Requirement for Conformational Flexibility in the Signal Sequence of ...

VOl. 269, No. 23, Issue of June 10, PP. 16029-16033,1994 Printed in U.S.A.

THEJOURNAL OF BIOLOGICAL CHEMISTRY 0 1994 by The American Society for Biochemistry and Molecular Biology, Inc.

Requirement for Conformational Flexibility in the Signal Sequence of Precursor Protein* (Received for publication, December 20, 1993, and in revised form, March 7, 1994)

Nico NouwenSOflll, Jan Tommassen*§,and Ben de Kruijffsfl From the Unstitute of Biomembranes, the §Department of Molecular Cell Biology, and the IDepartment of Biochemistry of Membranes, Utrecht University, Padualaan 8, Utrecht 3584 CH, The Netherlands

of OmpA and LamB ina memAccordingtothe"unlooping"model(deVrije, T., formation of the signal sequence The essential partsof t h e Batenburg, A. M., Killian, J. A, and de Kruijff, B. (1990) brane mimetic environment (13, 14). Mol. Microbiol. 4, 143-150), proposed to explain howsig- unlooping model are as follows. First, the positively charged nal sequences serve to target proteins into the secretory NH, terminus of the signal sequence interacts with the head pathway, the initial interaction of the signal sequence groups of negatively charged phospholipids in the membrane. with the membrane in a helix-turn-helix conformation Subsequently, the signal sequence inserts as a loop i n t h ecore (spanning half of the bilayer) plays important an role in of the bilayer leaving the NH, andCOOH termini at the cytothe initiation of the translocation reaction.To test this plasmicface of themembrane.Thishelix-turn-helixmotif, model we have introduced 2 cysteines (at positions -5 spanning halfof t h e bilayer, could serve as a recognition motif and -19) in the signal sequence of the Escherichia coli for proteinaceous components of the translocation machinery outer membrane protein PhoE. The mutations did not and/or might giverise to local changes in t h e lipid organization, influence the translocation of precursor PhoEin vivoor which possiblycould form a transient state in the translocation in vitro.The 2 cysteines were oxidized to form a disulfide bridge.In vitro translocation of the looped precur- process.Afterthisstage,thesignalsequenceunloopsand moves the NH, terminus of the mature sequence across the sor into inner membrane vesicles was disturbed. The bilayer. This unlooping of the signal sequence is thought to be looped precursor competed with translocation of wild the initiationof the translocation process.To test this model i n type precursor PhoE, and looped precursor that was first bound to inner membrane vesicles could be trans- the contextof a complete precursor protein, we have introduced of PhoE located after the additionof dithiothreitol.Apparently, cysteines at strategic positions in the signal sequence the mutant precursor with a disulfide bridge in sthe i g to enable the stabilization of the putative looped signal senal sequenceis arrested as a very early intermediate inquence by the formationof an intramolecular disulfide bridge. well the translocation process. All of these results are con- The behaviorof t h e looped precursor was investigated ain sistent with the proposed unlooping model and show defined in vitro translocation system. The results show that that, besides theprimary structure characteristicsof a conformational flexibility i n t h e signal sequence is needed for signal sequence, conformational flexibility is needed to the initiation of the translocation process. initiate the translocation reaction.

Signal sequences serve to target proteins into the secretory pathway both in prokaryotic and in eukaryotic cells. Their exact role in the export mechanism is still far from understood. Signal sequences appear to interact with proteins of the export apparatus such as SecA (1, 2), SecE and SecY (3), or the Ffh protein (41, which seems to be a component of the bacterial analogue of the signal recognition particle. Other lines of evidence, both in biophysical and biological systems, point to a direct interaction between signal sequences and membrane ids (5-8). In agreement with these data, biophysical studies revealed a strong interaction between a synthetic copy of t h e signal peptide of the outer membrane protein PhoE of Escherichia coli and lipids in model membrane systems (9, 10). Experiments with lipid monolayers resulted in the proposal of a mechanistic model for the involvement of signal sequencesin the translocation process, i.e. the "unlooping" model (11).A similar kind of model was proposed on the basis of the statistically frequent occurrenceof a-helix-destabilizing amino acids in the central region of the signal sequence (12) and can be extrapolated from nuclear magnetic resonance data the on con-

* The costs of publication of this article were defrayedin part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C.Section1734solely t o indicate this fact. 11 To whom correspondence should be addressed. Tel. 31-30-532592; Fax: 31-30-513655.

