 7). Mutations secY39 (ref. 19; Fig. 6, lane 9) and secY122(ref. 17; data not shown) that mapped in a different domainof SecY did not abolish the coimmunoprecipitation of SecE.Thus, the SecE association is specifically impaired by thesecY24 mutation.

-~~~~~~.74A, a ,I.

A

SecY * ph

BSecY24 * _l

FIG. 4. Lack of SecE-dependent accumulation of the overpro-duced SecY24 protein. Strain TW130 that carried pKY6 (secY+; A)was further transformed with pKY250 (secE; lane 1), pST30 (ydr;lane 2; unpublished data), or pKY237 (vector). Similar sets of strainswere constructed with the first plasmid pKY132, which contained thesecY24 mutation in its secY gene (B). Cells were grown at 37°C inminimal-glycerol medium to midlogarithmic phase, and 1 Pg ofwholecell proteins was subjected to SDS/PAGE and immunoblotting usinganti-SecY serum, as described previously (6).

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Cr

0 10 20 30 40Chase(min)

FIG. 5. Effects of SecE overproduction on stability of SecY.Cells of BB030 carrying the following sets of plasmids were pulse-labeled at 420C with [35S]methionine for 30 sec and chased withunlabeled methionine for the indicated periods. Labeled SecY wasimmunoprecipitated, electrophoresed, and quantitated (6). The ra-dioactivity without chase was taken as unity for each experiment andrelative radioactivities are plotted. The degradation of uncomplexedSecY was so rapid at 420C that the initial increase in radioactivity dueto polypeptide chain completion (m) was counterbalanced by thedegradation (n). Plasmids carried were as, follows: c, pKY248(secY+) and pKY225 (vector); *, pKY248 (secY+) and pKY271(secE); o, pKY316(secY24) and pKY225 (vector); *, pKY316(secY24) and pKY271 (secE).

DISCUSSIONAlthough dominant negative mutations do not always titrateout interacting component(s) (30), the present approach willbe applicable to systems Where active site domain andinteracting domain are separable. For instance, mutationsabolishing intracellular enzymatic activities of a cell surfacereceptor will sequester a ligand or an interacting subunit (31,32). The interaction site will then be dissected by the kind ofapproaches we used here.The -secY-dl mutation as well as several other cold-

sensitive mutations at the CS or the C6 domain are dominantnegative (11, 17). Dominant effects were also noted forseveral SecY-PhoA fusions in which C-terminal regions ofSecY were replaced by alkaline phosphatase (11). It was thussuggested that the region near the C terminus is important forthe translocation-facilitating activity of SecY, while the siteresponsible for the export interference is located more N-ter-minally (11).The present results provide a more refined model for the

functional organization of SecY. Deletion/insertion muta-tions that abolished the export-interfering phenotype ofsecY-dl but did not knock out the synthesis of the proteinitself were preferentially located in the C4-TM7 interval,mostly within C4. In contrast, in-frame insertions in severalother locations did not suppress secY-dl.The two mutations (X34 and X63) with rather extensive

sequence alterations in the TM7 segment may cause incom-plete insertion and/or incorrect topological arrangement ofthe product. However, given the results of the TnphoAanalysis, such a suppression mechanism does not seem tohold for the X4 mutation nor for the mutations X54, X213,and secY24 with less extensive sequence alterations; thesecY24 mutation itself does not abolish the SecY function atthe low temperature.The mutations did not affect the initial phase of degradation

kinetics of. the oversynthesized SecY dla molecules, sug-gesting that they did not abolish the dominant phenotype byeliminating the transient existence during which the mutantmolecules compete with wild-type molecules. We proposethat the insertion/deletion mutations within the C4 region

A 2 3 5 6 8 910 B 2 3 4 >

46- I; WINSecYm-.

20.3-

SecEmin- -.w-- 14.3-

12.3- .Antigen - - -..-.

L_.Y Yi4Y_Y + YV24 we

+ Y24t Y3L

Is

Ie

V2 .

