Proc. Natl. Acad. Sci. USAVol. 88, pp. 6545-6549, August 1991Biochemistry

Reconstitution of a protein translocation system containing purifiedSecY, SecE, and SecA from Escherichia coli

(membrane proteins/membrane assembly/proteoliposomes/ATP)

JIRO AKIMARU, SHIN-ICHI MATSUYAMA, HAJIME TOKUDA, AND SHOJI MIZUSHIMA*Institute of Applied Microbiology, the University of Tokyo, 1-1-1, Yayoi, Bunkyo-ku, Tokyo 113, Japan

Communicated by Gunter Blobel, April 15, 1991 (receivedfor review January 26, 1991)

ABSTRACT Reconstitution of the translocation machin-ery for secretory proteins from purified constituents wasperformed. SecY was solubilized from SecY/SecE-overpro-ducing Escherichia coli cells and purified by chromatographyon ion-exchange and size-exclusion columns. Proteoliposomesactive in protein translocation were reconstituted from thepurified preparations of SecY and SecE. The reconstitutedtranslocation activity was SecA- and ATP-dependent. Al-though the purified preparations of SecY and SecE were stillcontaminated with minute amounts of other proteins, theelution profiles of SecY and SecE on column chromatographiescoincided with the elution profres of reconstituted transloca-tion activity, indicating that SecY and SecE are the indispens-able components in these preparations. We conclude that SecY,SecE, and SecA are essential components of the protein secre-tion machinery and that translocation activity can be recon-stituted from only these three proteins and phospholipids.

Genetic studies have revealed that SecA (1), SecY (2), SecE(3), SecD (4), and SecF (4) play important roles in thetranslocation of secretory proteins across the cytoplasmicmembrane of Escherichia coli. All except SecA are integralmembrane proteins (2-5). Biochemical studies, therefore,require purification of the proteins involved and their recon-stitution into translocationally active proteoliposomes. Pro-teoliposomes that exhibit protein translocation activity havebeen reconstituted from unfractionated components (6-8).Brundage et al. (9) have reported the reconstitution ofproteoliposomes active in protein translocation from a frac-tion containing SecE, SecY, and an uncharacterized protein.It was not clear, however, which components in the fractionwere essential for reconstitution.SecA has been overproduced and purified (10, 11) and then

characterized in detail (12). SecE has been purified fromSecE-overproducing cells and shown to be essential for thereconstitution of active proteoliposomes (13). Furthermore,SecY could be overproduced when SecE was simultaneouslyoverproduced (14). We report here the purification of SecYfrom the cells overproducing both SecY and SecE and thereconstitution ofa protein translocation system from purifiedpreparations of SecY, SecE, and SecA.

MATERIALS AND METHODSBacterial Strains and Plasmids. E. coli W3110 M25 (ompT-)

(15) was transformed with pMAN809 for the overproductionof SecE or with pMAN809 and pMAN510 for the simulta-neous overproduction of SecE and SecY (14). The overpro-duction was induced by isopropyl -D-thiogalactopyranosideas reported (14). pOAD26 carries the ompA-D26 gene en-coding proOmpA-D26, which is a derivative of proOmpA

that lacks about 250 amino acid residues at its C terminus(K. Kanamaru, H. Yamada, and S.M., unpublished data).pSI053 (16) carries the ompA gene that codes for intactproOmpA.

Preparation of SecE, SecA, and Phospholipids. SecE waspurified from the cytoplasmic membrane fraction preparedfrom SecE-overproducing cells as described (13). SecA waspurified from SecA-overproducing cells as described (17). E.coli phospholipids were- prepared as reported (8).

