Two Distinct Regions of the LamB Signal Sequence Function in ...

THEJ o w u . OF BIOLLGICAL CHEMISTRY 0 1994 by The American Society for Biochemistry and Molecular Biology, Inc.

Vol. 269,No. 3,Issue of January 21,pp. 1648-1653, 1994 Printed in U.S.A.

T w o Distinct Regionsof the LamB Signal Sequence Functionin Different Steps in Export* (Received for publication, March 19, 1993, and in revised form, August 30, 1993)

Shui-QingWei and Joan StaderS From the School of Biological Sciences, University of Missouri, Kansas City, Missouri 64110-2499

The hydrophobic core of the Escherichia coli LamB signal sequence mutation suppressor (Prl) allele. Hence, prosignal sequence contains two structurally distinct reteins that actupon the precursor are SecA (PrlD), SecB, SecE gions. One region forms a helix in nonpolar environ(PrlG), and SecY (PrlA) (for review, see Bieker et al. (1990)). ments, and the other is less structured. These regions The only protein of this group for which there is no signal seem to be of special importance for export, asjudged by sequence mutation suppressor allele is SecB. The multiplicity the magnitude of the defect caused by their mutational of signal sequence mutation suppressor alleles suggests that inactivation. To gain insiqht into the mechanistic importhe signalsequence functionsin more than one step within the tance of these two regions, we examined the ability of export pathway, and in vivo biochemical studies with signal precursors to pass partially through the export pathway sequence mutants have supported this concept (Stader et al., when each region is mutated. The results demonstrate 1986; Thom and Randall, 1988). To characterizesignal sethat mutations in the helical and unstructured regions quence functionin detail, itis necessary to establishfunctional of the signal sequence block different steps in the export associations between subregions of the signal sequence and pathway. extragenic export components. The most widely used assay for LamB signal sequence function is the measurement of processing by signal peptidase using Many proteins whose site of function is extracytoplasmic pulse-chase analysis. Since signal sequence removal presumutilize a general export pathway for their localization. Al- ably marks the last function of the signal sequence, the procthough differences in theprokaryotic and eukaryotic pathways essing assay measures the functionality in sum, but does not exist, both require the presence of a n NH2-terminal “signal provide information related to individual steps of the pathway. sequence” on the nascent polypeptide, which is endoproteoWe are interested in determining the relationship between lytically removed by asignal peptidase during localization. signal sequence structure and functions as they relate to the Although there is little amino acid homology among the nu- individual steps in theexport pathway. Do specific domains or merous signal sequences examined, severalcommon character- residues function in distinct steps that can be identified by isticsexist: ( a ) apolar NH2 terminus, primarily positively accumulation of characteristic intermediate forms of the precharged; ( b ) a hydrophobic core; and ( c ) a signal peptidase cursor molecule? Can their accumulation be affected by marecognition site, Ala-X-Ala (for a current review, see Gierasch nipulating extragenic export components? For this study, we (1989)). Mutational studies have shown that all of these char- chose to do a comparative study between two point mutations acteristics impinge on export efficiency (Stader et al., 1986; in lamB that have been assigned to two genetically different Ryan et al., 1985; Fikes et al., 1987, 1990). classes (Stader et al., 1986) and that cause amino acid substiThe studyof signal sequence structure andfunction has been tutions in structurally distinct regions of the signal sequence approached from biophysical, biochemical, and biological per- (Fig. 1).lamBl4D is representative of a class of signal sequence spectives. The most broadly studied model system in this re- mutations that result inamino acid substitutions that place a gard is that of the LamB signal sequence (see Fig. 1).I n vivo charge in the helical portion of the hydrophobic core. Other function seems to requirea helical structure (Emr andSilhavy, members of this class are mutations at codons 12, 13, 15, and 1983), an observation consistent with biophysical studies mea- 16. The class that includes lamBl9R isalso in thehydrophobic suring the insertion of synthetic LamB signal peptides into core, but outside and at theCOOH-terminal side of the helix. artificiallipids andthe accompanying secondary structure Only two members of this class have been characterized, and changes (Briggs et al., 1986). Both genetic and biophysical ap- both are at residue 19. proaches indicate that thehelix lies within the firsthalf of the EXPERIMENTAL.PROCEDURES hydrophobic core and includes residues 10-17 (Emr and Silhavy, 1983; Bruch et al., 1989). Residues 19-25 compose a less Bacterial Strains-All strains are derivatives ofEscherichia coli K12 structured, less hydrophobic region (Bruch et al., 1989; Bruch and are listed in Table I. The SW series of strains were constructed by P1 transduction. To introduce the prL44 allele into the STA strains, and Gierasch, 1990). The nascent precursor is acted upon by at least four protein recipients were transduced to TetRwith a P1 lysate grown on NTlll (zch::TnlO). The resultingstrains (SWY14D and SWY19R) are AS and components of the export machinery. Most of these proteins are Dex’. The secB null strains were constructed using NT197 (secB::TnS) referred to by dual nomenclature arising from whether the as the donor and selecting KanRtransductants. The resulting strains, corresponding genecan possess a loss-of-function (Sec) and/or a SWB14D, SWBlSR, SWYB14D,and SWYBlSR, are phenotypically sensitive to rich media. The SWL strains carry a ZumB signal sequence and lon null mutation. The latter comes from a P1 lysate * This work was supported by National Institutes of Health Grant mutation GM43514 (to J. S.). The costsof publication of this article weredefrayed grown on SG1117 (lonI46::ATnlO).The SWL strains are “etR,mucoid, as in part by the payment of page charges. This article must therefore be andUV-sensitive(Gottesman,1990).SWAL14Dwasconstructed hereby marked “aduertisement”in accordance with 18 U.S.C. Section described (Gottesman,1990) and is also mucoid and W-sensitive, but without an antibiotic marker.The SWL strains were also transduced to 1734 solely to indicate this fact. $To whom correspondence should be addressed.Tel.: 816-235- SecB- as described above. The resulting SWLB strains are KanRTetR and sensitiveto rich media. pDC2 (Clark et al., 1980;obtained from Phil 2593; Fax: 816-235-5158; E-mail: [email protected].