EXPERIMENTALPROCEDURES Bacterial Strains-The E. coli K12 strains used were CE1344 (F-, AlacU169 araD139 rpsL thi reZA 0mpR::TnlO) (151,KO03 (Lpp-, AuncBC::TnlO) (161, CE1224(F-, thrleu AproAB-phoE-gpthis thi argE lacy galKxy1 rpsL supE ompR) (171, and "52 (F-, AlacU169 araD139 rpsL thi relA sed51 (18). DNA Manipulations and Plasmids-Plasmid DNA was purified as described (191, followed by anion-exchange chromatography on Qiagen columns (Diagen, Diisseldorf, Germany). Recombinant DNA techniques were performed essentially as described (20). Restriction endonuclease reactions and T4 DNA ligase treatments were performed as described by the manufacturers of the enzymes (Pharmacia Biotech Inc.and New lipEngland Biolabs Inc.). The mutagenic oligonucleotidesd(5"GCAGCCTGTACAGAGCMGATGCCACMT-3') and d(5"GCCAGAGTGTGCTACATTTTTCATTR"3'), which were used for site-specific mutagenesis, were synthesized on a Biosearch 8600 DNA synthesizer. Mutagenesis a two-step polymerase chain reaction (21).Mutations was performed via were confirmedby double-stranded DNA sequencing using the T7 DNA polymerase sequencing kit (Pharmacia). Plasmids pJP29 (22) and pJP320 (23) contain the phoE gene. Plasmid pNNlOO was created by cloning the BamHI-EcoRV fragment of pJP320 containingphoE into the expression vector pJFll8 EM (241, digested with Hind111 and (after filling in the protruding ends with Klenow fragment of E. coliDNA polymerase) with BamHI. In the resulting plasmid, PhoE is expressed under control of the tac promoter. Plasmids pNNl and pNN3were created by site-specific mutagenesis of pJP29, and the double mutant plasmid pNN4 wassubsequently obtained from pNN3.The corresponding mutations are listed in Fig. 1. To achieve overproduction of the mutant PhoE proteins, the PacI-ClaI fragments containing the mutations were substituted for the corresponding fragment of plasmid pNN100, creating plasmids pNN101, pNN103, and pNN104 and placing the mutantphoE genes under control of the tac promoter.

16029

16030

Conformational Flexibility

in Signal Sequences

STRUCTURE

FIG.1. Primary structureof the signal sequences of wild type and mutantprePhoE. Thecysteineresidues that were introduced by site-directed mutagenesis are underlined. The names of theplasmidsthat encode thedifferent PhoE forms and the corresponding proteins are indicated at the right.

-20 -10 -1 MetLysLysSerThrLelalValMetGlylleVaJAlaSerAlaSmValGlnAla

PLASMID

PROTEIN

-29

WT

-20 -10 -1 M e t L y s L y s S e r T h r ~ u A l a L e u V a l V a l M e t G l y n e V a J A l a S e r ~ r V ~ G ~ apNNl

A-5C

-20 -10 -1 M e t L y s L y s C v s S e r T h r L e u A l a L e u V a l V a l M e t G l y n e V ~ a S e r ~ ~ V ~ GpNN3 ~a