Purification of SecY. Cytoplasmic membrane fractions pre-pared from SecY/SecE-overproducing cells were solubilizedat 1 mg of protein per ml on ice for 10 min with 2.5% (wt/vol)n-octyl ,f-D-glucopyranoside (octyl glucoside) (Dojindo Lab-oratories, Kumamoto, Japan) containing 50 mM potassiumphosphate (pH 6.95), 150 mM NaCl, 10% (wt/vol) glycerol,and E. coli phospholipids (2.5 mg/ml). After ultracentrifu-gation at 140,000 x g for 30 min in a Beckman TLA 100.3rotor, the supernatant containing 11.5 mg of protein wasapplied on a Mono S cation-exchanger column (1 cm x 10 cm;Pharmacia), which had- been equilibrated with 2.5% octylglucoside containing 50 mM potassium phosphate (pH 6.95),10% glycerol, and 150 mM NaCl. The column was thendeveloped at the flow rate of 4 ml/min with a linear gradientof NaCI (0.15-1 M) in the same buffer. The amount of SecYin each fraction (2 ml) was determined by SDS/PAGEfollowed by immunoblot analysis with anti-SecY antiserum.The fraction that-contained most of the SecY was concen-trated by membrane filtration. A sample (0.5 ml) of theconcentrated fraction was further purified by size-exclusionchromatography on a Superose 12 HR column (1 cm x 30 cm;Pharmacia) that had been equilibrated with 2.5% octyl glu-coside containing 50 mM potassium phosphate (pH 6.95),10%O glycerol, and 150mM NaCI. The column was developedwith the same buffer at the flow rate of 0.4 ml/min. Theamount of SecY in each fraction was determined by densit-ometric scanning of the silver-stained gels. Size-exclusionchromatography was performed 10-20 times, and fractionscontaining SecY with a purity of =70% were combined andconcentrated.

Reconstitution of Proteoliposomes Exhibiting Protein Trans-location Activity. Proteoliposomes were reconstituted by theoctyl glucoside dilution method (13). Samples of fractionsobtained by cation-exchange chromatography or size-exclusion chromatography of the solubilized membrane frac-tion derived from SecE/SecY-overproducing cells weremixed with 1.25 mg of E. coli phospholipids in 2.5% octylglucoside. Where specified, purified SecE was also added tothe mixture. After a 20-min incubation on ice, the mixturewas rapidly diluted with 4.6 ml of 50 mM potassium phos-phate (pH 7.5) containing 150 mM NaCI and then incubatedat room temperature for 5 min with stirring. The proteolipo-somes formed were recovered by centrifugation at 160,000 xg for 2 h, suspended in 100 Al of50mM potassium phosphate

*To whom reprint requests should be addressed.

6545

The publication costs of this article were defrayed in part by page chargepayment. This article must therefore be hereby marked "advertisement"in accordance with 18 U.S.C. §1734 solely to indicate this fact.

Dow

nloa

ded

by g

uest

on

Apr

il 2,

202

0

6546 Biochemistry: Akimaru et al.

(pH 7.5) containing 150 mM NaCI, frozen, thawed, andsonicated as described (8).In Vitro Transcription and Translation. In vitro transcrip-

tion of the ompA-D26 and ompA genes was performed asdescribed (18). The translation reaction was carried out in thepresence of Tran35S-Label (0.46 mCi/ml; 1 Ci = 37 GBq;ICN) as described (19). [35S]Methionine-labeled proOmpA-D26 and proOmpA were partially purified as reported (20).

Protein Translocation by Reconstituted Proteoliposomes.Samples (15 jul) of the reconstituted proteoliposomes weremixed with 1 Al of SecA (1.5 mg/ml) and 5 A.l of 50 mMpotassium phosphate (pH 7.5) containing 10 mM MgSO4, 10mM ATP, 150 mM NaCl, and an ATP generating systemcomposed of 50 mM creatine phosphate and creatine kinase(1.25 mg/ml). After a 3-min preincubation at 370C, the assaywas started by the addition of 4 ,.l of [35S]methionine-labeledproOmpA-D26 or proOmpA (2 x 105 cpm). The translocatedprotein, which was proteinase K-resistant, was detected onan SDS/polyacrylamide gel by means of fluorography, asdescribed (21). Densitometric quantification of band materi-als was carried out with a Shimadzu (Kyoto) CS-930 chro-matoscanner. The amounts of translocated proOmpAs wereexpressed as percentages of the input precursor protein.

Preparation of Anti-SecE and Anti-SecY Antisera. Peptidescorresponding to the region from Lys-64 to Lys-81 of SecE(3) and the region from Met-1 to Arg-22 of SecY (22) weresynthesized and used to raise antisera as described (8).SDS/PAGE and Immunoblot Analysis of SecE and SecY. A

gel containing 13.5% acrylamide and 0.36% N,N'-methyl-enebisacrylamide was used as described by Laemmli (23). Allsamples were applied to the gel without boiling. Immunoblotanalysis was carried out as described (24).

Determinations of Phospholipids and Proteins. Amounts ofphospholipids and proteins were determined by the methodsof Bartlett (25) and Lowry et al. (26), respectively.