FIG. 6. Immunoprecipitation of SecY from mild detergent ex-tracts of membranes. (A) Cells of AD202 (secY+; lanes 1, 2, 5, and6), AD206 (secY24; lanes 3, 4, 7, and 8), and AD208 (secY39; lanes9 and 10) were grown on amino acid (other than Met and Cys)-supplemented M9 medium (14, 19) at 300C (lanes 1-4) or at 3rC(lanes 5-10) and pulse-labeled with [35S]methionine for 5 min. Weused these ompT-deficient strains to avoid in vitro cleavage of SecY(16). Membrane fractions were solubilized under nondenaturingconditions (27) and treated with anti-SecY (28) in the presence(even-numbered lanes) or absence (odd-numbered lanes) of syntheticpeptide (4 Ag/ml) of the N-terminal 20 residues of SecY. Antigen-antibody complexes were subjected to SDS/PAGE, using a 19.6%acrylamide/0.077% methylene-bisacrylamide gel containing 6 Murea (23). Results with AD208 were unchanged when cells incubatedat 20"C( (nonpermissive temperature) for 2 hr were used. Near the topof the gel (not shown here) smearing bands were observed, presum-ably representing aggregated forms of SecY (29). (B) Unlabeledsamples corresponding to lanes 1-4 in A were nimunoprecipitated,electrophoresed, and then transferred to a ZetaProbe membranefilter (23). The upper part of the filter was stained with anti-SecY,whereas the lower half of the filter was stained with anti-SecEantibodies (5). Immunoglobulin heavy chains were electrophoresedabove SecY and heavily stained. The positions of appropriatemolecular mass standards are indicated in kDa.

suppress the dominant secY-dl mutation by impairing theSecY-dl's ability to complex with and sequester SecE.SecY-dl never complemented the chromosomal secY muta-tions, whether it was suppressed intragenically or extragen-ically (data not shown), eliminating a "reactivation" as amechanism of suppression.The secY24 mutation impairs the SecY-SecE interaction.

The overproduced SecY24 protein cannot be stabilized bysimultaneous overproduction ofSecE. The SecY24 protein ofthe basal expression level is destabilized at 42°C. Further-more, SecE cannot be coimmunoprecipitated with SecY24.This was true even when cells had never been exposed to atemperature higher than 30°C. SecY24's affinity for SecEappears to be reduced even at the permissive temperaturesuch that the complex cannot survive the subsequent oper-ations of immunoprecipitation. A histidine-tagged SecE canbring down SecY+ but not SecY24 (T.Y., unpublished re-sults).Degradation of the chromosomal SecY24 protein at 42°C

was slower than that of uncomplexed SecY. The slow deg-radation could be due to a residual SecY24-SecE interactionat 42°C. Indeed, the temperature-sensitive phenotype ofsec Y24 can be suppressed partially by a SecE-overproducingplasmid (T.T. and M. Fujita, unpublished observation). Theslow degradation of SecY24 starts immediately upon tem-perature shift (data- not shown), whereas the onset of exportdefects delays (12). The impairment of SecY-SecE interac-tion may be the primary event, whereas the export defectitself could be a consequence ofthe gradual proteolysis oftheSecY24 protein.SecE can function in a truncated form that contains only

the C-terminal portion including TM3 (33, 34). Osborne andSilhavy (9) proposed that TM10 of SecY interacts with TM3of SecE. If the SecY-SecE interaction is a TM-TM interac-tion, how can the C4 domain of SecY be important? Theactive SecE fragments still contain significant portions of thepreceding cytoplasmic domain (C2) that is significantly con-

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served among prokaryotes (35, 36). Interaction of this seg-ment with the C4 region ofSecY could induce conformationalchanges to the SecY and/or SecE TM segments and theirassociation. Alternatively, the putative C4-C2 interaction isitself directly responsible for the stable association betweenSecY and SecE and for the integrity of the translocationmachinery.

We thank J. Beckwith for the secA-IacZ fusion and for commentson the manuscript, W. Wickner, S. Mizushima, and Y. Anraku forantisera against SecY, SecE, and OmpA, respectively, K. Cannonfor reading the manuscript, and K. Mochizuki and J. Kataoka fortechnical and secretarial assistance. This work was supported bygrants from the Ministry of Education, Science and Culture, Japan,from Naito Foundation, and from Yamada Science Foundation.

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