6-19

-20 -10 -1 MetLysLys~&rThrLeeuAlaLeuVaJValMetGlyIleVL4laSer&g3erVdGlnAla

A-5C OC-19

pNN4

Pulse Labeling Experiments-Cells, grown under phosphate limita- photometrically determined (30) and compared with a standard curve tion to inducethe expression of PhoE protein, were labeled with pCi 2.5 prepared with potassium hydrogen phosphate. I n a typical experiment, of [35Slmethioninefor 30 s at 30 "C and subsequently chased with a n the reactions were performedin triplicate, and, after subtraction of the excess of nonradioactive methionine as described (22). Radiolabeled ATPase activity without the addition of precursor protein, the results proteins were analyzed by sodium dodecyl sulfate-polyacrylamide gel were compared with the translocation ATPase activity of wild type electrophoresis (PAGE)' ( 2 3 , followed by autoradiography. PhoE in the presence of D m . The standard deviation was approxiProteins-Wild type and mutant formsof prePhoE were purified as mately 10%. described (26) with some modifications. Overexpression of prePhoE was Danslocation Competition Experiment-[35SlMethionine-labeled achieved in s e d mutant strain "52 containing plasmids with phoE wild type prePhoE (0.5 pg/pl) was mixed with a n equal amount of under control of the tac promoter. Cells were grownat 30 "C in Luna nonradioactive mutant prePhoE. As controls, radiolabeled prePhoE was broth untila n optical densityat 660 nmof 0.7 was reached. Then, PhoE mixed with a n equal amount of bovine serum albumin, which was expression was inducedby the addition of isopropyl-1-thio-P-D-galacto-dissolved in 8 M urea, 50 m TridHC1, pH 8.0, or wild type prePhoE. pyranoside (1 mM end concentration). After a 2-h incubation at 30 "C, These different mixtures were used in a translocation reaction. The cells were harvested, and the PhoE precursors were purified as de- translocation mixture(40 IMITris acetate, pH 8.0, 10.8 m magnesium scribed (26). For purification of the cysteine-containing mutant proacetate, 28 m potassium acetate, 4mM ATP, 0.40 pg/ml inner membrane vesicles, 100 pg/ml SecA, and 13 pg/ml SecB) was prewarmedfor teins, 2 mM dithiothreitol (DTT) was added to the buffers to prevent oxidation of the cysteines. SecA (27) and SecB (28) were purified as 3 min at 37 "C, and the reaction was initiated by the addition of the described. prePhoE mixture. After 3 min, the reaction was terminated by chilling Ferricyanide Deatment of Precursor PhoE-Purified prePhoE (1 the sample in an ice water bath. Immediately, proteinase K was added, mg/ml in 8 M urea, 50 mM Tris/HCl, pH 8.0, 2 mM Dm) was diluted and, after inactivationof the protease with phenylmethylsulfonylfluo8-fold into 10 mM potassium ferricyanide and incubated for 1h at room ride and trichloroacetic acid precipitation, the samples were analyzed temperature. Trichloroacetic acid was added toa final concentration of by SDS-PAGE. 10%.After a 30-min incubation on ice, the mixture was centrifuged a t 12,000 x g. The resulting precipitate was washed twice with acetone RESULTS and dissolved in 8 M urea, 50 mM TridHCl, pH 8.0. The material was Danslocation of Cysteine-containing Mutant Precursors in repurified by chromatography on a MonoQ column as described (26). Vivo-To examine the role of the signal sequence in the transDanslocation of prePhoE-Translocation of purified prePhoE into location process, we decided to introduce cysteinesin the signal inner membrane vesicles was carried out as described (26) with the modification that DTT (10 mM end concentration) was only present sequence of PhoE by site-directed mutagenesis (Fig. 1).The when reducing circumstances were essential. After proteinaseK treat- selective introduction of cysteine residues gives unique properment todigest nontranslocated precursors, the samples were analyzed ties to the mutant precursor due to the high chemical reactivity by SDS-PAGE under nonreducing conditions. prePhoE and PhoE proof the aminoacyl side chain and to the fact that wild type teins were detectedby Western immunoblotting (29) usingpolyclonal a prePhoE does not contain any cysteine. The positions of the antiserum directed against PhoE and a n alkaline phosphatase-conjugated secondary antibody(Tago, Inc.). The alkaline phosphatase activ- cysteines in the double mutant protein were chosen such that ity was visualized with a solution of 0.1 M diethanolamine, pH 9.8,1 mM the side chains are in close proximity when the signal sequence MgCI,, 0.35 mM 5-bromo-4-chloro-3-indolyl phosphate, 0.37 m nitro is in a helix-turn-helix conformation according to thecomputer blue tetrazolium. model for the PhoE signal sequence (31).To test whether the Uncoupling of Binding and Danslocation of prePhoE into Inner introduced cysteines influence the translocation of PhoE i n Membrane Vesicles-prePhoE was mixed with SecA, SecB, ATP, and vivo, pulse-chase experiments were performed. All cysteineinner membranevesicles in a standard translocation reaction (26). After a 20-min incubation at 0 "C to allow binding of the precursor, the mix- containing mutant proteinswere processed with similar kinetture was layered on a sucrose cushion (0.5 M sucrose, 40 mM Tris acetate, ics as the wild type protein (Fig. 2). Since processing takes pH8.0,4 mM magnesiumacetate,28 mM potassiumacetate),and place at the periplasmic face of the membrane, these mutations vesicles were pelleted by centifugation for 30 min at 140,000 x g in a apparently had no deleterious effect on the translocation of TLA100.2 rotor. The pellet was dissolved in translocation buffer and PhoE i n vivo. split into three fractions. One was used t o determine the amount of Mutant Precursor i n Danslocation of OxidizedDouble pelleted material.To the other two fractions, ATP (4 mM end concentraVitro-The purified mutant precursors translocated with wild tion) was added, and a translocation reaction was performed in the presence or absence of DTT. After a 20-min incubation at 37 "C, pro- type efficiency into inner membrane vesicles in a standard i n teinase K was added, and the protected material was analyzed by SDS- vitro translocation reaction (results not shown). To introduce PAGE andWesternimmunoblotting.Forquantification of protein an intramolecular disulfide bridge in the signalsequence, the bands, the blots were scanned with a densitometer (LKB Ultrascan XL). purified double mutant precursor containing 2 cysteines was Danslocation ATPase Actiuity-To assay translocation ATPase activity, the same conditions were applied as in the translocation reaction, oxidized with potassiumferricyanide and, after reisolation, used in ani n vitro translocation reaction. The oxidized precurwith themodification that urea-stripped inner membrane vesicles from strain KO03 were used. After mixing the componentsat 0 "C, samples sor did not translocate into inner membrane vesicles under were incubatedat 37 "C for 10 min ina water bath. The reactions were nonreducing circumstances (Fig. 30,lane 7).However, the prestopped by the subsequent additionsof 800 pl of color reagent (0.034% cursorwas translocated when DTT was added (lane 8). To malachite green (Sigma) and 10.5 g/liter ammonium molybdate in 1 N exclude the possibility that a nonspecific modification, which HCl and 0.1%Triton X-100) and 100 pl of 34%citric acid. After 30 min might occur during the ferricyanide treatment, directly abolat room temperature, the amountof released inorganic phosphate was