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stability between the two mutants. When the half-lives of LamB14D and LamB19R are measured at 37 "C,a marked T difference in their stabilities is indeed observed (Fig. 2b). The 6 zo ++ twz of LamB14D is 1.9 whereas that min, of LamB19R is -2MMITLRKLPLAVAVAAGVMSAQAMAVDF fold higher at 3.7 min. & 4 D R Because neither precursor is processed by signal peptidase, + integral anmembrane protein whose active issite located at the periplasmic side of the inner membrane, it seemed likely 1. LamB sequence (see telrtfor details). The numberthat degradation would be occufing in the CfloPlasm. Since ing proceeds from the NH, to COOH terminus. The siteof cleavage by signal peptidase is indicated (V).The substitutions shown below the Lon is one of the major cytoplasmic proteases, the effect of a lon sequence refer to the two mutant signal sequences used in this study. null mutationon precursor stability was determined. It can be seen by comparison of Figs. 2a and 3 that LamB14D is stabiTABLEI Bacterial strains lizedthe in absence of Lon. Quantitation of the band intensities by densitometric analysis shows a dramatic increase (see Fig. E. coli strains Description Source 6, right panel). The stability of LamB19R also increases in the Stader and F- araD139 A (argF-lac), STAlOOO ion- background, but only by a small amount (see Fig. 6, left Silhavy (1988) U169 rpsL 150 relAl panel). The most outstanding observation is that the relative flbB-5301 p t ~ F 2 5 &oC '1thiAl stabilities of these two precursors are reversed in theabsence of Stader and STAlOOO lamB14D STA14D Lon. Whereas LamB19R is more stable than LamB14D in the Silhavy (1988) Lab stock STAlOOO lamB19R STA19R wild-type background, LamB14D is more stable in the lonLab stock STAlOOO lamB1021 STA1021 background. T h s study STA14D secB::Tn5 SWBl4D These data demonstrate that Lon is the primary protease This study STA19R secB::Tn5 SWB19R responsible for the degradation of LamB14D. Moreover, since T h s study STAl4D prlA4 zch::TnlO SWY14D This study STA19R prlA4 zch::TnlO Lon is cytoplasmic, it can be concluded that the LamB14D SWY19R T h s study SWY14D secB::Tn5 SWYB14D precursor either is in thecytoplasm or is reversibly associated T h s study SWY19R secB::Tn5 SWYB19R with the inner membrane. The relativelysmallincrease in This study STA14D 1on::mini Tet SWLl4D stability of LamB19R in thelon- background indicates that this T h s study STAl9R 1on::mini Tet SWL19R This study mutant precursor either is not a good substrate for Lon or is SWAL14D STA14D AZon-510 This study SWLl4D secB::Tn5 SWLBllD inaccessible to this protease. SWL19R secB::Tn5 This study SWLB19R Interaction between Mutant Precursors and SecB-It has STAlOOO lamBl3D prlA4 secB::Tn5 Trun et al. (1988) NT197 been shown previously with MBP that SecB binding preserves STAlO2l prlA4zch::TnlO N. J. "run NTlll precursor, thereby maintainingexport Gottesman (1990) the unfolded state in the HBlOl &a1 Alac lon146:A SG1117 a10 leu sup+ rec+ competence (Collier et al., 1988). An additional effect of SecB SG1039 Gottesman (1990) binding is to cause theprecursor to be more sensitive to proteAlac proCYA221 zaj-403::TnlO SG4144 Gottesman (1990) Alon-510 pro+ olysis (Collier et al., 1988; Weiss et al., 1988; Kumamoto and Gannon, 1988). We investigated the possibility that the differBassford) is a plasmid carrying thesecB gene, and it was transformed ent stabilities of LamB14D and LamB19R may result