A

E 0.1c

0co04

-co0

c 0.050m0

.04

0

20 30 40 50Fraction

Br..C^ ^ 2 9

.~~~~~~~~~~~~* j _ _ _,4.-.34.~o

C0

U)

0CC-

50

40 /

20~~~~

10

27 29 31 33 35 37 39Fraction

D1 2 3 4

__r -

A

E 0.80GoN 0.6

.0

0 0.4-

O 0.2

' n

BSecY -p

SecE -

Fraction

I I.

I -§

|I _-" lna

10 15

Fraction

20 25

kDa- 432918.4

-14.3- 6.2

Anti-SecY

Anti-SecE

FIG. 1. Separation of SecE and SecY by cation-exchange chro-matography. (A) The cytoplasmic membrane prepared from SecE/SecY-overproducing cells was solubilized with 2.5% octyl glucoside.A supernatant was obtained and fractionated on a Mono S column.The column was developed with a linear gradient of NaCI (---).

Fractions of 2 ml were collected and Ano was recorded (-). (B) Asample (50 p1) of each fraction in A was analyzed by SDS/PAGE.The gels were either stained with Coomassie brilliant blue or immu-noblotted with anti-SecE or anti-SecY antiserum.

FIG. 2. Purification of SecY by size-exclusion chromatography.Fractions 11-20 in Fig. 1A were combined and then concentrated bymembrane filtration. (A) A sample (500 p1) of the concentratedfraction was purified on a Superose 12 HR column. The elutionpositions of the following molecular mass markers are also indicated:ferritin (440 kDa), aldolase (158 kDa), bovine serum albumin (67kDa), ovalbumin (43 kDa), and ribonuclease A (13.7 kDa). VOdenotes the void volume. (B) SDS/PAGE was performed with 10 ,ulofeach fraction inA followed by silver staining. (C) Proteoliposomeswere reconstituted from samples (100 ,ul) of the fractions in A and1.25 mg of E. coli phospholipids with (e) or without (o) 8 tzg ofpurified SecE. The translocation of partially purified [35S]methio-nine-labeled proOmpA-D26 into the proteoliposomes was assayed at37°C for 20 min. (D) SecY-containing fractions obtained at each stepof purification were analyzed by SDS/PAGE, followed by stainingwith Coomassie brilliant blue. Lanes: 1, membrane fraction (8.0 ,ug);2, octyl glucoside supernatant (7.6 ,ug); 3, Mono S fraction (1.9 ,ug);4, Superose 12 HR fraction (0.5 jig).

RESULTS

The cytoplasmic membrane fraction was prepared from cellsoverproducing both SecY and SecE and solubilized withoctyl glucoside. The soluble fraction, which accounted for95% of the membrane protein, was recovered by centrifuga-tion and then subjected to cation-exchange chromatographyon a Mono S column. The amounts of SecY in the eluateswere determined on SDS gels with Coomassie brilliant bluestaining and immunoblot analysis with anti-SecY antiserum(Fig. 1). SecY was preferentially eluted immediately after thepass-through fraction in which SecE and most of the otherproteins appeared. Proteoliposomes were then reconstitutedfrom these eluates in the presence of purified SecE andanalyzed for protein translocation activity in the presence ofSecA. The highest activity was reconstituted with fractions

15867 43 13.7

Vo 440

kDa

43- SecY- 29

18.4

14.3-6.2

Proc. Natl. Acad. Sci. USA 88 (1991)

- .,IF

I-

Dow

nloa

ded

by g

uest

on

Apr

il 2,

202

0

Proc. Natl. Acad. Sci. USA 88 (1991) 6547

Fraction

B

SecE -.

kDa

- 43

-29

- 18.4

-14.3- 6.2

1415 1617 18 19 20 21 22 23

Fraction Fraction

FIG. 3. Purification of SecE by size-exclusion chromatography.(A) Partially purified SecE was obtained by anion-exchange chro-matography as described (13) and then subjected to size-exclusionchromatography under the conditions described in Fig. 2 for SecY.The molecular mass markers used were the same as those in Fig. 2.(B) A sample (25 Al) of each fraction in A was analyzed by SDS/PAGE, followed by staining with Coomassie brilliant blue, as de-scribed in Fig. 1B. (C) Proteoliposomes were reconstituted from 2041A of each fraction in A and 1.25 mg of E. coli phospholipids with (e)or without (o) 1.9 ,ug of purified SecY and then assayed for trans-location of proOmpA-D26.

11-20 (data not shown). These fractions were combined andconcentrated.