' The

abbreviations used are: PAGE, polyacrylamide gel electrophoresis; DTT, dithiothreitol; prePhoE, precursor PhoE.

ished the translocation reaction, the same oxidation procedure was carried out with thewild type precursor. Translocation of wild type prePhoE was not affected by the ferricyanide treat-

in Signal Sequences

Conformational Flexibility plasmid:

PR29

pNN3

PNNl

pNN4

WT

F:

st.

A-5C

1

-

2

*

6 4 5 6

chase:

0'30-1' 2' 5'

0' 301'

2' 5'

0'30.1' 2' 5'

st.

3

-

4

9

62 efficiency%

DTT

FIG.4. Functional bindingof looped precursor toinner membrane vesicles. Oxidized prePhoEwas preincubated in a translocation

0'30-1' 2' 5'

FIG.2. Processing kinetics of the cysteine containing mutant proteins in vivo. Plasmid-containing derivatives of strain CE1224 were grown under phosphate limitation and pulse-labeled for 30 s. The pulse was followed by the indicated chase peri0ds.p. precursor PhoE;m, mature PhoE.

A. WT

16031

B. A-SC

mixture with inner membrane vesicles. The vesicles were then pelleted through a 0.5 M sucrose cushion, dissolved in translocation buffer, and, after addition of Dl"l' (lanes 2 and 4 ) , further incubated for 20 min at 37 "C. After proteinase K treatment, the amount of translocated material was analyzed by SDS-PAGE and Western immunoblotting.Lanes 1 and 3 show the amount of material that can be translocated without the addition of D m . The efficiency of translocation after binding is also indicated. st, 25%standard of the pelleted material;p, precursor PhoE; m, mature PhoE.