from a into SWAL14D by selection for TetR. variance in their ability to bind SecB. Pulse Labelinga n d Immune Precipitation-Cell cultures were grown One way to demonstrate SecB binding to a mutant LamB and pulse-labeled a t 37 "C as described previously (Stader and Silhavy, precursor is to measureLamB exportunder conditions ofprlA4 1988), except that the cells were killed with 5% trichloroacetic acid. suppression in either the presence or absence of SecB ("run et ProteinA(extrace1lular)-agarose(20 mg/ml) was used instead of protein A whole cells for immune precipitation. al., 1988). The suppression is necessary because without it, the SDS-Polyacrylamide Gel Electrophoresis and Autoradiographymutant precursor is not processed, and export is impossible to Polyacrylamide (10%) gelelectrophoresisandautoradiographywere assay biochemically using presentlyavailabletechniques. camed out asdescribed (Stader etal., 1986). Thus, export of the mutantprecursors can be demonstrated in Data Analysis-The autoradiograms were scannedon a Bio-Rad 620 video densitometer, and the areaof peaks corresponding to LamB and the prlA4 background genetically by partial restoration of the wild-type LamB phenotypes Dex+ and AS and biochemically by MBP' was analyzed by Bio-Rad 1-D Analyst I1 software. Normalized LamB was determined as the ratio of LamB to total MBP using the an increase in signal sequence processing during pulse-chase following equation: normalized LamB= LamBMpMBP + 1.5MBP), since analysis. there are 3 of the 9 methionine residues in the MBP signal sequence Isogenic strains carrying either lamBl4D or lamB19R and (Randall and Hardy, 1986). The decay curve indicates the amount retheprlA4 suppressorallele in a secB' or secB- background were maining at later time points (1,3, or 5 min) a s a percentage of the early constructed as described under "Experimental Procedures" and time point (15 s). The tl,z is the time a t which only 50% normalized assayed for processing (Fig. 4). There is an increase in signal LamB remains. sequence processing in the suppressor strain when SecB is RESULTS present, butno processing when SecB is absent, indicating that Precursor Stability-Previous studies involving lamB signal SecB is required for the export of LamB14D and LamB19R in the prlA4background. In other words, both LamB14D and sequence mutations revealed thatthemutantprecursors LamB14D and LamB19R possess a kinetic defect in an export LamB19R can bind to SecB. Another way to demonstrate interaction between a mutant step thatoccurs prior to processing by signal peptidase (Stader precursor and SecB is through thephenomenon of interference et al., 1986). Since both mutant precursors behave similarly when assayed for signal sequence processing (Fig. %), it be- (Bankaitis and Bassford, 1984). Mutant LamB precursors able came necessary to develop assays that would distinguish pre- to bind SecB but unable to be efficiently exported by the cell cursor intermediates proximal to processing within the export titrate out cellular SecB, making it unavailable for binding pathway. One logical approach is to look at the differences in MBP. Interference is observed as the decreasedexport eficiency of MBP in theLamB signal sequence mutant strains due The abbreviations used are: MBP, maltose-binding protein; pMBP, to lack of available SecB. Interference can be reduced by reprecursor of M B P pLamB, precursorof LamB. moving the SecB-binding sites in the mature region of the I*