Further purification of SecY was carried out on a Superose12 HR column (Fig. 2A) and the individual fractions obtainedwere analyzed by SDS/PAGE and silver staining (Fig. 2B).Reconstitution was then performed with fractions in thepresence of purified SecE. The translocation activity peak

A BSecY SecE SecA

kDa

43 @-- 2 0

a29 0

C

Ie.

only coincided with the SecY peak (Fig. 2C). No transloca-tion activity was reconstituted in the absence of SecE. Weconclude, therefore, that SecY is the component of thisfraction that is essential for the translocation reaction. Sam-ples obtained at each stage of the purification were analyzedby SDS/PAGE followed by Coomassie brilliant blue staining(Fig. 2D). The purity of the final SecY preparation was>70%o. The band near the top of the gel was more prominentin Fig. 2B. This may be due to a lack ofuniformity in the silverstaining procedure.The SecE preparation used in this and previous (13) studies

was still contaminated by minute amounts of other proteins.To exclude the possible involvement of these minor proteinsin the translocation, fractions from the Superose 12 HRcolumn (the final step ofthe SecE purification) were analyzedfor SecE and translocation activity (Fig. 3). The reconstitu-tion was carried out with a fixed amount of purified SecY.The elution profile of SecE and the profile of reconstitutedactivity coincided closely, whereas the elution profile ofother proteins was different from the reconstitution profile.We conclude, therefore, that SecE is the essential componentin the final preparation. No translocation activity was recon-stituted in the absence of SecY.The reconstitution of protein translocation activity from

the purified preparations of SecY, SecE, and SecA was thenperformed, and the translocation kinetics were studied moreprecisely. The Sec proteins used were of the highest purityobtained (Fig. 4A). Proteoliposomes were reconstituted from1.8 t.g of SecY, 1.9 ,Ag of SecE, and 1250 Ag of E. coliphospholipids. The reconstituted proteoliposomes recoveredby centrifugation contained 1.3, 1.5, and 530 ug of SecY,SecE, and phospholipids, respectively. A time course studywith proOmpA-D26 showed that the reconstituted activitywas SecA- and ATP-dependent, and the reaction proceededquite steadily for 15 min (Fig. 4B). The activity was as highas the activity of the proteoliposomes reconstituted from theSecY/SecE-overproducing membrane. The rate of translo-cation of proOmpA-D26 into everted membrane vesicles ismuch faster than that of intact proOmpA (M. Kato and S.M.,unpublished observation). This was the reason we usedproOmpA-D26 in the present study. We found, however,that the reconstituted proteoliposomes were appreciablyactive in the translocation of intact proOmpA, a naturalpresecretory protein (Fig. 4C). It should be mentioned that

C

18.4 0'

10

0 05 10 15 20 5 10 15 20

Tune (m-) rue (m-)

FIG. 4. Reconstitution of a protein translocation machinery from purified SecY, SecE, and SecA. (A) SDS/PAGE profiles of the purifiedcomponents used for the reconstitution. (B) Proteoliposomes were reconstituted from 1.8 Ag (0.037 nmol) of SecY and 1.9 Ag (0.14 nmol) ofSecE and then assayed for translocation of proOmpA-D26 for the indicated times in the presence of SecA plus ATP (e), SecA alone (A), orATP alone (o). Reconstitution was also performed using 20 A.l of the unfractionated octyl glucoside extract, containing 1.2 ,ug (0.024 nmol) ofSecY and 1.6 ,g (0.12 nmol) of SecE, prepared from the SecY/SecE-overproducing membrane preparation, and the resultant proteoliposomeswere assayed for protein translocation activity (a). (C) Proteoliposomes were reconstituted from 2.0 jig (0.041 nmol) of SecY and 2.8 EAg (0.21nmol) of SecE and then assayed for translocation of intact proOmpA for the indicated times in the presence of SecA plus ATP (A), SecA alone(a), and ATP alone (A). The translocation profile of proOmpA-D26 in the presence of SecA plus ATP is also presented (-).

AE000CO

04-W

I0o0.0

Biochemistry: Akimaru et al.