that upon oxidation of the double mutant precursor an intramolecular disulfide bridge was formed that prevented translocation. Binding of Looped Precursor to Inner Membrane Vesicles-At what stage is the precursor with an intramolecular disulfide st. 1 2 st. 3 4 DTT bridge blocked in the translocation process? First, we studied the interaction of the precursor with SecA. It hasbeen shown that theprecursor of OmpA stimulates theSecAATPase activity (1)and increases the sensitivity of SecA to V8 protease (32) in a signal sequence-dependent way. However, even with wild c. x-19 D. A-5CvC-19 type prePhoE, no similar effects on SecA could be detected.' Therefore, the interactions of the looped precursor with the export apparatus had tobe tested ina more indirect assay, i.e. - t P the functional binding to inner membrane vesicles. The oxit P t m cm dized precursor was incubated under nonreducing circumstances with inner membranevesicles a t 0 "C.The mixturewas centrifuged through a sucrose cushion. Approximately 20% of st. 5 6 st. 7 8 the total amountof precursor was found in thepellet fraction. + + DTT A similar amount of precursor was found in the pellet when FIG.3. 'banslocation of mutant precursors in vitro. prePhoE DTT was present during the incubation showing that the ditranslocation into inner membrane vesicles was carried out by dilution sulfide bridge formation did not inhibit the binding to the of the different oxidized mutant prePhoE forms solubilized in 8 M urea into a translocation mixture without or with D'IT as indicated. Panels vesicles. To determine whether thebinding to thevesicles was A-D show the translocation of the wild type (WT), A-5C, VC-19, and functional, the pellet was dissolved into translocation buffer, A-5CVC-19 proteins, respectively. st, 10% standard of total added ATP was added,and translocation reactionswere performed. In material; p , precursor PhoE; m, mature PhoE. the case of the looped precursor, the addition of DTT to the translocation buffer gave a @-fold stimulation of translocament, and DTT did not enhance the translocation efficiency tion (Fig. 4). The small amountof translocation in the absence (Fig. 3A). Hence, ferricyanide specifically acted on the cys- of DTT is probably due to some reduction of the intramolecular teines in the mutant prePhoE, and DTT could reverse this disulfide bridge during the reisolation procedure. DTT had no action. Oxidation of the mutant prePhoE may not only lead to similar effect on the translocation of wild type prePhoE. It a n intramolecular disulfide bridge but also to intermolecular could be, in thecase of the double mutant, that the translocated disulfide bridges, resulting in oligomeric structures. To assess material in the presence of DTT is derived from a precursor the amount of oligomeric protein, SDS-PAGE was always per- population that was aspecifically bound to the vesicles and formed in the absence of the reducing agent P-mercaptoetha- released upon dissolving the vesicles in translocation buffer. nol. Oxidation of the cysteine-containing mutant protein, in- However, a 10-fold dilution of the reisolated vesicles compared deed resulted in dimerformation (approximately 5-10% of the with the volume in the binding reaction had no effect on the total amountof protein). However, boiling of the samples in the efficiency of translocation (data not shown). Furthermore, since presence of a reducing reagent was necessary to convert the neither SecA nor SecB was added after reisolation of the dimers into monomers (data not shown). Therefore, the im- vesicles, such precursors would not have been targeted effiproved translocation of the double mutant precursor in the ciently to the translocation apparatus. Apparently, the transpresence of DTT (Fig. 3 0 , lane 8) could not have resultedfrom location had occurred from a complex that was functionally the dissociation and the subsequenttranslocation of these di- bound to the inner membrane vesicles during the preincubameric forms. This conclusion was further supported by the re- tion at 0 "C.In conclusion, the binding of the precursor to inner sults obtained with the mutantproteins containing single cys- membrane vesicles was not disturbed by the disulfide bridge, teine residues (Fig. 3, B and C). Oxidation of these mutant and thisbinding was functional. Furthermore, the data suggest precursors led to approximately the same amountof dimers as that the looped precursor is a very early intermediate in the in the case of the double mutant precursor. However, the mo- translocation process. nomeric form translocated with the sameefficiency as the wild type precursor under nonreducing circumstances, and theeffiN. Nouwen, J. Tommassen, and B. de Kruijff, unpublished observaciency was not further enhanced by DTT. These results imply tion. n

t P t m

t P t m

Conformational Flexibility

16032

in Signal Sequences

-

"

st.

wt

"loop"

1

2

3

4 P 4 m

4

FIG.6.Competitionexperiment.[S5S]Methionine-labeledwild type prePhoE wasmixed with no competing precursor(lane l), with an equal amount of nonradioactive wild type (lane 2) or looped precursor (lane 3), or a s a control with bovine serum albumin (lane 4 ) . The mixtures were used to initiate the translocation reaction. Lunes 1 4 show the amount of radiolabeled translocated PhoE after a 3-min incubation at 37 "C.st, 10% standard of radiolabeled prePhoE used in the translocation reaction; p , precursor PhoE; rn, mature PhoE.

precursor FIG.5. Stimulation of translocation ATPase activity. Wild type (rot)and looped precursor weremixed with inner membranevesicles in a standard translocation mixture in the absence or presence of D m , and disulfide bridge, or (ii) interaction of the signal sequence with the amount of inorganic phosphate released by ATP hydrolysis was determined. The amount ofATP hydrolysis by wild type prePhoEin the SecA, SecY, or SecE is disturbed by the disulfide bridge. The presence of D m was taken as 100%. accumulation of a translocation intermediate of proOmpA, of