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LamB Signal Sequence Structure and Function

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Time (sec) FIG.2. a, autoradiography of pulse-chase assay withSTAlOOO (lamB+),STA14D (ZamBI4DD),and STA19R (lamBI9R).Cultures weregrown and pulsed for 20 s with ["sSlmethionine, andthe label was chased for the times indicated. LamB and MBP were immunoprecipitated from whole cell extracts, subjected toSDS-polyacrylamide gel electrophoresis,and autoradiographed.Arrows indicate the positions of (from top to bottom) pLamB, LamB19R. Autoradiograms fromfive separate experiments LamB, pMBP, and MBP. WT,wild-type. b, stability of mutant precursors LamBl4D and were scanned as described under "Experimental Procedures." MBP was used as an internal control for induction and protein levels. LamB levels a t each time point were standardized againstMBP. A, LamB19R 0, LamB14D.

plasmid (Fig. 5). It can be concluded that both mutant precursors functionally interact withSecB based on two criteria. First,SecB is required for export of the mutant precursors under prlA4 suppression; and second, under nonsuppressing conditions, LamB14D and LamB19R interfere with the export of wild-type MBP. Therefore, the different stabilities of the two mutant precursors are not due to differences in SecB binding. Export-specific Sequestration of LamBlSR-One possible explanation for the stabilityof LamB19R is that this precursor is able to proceed partially through the export pathway to an FIG.3. Autoradiogram of pulse-chase assay withLamBl4D in lon- genetic background. Samples were preparedas described in the intermediate stage inwhich it becomes inaccessible to Lon. If of several this explanation is correct, then blocking progression of legend to Fig. 2 a . The autoradiogram is a representative similar experiments. Markers indicate the positions of (from top to LamB19R to theinaccessible state should render precursor Lon bottom) pLamB, pMBP, and MBP. sensitive. Such a blockage can be accomplished by measuring LamB19R stability in a secB null strain. We compared the mutant precursor, or it canbe increased by raising theexpres- stability of LamB19R in a secB-lon+ background to that in a sion of the mutant precursor (Altman et al., 1990). It can be secB-lon- background. The half-life of LamB19R is 2.8 min in secB-lon- background, all seen in Fig. 2a that interference with MBP export occurs with the secB-Lon+ background, but in the LamB19R (compare MBP export in the lamBl9R and lamB+ of LamB19R remains even at the 5-min chase (Fig. 6, left strains). LamB14D appears not to interfere withMBP export in panel 1. Hence, resistance to Lon proteolysis is not a n intrinsic a wild-type background; but if it isstabilized in thelon- back- characteristic of LamBlgR, but is related to partial export of ground, interference from LamB14D is strong. Indeed, inter- this mutant precursor. In contrast, when the same genetic ference can be released when SecB is overproduced from a background (secB-lon+) is introduced into lamB14D, the sta15"

1'

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LamB Signal Sequence Structure and Function

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pdA4 secB::Tn5

prlA4 1'

FIG.4. Dependence of mutant precursors LamB14D and LamB19R on SecB for export underprlA4 suppression. Top panels, strains SWY14D (SecB+) and SWYB14D (SecB-), respectively; bottom panels, strains SWY19R (SecB') and SWYB (SecB-), respectively. Samples were prepared as described in the legend to Fig. 2 u . Markers in both panels indicate the positions of (from top to bottom) pLamB, LamB, pMBP, and MBP.

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(the mutation used in this study) or deleted (Altman et al., 1990).