Dow

nloa

ded

by g

uest

on

Apr

il 2,

202

0

6548 Biochemistry: Akimaru et al.

A 'B50e 500

'O40-u40 4

0030 0. 0C

IP 20-20-

10 10

0~~~~~~0.04 0.08 0.12 0 0.1 0.2 0.3 0.4SecY (nmol) SecE (nmol)

FIG. 5. Effects ofthe amounts ofSecY and SecE on the translocation activity ofreconstituted proteoliposomes. (A) The translocation activityofproteoliposomes reconstituted from 1.9 ,ug (0.14 nmol) ofSecE and the indicated amounts ofSecY was assayed in the presence (e) or absence(o) ofSecA for 20 min. (B) The translocation activity ofproteoliposomes reconstituted from 3.0 ,ug (0.06 nmol) ofSecY and the indicated amountsof SecE was assayed for 20 min with (0) or without (o) SecA.

the rate oftranslocation ofproOmpA was still 180 times lowerthan that exhibited by native membrane vesicles on the basisof the amount of SecY.When reconstitution was carried out with various amounts

of SecY and a fixed amount of SecE (0.14 nmol), thereconstituted activity exhibited saturation at about 0.03 nmolof SecY (Fig. SA). Reconstitution was also carried out withvarious amounts of SecE and a fixed amount of SecY (0.06nmol) (Fig. 5B). The reconstituted activity increased as theamount of SecE increased. The increase was observed evenwhen SecE was >5-fold greater than SecY. These resultssuggest that SecE is present in excess to SecY in functionalstoichiometry. Bieker and Silhavy (27) indicated that SecEprecedes SecY in the protein translocation pathway. Theimportance of SecA in the initial stage of the secretionpathway has been suggested (17, 20, 28). Thus by taking theseresults (17, 20, 27, 28) and our results (Fig. 5) into consider-ation, it is likely that SecE functions as a shuttle betweenSecA and SecY in the secretory pathway.The results shown in Fig. 5 also indicate that SecY, SecE,

and SecA are all indispensable components of the proteintranslocation machinery. This conclusion is inconsistent witha recent report (6) that SecY may be dispensable for thereconstitution of a translocation system. The reasons for thisdiscrepancy are unclear.

DISCUSSIONIn the present work, a translocation system for secretoryproteins was reconstituted from purified protein components(SecY, SecE, and SecA) and phospholipids. The reconsti-tuted activity was ATP-dependent. In the present study,proteinase K resistance was the sole index of protein trans-location. Cleavage of the signal peptide, another index oftranslocation, was not demonstrated, since signal peptidasewas not included. The possibility that the observed activityrepresents only a partial reaction of translocation has notbeen excluded completely.Although the SecY and SecE preparations used were still

contaminated by minute amounts ofother proteins, SecY andSecE were the only proteins whose elution profiles coincidedwith profiles of the reconstituted translocation activity (Figs.2 and 3). The results strongly suggest that SecY and SecE arethe essential components in these preparations. Brundage etal. (9) have demonstrated the reconstitution of translocation-ally active proteoliposomes from a solubilized membranefraction containing SecY, SecE, and an uncharacterizedprotein as major components. We demonstrated that bothSecY and SecE are essential but that the third protein is not;

the third protein was not detected in our purified Sec prep-arations on SDS gels. We also demonstrated that SecY andSecE were not cofractionated by column chromatography.On the basis of translocation activity per unit amount of

SecY or SecE, proteoliposomes reconstituted from the pu-rified components were as active as those reconstituted fromthe crude solubilized membrane from which the purifiedsamples were derived. The activity was, however, far lowerthan that exhibited by native everted membrane vesicles asmentioned above. Possible reasons for this are as follows. (i)Only small fractions of SecY and SecE might have beenfunctionally reconstituted into proteoliposomes, (it) the re-constituted proteoliposomes were unable to generate theproton motive force that generally enhances the translocationactivity (29), and (iii) the reconstitution was carried out in theabsence of SecD and SecF, both of which are geneticallysuggested to be involved in the translocation (4). The absenceof SecD and SecF may not be crucial, however, since theymight function on the periplasmic side of the cytoplasmicmembrane (i.e., inside the everted membrane vesicles) (4)and the translocation was assayed by resistance to proteinaseK, which was added externally to the membrane vesicles.

Finally, it should be mentioned that the present biochem-ical study is consistent with a previous genetic study (27) inthat both suggest that SecY and SecE interact but can easilybe separated and SecE appears to be in functional excess toSecY. Such consistencies support the physiological signifi-cance of the translocation activity reconstituted in the pres-ent study.

Reconstitution techniques have been applied to structure-function analysis of organelles and complex biological sys-tems. The reconstitution of a secretory machinery frompurified components will greatly facilitate elucidation of themolecular mechanisms underlying protein secretion acrossthe cytoplasmic membrane and the mechanisms underlyingprotein translocation across membranes in general.