Danslocation ATPase Activity-Since initiation of the translocation reaction could be accompanied by ATP hydrolysis, a translocation ATPase assay wasperformed. Translocation ATPase activity is defined as hydrolysis of ATP during the translocation of a precursor proteininto inner membrane vesicles (33). The translocation ATPase activity of the wild type precursor was not influenced by the presence of DTT (Fig. 5). However, this activity was strongly reduced in the case of the looped precursor in theabsence of DTT and wasincreased to wild type level upon the addition of DTT. This result indicates that the translocation of the looped precursor is indeed disturbed at a very early stage in the translocation process, i.e. before the hydrolysis of ATP. Competition BetweenLooped Precursor and Wild Type prePhoE-To analyze further whether thelooped precursor is directed into the correct translocation pathway, a competition experiment wasperformed. A fwed amount of [35S]methioninelabeled wild type prePhoE was mixed with nonradioactive wild type or looped precursor, and the translocation of the labeled precursor was determined. To be sure that competition at the level of the membrane was determined and not at the level of SecA or SecB, the experiments were performed with a ratelimiting amount of vesicles and an excess of SecA and SecB. The results showed that nonradioactive wild typeprePhoE as competed with theradiolabeled precursorto the same extent the looped precursor, whereas bovine serum albumin, as a nontranslocatable control, had no effect (Fig. 6). Apparently, the looped precursor entered the same pathway as the wild type precursor.

which a large portion of the mature sequence had been translocated into inner membrane vesicles, has been reported to enhance ATP hydrolysis by SecA(33). The looped precursor did not stimulate theATPase activity, but theATPase activity could be restored to wild type levels by the addition of Dm. This result implies that translocation is already blocked at a stage before the hydrolysis of ATP. Because SecA has ATPase activity and theprecursor of OmpA stimulated both this activity (1)and sensitivity of SecA to V8 protease (32) in a signal sequencedependent way, we attempted to investigate prePhoE-SecA interactions ina similar way. However, no similar effects of wild type prePhoE on SecA were detected. The reason for this discrepancy is unknown. The data obtained so far are consistent with the unlooping model where the disulfide bridge would prevent theunlooping of the signal sequence in thebilayer, but alternativeinterpretations, i.e. incorrect interaction of the looped precursor with SecA, SecY,or SecE, cannot beexcluded. However, the results clearly show that, besides the primary (positively structure characteristics of a signalsequence charged NH, terminus, hydrophobic core, and cleavage site), which are all present in thelooped signal sequence, conformational flexibility is necessary to initiate thetranslocation process. That conformational flexibility is probably a n important feature of signal sequences was previously suggested by the statistically frequentoccurrence of a-helix-destabilizing amino acids in the center of the hydrophobic core (12). Batenburget al. (31) have proposed that theglycine in position -10 of prePhoE gives this conformational flexibility. Future experiments will be directed to determine whether thisglycine is indeed giving the necessary flexibility to the signal sequence.