Signal Sequence Structure It hasbeen suggested that helix formation in the region beFIG.5. Interference inlamB14D strains withexport efficiency tween residues 9 and 17 is importantfor the efficient export of of MBP. Samples were prepared as described in the legend t o Fig. 2 u , and the 15-s chase is presented for strains STAlID, SWL14D, and LamB in vivo (Emr and Silhavy, 1983). Biophysical studies carried out by Gierasch and co-workers on synthetic LamB SWAL14D pDC2 (relevant genotypes are indicated). Arrows indicate the positions of (from top to bottom) pLamB, pMBP, and MBP. signal sequence peptides have determined that the conformation is random in a hydrophilic environment, but that helix bility of LamB14D increases slightly ( t l j 2= 2.1 min) (Fig. 6, formation is favored in a hydrophobic environment(Bruch right panel) due to protein folding in theabsence of SecB, which and Gierasch, 1990; Bruch et al., 1989; Briggs et al., 1986). In makes the protein less sensitive to protease. These data con- contrast, theregion following residue 18 is less structured,exfirm thatLamB19R is inaccessible to proteolysis in a wild-type cept under certain in vitro conditions, when the helix can be background due to export-specific sequestration. Moreover, this propagated throughthis region. If thesestudieshave relevance to the in vivo situation, one could envision it as a way sequestration is dependent upon SecB activity. for the signalsequence to become properly oriented in the DISCUSSION membrane, as proposed by Briggs et al. (1986). Their model We have analyzed two different LamB signal sequence mu- proposes that residues 14 and 19 lie within the two structurtant precursors and found that theyare similar in theirability ally distinct regions. If helix formation is favored in a hydroto bind SecB and to be exported in the presence of the prlA4 phobic environment, then aspartic acid and arginine residues, suppressor allele, yet they are biochemically distinguishable in which should not interfere with helix formation per se, may vivo based on a difference in stability. LamB14D is accessible to prevent partitioning of the signal sequence into such a n enviLon protease, suggesting that this mutant precursor is primar- ronment and preclude helix formation. In the cell, the hydroily in thecytoplasm. However, crude fractionation studies have phobic environment canbe provided by the lipid bilayer, a proshown that the majority of both LamB14D and LamB19R is tein, or a combination of the two. If initial helix formation associated with the membrane.2 The ability of these mutant requires the insertion of residue 14 into a hydrophobic enviprecursors to associate with the membrane is consistent with ronment and if propagation of the helix requires the transfer its prlA4 and prlGl suppression since both SecY and SecE are of residue 19 into thehydrophobic environment, then itis posintegral membrane proteins. These data suggest that mem- sible that these are themechanistic steps inactivated by 14D and helix brane association of LamB14D is partialor reversible and that and 19R, respectively. Signalsequenceinsertion propagation may involve the catalytic activity of one of the stable association of the precursor withthemembraneis blocked by the 14D mutation, causingequilibrium to favor the cellular export components, or perhaps the signal sequence is competing pathway of Lon degradation. LamB19R becomes in- capable of undergoing substrate modulation once it reaches a accessible to the cytoplasmic protease Lon in anexport-depend- hydrophobic environment. ent manner, suggesting that this mutant precursor is stably Cellular Export Components associated with the innermembrane. It could be argued that the different behavior of the two Parts of the export machinery that are known to interact mutant precursors is dueto the polarity of the charged amino with the precursor are SecB, S e d , SecE, SecY, and signal acid residue resulting from the mutation (LamB14D cames a peptidase. In this study, we have not addressed the issue of negative charge, whereas LamB19R cames a positive charge). interaction between either mutantprecursor and SecA or SecE, However, evidence obtained over recent years indicates that but we have gained insight into interactions with the other the position of the mutation ismore important than thepolar- components. ity. First, both 14D and 19R confer a strong kinetic export deSecB-It had been previously suggested that residue 14 may fect, whereas substitution of either a positively or negatively contribute to the interactionwith SecB based on the fact that charged amino acid at position 17 confers a very weak defect interference is reduced in the lamBl4D mutant compared to (Stader et al., 1986). Second, SecB interference is the same the lamBl9R mutant (Altman et al., 1990). In this study, we whether the wild-type Met residue is substituted with Arg have demonstrated that the apparent lack of interference is due to precursorinstability. When stabilityis restored to LamB14D in a lon- background, interference is also restored, S.-Q.Wei and S. Justice, unpublished data.