We thank Y. Kabuyama for technical help and I. Sugihara forsecretarial support. This work was supported by grants from theMinistry of Education, Science and Culture of Japan (61060001,02404013, 02680153, and 02780170).

1. Oliver, D. B. & Beckwith, J. (1981) Cell 25, 765-772.2. Ito, K., Witterkind, M., Nomura, M., Shiba, K., Yura, T.,

Miura, A. & Nashimoto, H. (1983) Cell 32, 789-797.3. Schatz, P. J., Riggs, P. D., Jacq, A., Fath, M. J. & Beckwith,

J. (1989) Genes Dev. 3, 1035-1044.4. Gardel, C., Johnson, K., Jacq, A. & Beckwith, J. (1990)EMBO

J. 9, 3209-3216.

Proc. Nad. Acad. Sci. USA 88 (1991)

Dow

nloa

ded

by g

uest

on

Apr

il 2,

202

0

Biochemistry: Akimaru et al.

5. Akiyama, Y. & Ito, K. (1985) EMBO J. 4, 3351-3356.6. Watanabe, M., Nicchitta, C. V. & Blobel, G. (1990) Proc. Natl.

Acad. Sci. USA 87, 1960-1964.7. Driessen, A. J. M. & Wickner, W. (1990) Proc. Natl. Acad.

Sci. USA 87, 3107-3111.8. Tokuda, H., Shiozuka, K. & Mizushima, S. (1990) Eur. J.

Biochem. 192, 583-589.9. Brundage, L., Hendrick, J. P., Shievel, E., Driessen, A. J. M.

& Wickner, W. (1990) Cell 62, 649-657.10. Kawasaki, H., Matsuyama, S., Sasaki, S., Akita, M. & Mizu-

shima, S. (1989) FEBS Lett. 242, 431-434.11. Cunningham, K., Lill, R., Crooke, E., Rice, M., Moore, K.,

Wickner, W. & Oliver, D. (1989) EMBO J. 8, 955-959.12. Oliver, D. B., Cabelli, R. J. & Jarosik, G. P. (1990) J. Bioen-

erg. Biomembr. 22, 311-336.13. Tokuda, H., Akimaru, J., Matsuyama, S., Nishiyama, K. &

Mizushima, S. (1991) FEBS Lett. 279, 233-236.14. Matsuyama, S., Akimaru, J. & Mizushima, S. (1990) FEBS

Lett. 269, 96-100.15. Sugimura, K. (1988) Biochem. Biophys. Res. Commun. 153,

753-759.16. Yamada, H., Tokuda, H. & Mizushima, S. (1989) J. Biol.

Chem. 264, 1723-1728.

Proc. Nati. Acad. Sci. USA 88 (1991) 6549

17. Akita, M., Sasaki, S., Matsuyama, S. & Mizushima, S. (1990)J. Biol. Chem. 265, 8164-8169.

18. Melton, D. A., Krieg, P. A., Rebagliati, M. R., Maniatis, T.,Zinn, K. & Green, M. R. (1984) Nucleic Acids Res. 12, 7035-7056.

19. Yamane, K., Matsuyama, S. & Mizushima, S. (1988) J. Biol.Chem. 263, 5368-5372.

20. Tani, K., Shiozuka, K., Tokuda, H. & Mizushima, S. (1989) J.Biol. Chem. 264, 18582-18588.

21. Yamane, K., Ichihara, S. & Mizushima, S. (1987) J. Biol.Chem. 262, 2358-2362.

22. Ceretti, D. P., Deau, D., Davis, G. R., Bedwell, D. M. &Nomura, M. (1983) Nucleic Acids Res. 11, 2599-2616.

23. Laemmli, U. K. (1970) Nature (London) 227, 680-685.24. Yamada, H., Matsuyama, S., Tokuda, H. & Mizushima, S.

(1989) J. Biol. Chem. 264, 18577-18581.25. Bartlett, G. R. (1959) J. Biol. Chem. 234, 466-468.26. Lowry, 0. H., Rosebrough, N. J., Farr, A. L. & Randall, R. J.

(1951) J. Biol. Chem. 193, 265-275.27. Bieker, K. L. & Silhavy, T. J. (1990) Cell 61, 833-842.28. Hartd, F.-U., Lecker, S., Shiebel, E., Hendrick, J. P. & Wick-

ner, W. (1990) Cell 63, 269-279.29. Mizushima, S. & Tokuda, H. (1990) J. Bioenerg. Biomembr.

22, 389-399.

Dow

nloa

ded

by g

uest

on

Apr

il 2,

202

0