DISCUSSION

REFERENCES

In this study, we have shown that prePhoE with an intramo- 1. Lill, R.,Dowhan, W., and Wickner, W. (1990)Cell 60,271-280 2. Akita, M., Sasaki, S., Matsuyama, S., and Mizushima, S.(1990)J. Bwl. Chem. lecular disulfide bridge in thesignal sequencecannot be trans265,8164-8169 located into inner membrane vesicles. To gain insight in the 3. Hartl, F.,-U., Lecker, S., Schiebel, E., Hendrick, J. P., and Wickner, W. (1990) Cell 63,269-279 stage at which the translocation is blocked, the interaction of J., High, S., Wood, H., Giner, A,, Tollewey, D., and Dobberstein, B. the looped precursor with theexport apparatus was tested. "he 4. Luirink, (1992)Nature 359,741-743 5. Gierash, L. M. (1989)Biochemistry 28,923-930 looped prePhoE wasstill able to bind to innermembrane Geller, B. L., and Wickner, W. (1985)J. Biol. Chem. 260,13281-13285 vesicles and could be translocated into these vesicles after re- 6. 7. De Vriie, T.,de Swart, R. L., Dowhan, W., Tommassen, J., and de Kruijff, B. duction of the disulfide bridge. Furthermore, the looped pre(1988)Nature 334,173-175 cursor competed as efficiently as wild type nonradioactive pre- 8. Phoenix, D.A., Kusters, R., Hikita, C.,Mizushima, S.,and deKruijff, B. (1993) J. Biol. Chem. 268,17069-17073 cursorwithradioactive wild typeprePhoE in translocation 9. Killian, J. A,, de Jong, A. M. P., Bijvelt, J., Verkleij, A. J., and de Kmijff, B. (1990)EMBO J . 9,815-819 experiments. These results imply that the looped precursor is A. M., Demel, R. A,, Verkleij, A. J., and de Kruijff, B. (1988) targeted to theexport apparatus and accumulates as a n early 10. Batenburg, Biochemistry 27.5678-5685 translocation intermediate at the level of the membrane. This 11. De Vrije, T.,Batenburg, A. M., Killian, J. A., and de Kruijff, B. (1990)Mol. Microbiol. 4, 143-150 leads to the intriguing question of what stage is inhibited after 12. Shinde, U. P., Gunn Row, T.N., and Mawai,Y. R.(1989)Biosci. Rep. 9,737-745 this initial binding step. The possibilities are (i) unlooping of 13. Rim, J., Blanco, F. J., Kobe,B., Bruch, D. M., and Gierasch, L. M. (1993) Biochemistry 32,4881-4894 the signal sequence in the lipid bilayer is prevented by the

Conformational Flexibility 14. Wang, Z., Jones, J. D., Rizo, J., and Gierasch, L. M. (1993) Biochemistry 32, 13991-13999 15. DeCock, H., Overeem, W., andTommassen, J.(1992)J. Mol. Eiol. 224,369-379 16. Yamane, K., Ichihara, S., and Mizushima, S. (1987) J. EioZ. Chem. 262,23582362 17. Tommassen,J., van Tol, H., and Lugtenberg, B. (1983)EMEO J. 2,1275-1279 18. Oliver, D. B., and Beckwith, J. (1981) Cell 25, 765-772 19. Birnboim, H. C., and Doly, J.(1979) Nucleic Acids Res 7, 1513-1524 20. Sambrook, J., Fritsch, E. F., and Maniatis, T. (1989) Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor,

NY 21. Landt, O., Grunert, H., and Hahn, U. (1990) Gene (Amst.)96, 125-128 22. Bosch, D., Leunissen, J., Verbakel, J., de Jong, M., van Erp, H., and Tommassen, J. (1986) J. Mol. B i d . 189, 449-455 23. Tommassen, J., Koster, M., and Overduin, P. (1987)J. Mol. Eiol. 198,633-641 24. Furste, J. P., Pansegrau, W., Frank, R., Blocker, H., Scholz, P., Bagdasarian, M., and Lanka, E. (1986) Gene (Amst.)48, 119-131 25. Lugtenberg, B., Meijers, J., Peters, R., van der Hoek, P., and van Alphen, L.

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(1975) FEES Lett. M), 254-258 26. Kusters, R., de Vrije, T., Breukink, E., and de Kruijff, B.(1989)J. Eiol. Chem. 264,20827-20830 27. Breukink, E., Demel, R. A., de Korte-Kool, G., and de Kruijff, B. (1992) Eiochemistry 31, 1119-1124 28. Lecker, S., Lill, R., Ziegelhoffer, T., Georgopoulos, C., Bassford, P. J., Jr., Kumamoto, C. A,, and Wickner, W. (1989) EMEO J . 8, 2703-2709 29. Agterberg, M., Fransen, R.,and Tommassen, J. (1988) FEMS Microbiol. Lett. 50,295-299 30. Lanzetta, P. A., Alvarez, L. J., Reinach, P. S., and Candia, 0.A. (1979)Anal. Eiochem. 100,95-97 31. Batenburg, A. M., Brasseur, R., Ruysschaert, J. M., van Scharrenhurg, G. J . M., Slotboom, A. J., Demel, R. A., and de Kruijff, B. (1988) J. Biol. Chem. 263,42024207 32. Shinkai, A,, Mei, L. H., Tokuda, H., and Mizushima, S. (1991) J. B i d . Chem. 266.5827-5833 33. Schiebel, E.,Driessen, A. J. M., Hartl, E-U., and Wickner, W. (1991) Cell 64, 927-939 I

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