1652

LamB Signal Sequence Structure and Function

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FIG.6. Stabilities of LamB19R and LamBllD in various backgrounds. Samples were prepared and analyzed as described in the legend to Fig. 2. Each data point represents the average of at least three separate experiments. 0, wild-type;0, SecB-; A, Lon-; V, SecB-Lon-.

and overproduction of SecB from a plasmid can release the interference. “his observation confirms that residue 14 does not contribute to SecB binding. We have found that LamB14D is slightly more stable in theabsence of SecB (Fig. 61, suggesting thatSecB functions normally to prevent premature folding of the mutant precursor and to make it more sensitive toproteolysis (Collier et al., 1988; Weiss et al., 1988; Kumamoto and form of the Gannon, 1988). In thelamBl9R mutant, the stable precursor that accumulates remainsbound to SecB, indicating that this mutationblocks a step prior to SecB dissociation and that the mutant precursor most likely remains unfolded. If SecB can perform its antifolding activity on both mutant precursors, but is also required for LamB19R to reach its stable form, then SecB must also be providing another function. It has been recently proposed that SecB has an additional function besides antifolding in the localization of another outer membrane protein, PhoE (De Cock and Tommassen, 1992). At this point, it is premature to speculateon what that function may be. Whether or not LamB14D and LamB19R are in the same intermediate form in the absence of SecB is unknown. Since there is also a SecB-independent pathway that requires the presence of a functional signal sequence (Trun etal., 19881, it is possible that thetwo signal sequence mutants utilize this pathway with different efficiency as well. SecY-The restoration of export of LamB14D and LamB19R in a prlA4 background, as measured by the signal peptidase processing activity, is equivalent. This observation presents an apparent paradox in light of our evidence that the two mutations block different steps in the export pathway. The prlA4 allele was originally isolatedas a suppressor of a 12-amino acid residue deletion in theLamB signal sequence (Emr etal., 1981) and has since been shown to be a general suppressor of mutations in thehydrophobic core of LamB (Stader etal., 1986) and in many other different signal sequences. In spite of its broad suppression activity, prlA4 has never been shown to facilitate the export of proteins that are notnormally substrates of the general export pathway. The lack of allele specificity suggests

FIG.7. Competing pathways of export and proteolytic decay for mutant and wild-type L a d precursors. The different shapes represent three distinct forms of LamB that appearduring export. Shading indicates the form that accumulates under each circumstance. Horizontal arrows represent export, and verticalarrows represent degradative pathways. Top panel, wild-type ( W T )LamB is exported with negligible proteolysis and concomitant accumulation of stable trimers (triangle). Middle panel, LamB19Rexport is blocked after SecB-dependent stable association with the membrane and accumulates in a form (circle) that is eventually removed by an unknown protease. Bottom panel, LamB14D fails to make a stable association with the membrane, and the precursor that accumulates (square) is rapidly degraded by Lon. See “Discussion”for further details.

that PrlA4 may by-pass signal sequence defects in some manner. The ability ofprlA4 to suppress LamB14D and LamB19R with equalefficiency does not preclude the possibility of isolating allele-specific suppressors of particular signal sequence mutations in secY or any other export locus. Indeed, we have isolated a prlA suppressor of lamB19R that does not export

LamB Signal Sequence Structure and LamB14D with equalefficien~y.~Thus,thesuppressorapproach can be very valuable, but one must be careful in assigning functions to export components solely on the basis of suppression. Kinetic Partitioning Model In Fig. 7, we have organized the information obtained in this study intoa kinetic model. It is likely that many intermediate forms of LamB appear during export, but here, only the three forms relevant to this work are depicted. We propose that the precursor can go into either theexport pathway or a degradation pathway. In thewild-type situation under optimal growth conditions (Fig. 7, top panel), the export pathway is favored because it is the most efficient. If export is blocked at an early step by signal sequence mutation 14D (Fig. 7, bottom panel), the majority of the defective precursors enter the degradation pathway. If the degradation pathway blocked is by introduction of a null mutation into Lon, one would expect to observe increased export of LamB14D by a mass action effect. Indeed, LamB14D lon null strainas a small but this effect is seen in the measurable increase in the Dex+ and AS phenotypes (data not shown). This observation also demonstrates that the accumulated precursor is a bona fide export intermediate. Presumably, export is possible in spiteof the mutationbecause other export signals (for example,SecB-binding sites) on the precursor molecule are stillactive. Consistent with our model, further export of LamB14D, as under RIA4or PrlGl suppression, does make LamB14D more resistant to proteolysis (data not shown). If export is blocked after stableassociation with the membrane by signal sequence mutation 19R (Fig. 7, middle panel),the precursors areforced into an alternate pathway of either proteolysis or aggregation, followed by proteolysis. One would also exS.-Q. Wei, unpublished data.

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pect to observe phenotypic suppression if this pathway were blocked. We are currently testing this prediction using a genetic approach. This model is analogous to the kinetic partitioning model describing the competing pathways of precursor folding and SecB binding (Hardyand Randall, 1991). Our export model relates to kinetic partitioning at later steps in the pathway. Acknowledgments-We thank Nancy 'Run and Susan Gottesman for strains. We also thank Sheryl Justice for critically reading this manuscript. REFERENCES Altman, E., Emr, S. D. & Kumamob, C. A. (1990)J. Biol. Chem. 266,18154-18160 Bankaitis, V.A. & Bassford, P. J., Jr. (1984) J. Biol. Chem. 269, 12193-12200 Bieker, K. L., Phillips, G. J. & Silhavy,T. (1990) J. Bioenerg. Biomembr. 22, 291-310 Briggs, M. S., Cornell, D. G., Dluhy, R. A. & Gierasch, L. M. (1986) Science 233, 206-208 Bruch, M. D. & Gierasch, L. M. (1990) J. Biol. Chem. 266,3851-3858 Bruch, M. D., McKnight, C. J. & Gierasch, L. M. (1989) Biochemistry 28, 85548561 Clark, D., Lightner, V., Edgar, R., Modrich, P., Cronan,J.E.,Jr. &Bell, R. M. (1980) J . Biol. Chem.266,714-717 Collier, D. N., Bankaitis, V. A., Weiss, J. B. & Bassford, P. J., Jr. (1988) Cell 63, 273-283 De Cock, H. & Tommassen, J. (1992)Mol. Microbiol. 6, 599-604 E m , S. D. & Silhavy, T.J. (1983) Proc. Natl. Acad. Sci. U. S. A. 80,45994603 E m , S. D., Hanley-Way, S. & Silhavy, T. J. (1981) Cell 23, 7 9 4 8 Fikes, J. D., Bankaitis, V. A,, Ryan, J. P. & Bassford, P. J.,Jr. (1987)J. Bacteriol. 169,23452351 Fikes, J. D., Barkocy-Gallagher,G. A., Klapper, D. G. & Bassford, P. J., Jr. (1990) J. Biol. Chem. 266,3417-3423 Gierasch, L. M. (1989) Biochemistry 28, 923-930 Gottesman, S. (1990) Methods Enzymol. 185, 119-129 Hardy, S. J. S. & Randall, L. L. (1991)Science 261, 439443 Kumamoto, C. A. & Gannon, P.M. (1988) J. Biol. Chem. 263, 11554-11558 Randall, L. L & Hardy, S. J. S. (1986) Cell 46, 921-928 Ryan, J. P. & Bassford, P. J., Jr. (1985)J . Biol. Chem. 260, 14832-14837 Stader, J. & Silhavy, T. J. (1988) J. Bacteriol. 170, 1973-1974 Stader, J.,Benson, S. A. & Silhavy, T. J. (1986)J. B i d . Chem. 261, 1507515080 Thom, J. R. & Randall, L. L. (1988)J. Bucteriol. 170, 5654-5661 Trun, N. J.,Stader, J., Lupas, A,, Kumamob, C. & Silhavy, T. J.(1988)J. Bacteriol. 170,592-930 Weiss, J. B., Ray, P. H. & Bassford, P. J., Jr. (1988)P m . Natl.Acad. Sci. U. S . A. 85,8978-8982