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Structural and functional characterization of the OmpF and OmpC porins of the Escherichia coli outer membrane: Studies involving chimeric proteins
Re$. Microbiot. 1989, 140, 177-190
~) INSTITUTPP.STEU~,/ELS~'~[E.~ Paris I989
STRUCTURAL A N D ]~UNCTIONAL CI~dtACTERIZATION OF THE OmpF A N D OmpC PORINS OF THE ESCHERICHLA COLI O U T E R MEMBRANE: STUDIES INVOLVING CHIMERIC PROTEINS C. Hikita (1), y . Saeake (2), H. Yamada (2), T. Mizuno (2) and S. Mizushima (l)(,} (J) Institute o f Applied Microbiology, The University of Tokyo, Yayoi, Bunkyo.ku, Tokyo 113, and (2) Laboratory o f Microbiology, School of Agricuituw, Nagoya University, Chitusa-ku, Nagoya 464 (Japan)
SUMMARY The roles of submolecular regions of OmpF and OmpC, lnajor outer membrane proteins of Escherichia coli, as concerns their biogenesis, structure and function were studied using a large number of chimeric genes constructed from the o m p F and o m p C genes through single or double homologous in vivo recomb'nation. When recombination between the two genes took place at ~ tain regions of their central regions, no chimeric protein was detected, irrespective of whether the amino-terminal and carboxy-terminal regions were derived from OmpF or OmpC. Biochemical studies revealed that these proteins were synthesized and exported across the cytoplasmic membrane normally, but that they were not properly assembled into the outer membrane and hence were degra~,~d rapidIy. Characterization o f th~se chimeric proteins, in which recombination between OmpF and OmpC took piece once or twice, suggeste~ that the central region of each of these proteins plays an important role in the respective assembly, whereas the roles of the amin~'...t¢~;m~naland carboxyterminal regions may be marginal. Functional character~ation of these chimeric proteins revealed the regions important for the receptor functions of O m p F and OmpC for phages Tula and TuIb, respectively.
Received April 18, 1989. (*) Correspondingauthor.
178
C. H I K I T A E T A L .
I~Y-WORDS" Escherichia coli, Outer membrane, Porin; OmpF, OmpC, Chimeric proteins.
INTRODUCTION
OmpF and OmpC are major outer membrane proteins vf Escherichia coli KI2. Although they are homologous proteins (Inokuchi et al., 1982; Mizuno et al., 1983, see also fig. 1), they function differently in several respects (Datta etal., 1977; Furukawa etal., 1979; Hantke, 1978; Nikaido et ate, 1983; Nikaido aud Vaara, 1985; Van Alphen et al,, 1978). As a means of elu._:dating the submolecular structures that determine the difference, a method was developed for constructing series of ompF.ompC and ompC-ompF chimeric genes through in vivo homologous recombination between these two genes (Mizuno et al., 1987; Nogami et al., 1985). Many of the chimeric genes thus constructed produced chimeric proteins which were stably assembled into the cell envelope. When homologous recombination took place within certain parts of their central regions, ht~wever, no chimeric proteins were observed, suggesting that the central regions play an important role in the biogenesis and assembly of the individual porin proteins. In the present work, we found, first of all, that these chimeric proteins are not accumulated because they cannot be assembled prol:rerly into the outer membrane and are hence degraded rapidly. We then coixsttucted a large number of ompF-ompC-ompF chimeric genes in which homologous recombination between the ompF and ompC genes had taken place twice, and studied the roles of the submolecular regions of the porins in their assembly into the outer membrane and their functions. The, results are also discussed in relation to the structures of these porin proteins.
MATERIALS AND METHODS Enzymes. Restriction endonuclease, 1"4 DNA polymerase and T4 DNA iigase were purchased from Takara Shuzo Co. Bacterial strains.
E. coii MHII60 (F-AiocUl#9 araD139 rpsL flbB relA ompBlOl) (Hall and Silhavy, 1981) was used for the construction of plasmids carrying omT~F-ompC-ompF chimeric genes. E. coli SM1005 (F-AlacU169rpsL :~lA thiA flbB syrA ompC
SDS-PAGE = sodium dod~'yl sulpbalc-polyacrylamid¢gel electrophoresis.
P O R I N A S S E M ~ L Y I N E. COLI
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ompFl4) ( M a t s u y a m a , 1984) was used for identification o f the p r o d u c t s o f t h e chimeric genes. E. coli M M I 8 opmF- (F-AtacU169araDt39rpsLre~4 thiA 0,p72-47) ompF::TnS) was constructed t h r o u g h P 1 t r a n s d u c t i o n o f ompF::Tn=; f r o m M H 4 5 0 (Hall a n d Siihavy, 1981)to M M I I ; (Ito el ai., 1981), a n d used f o r t h e detection or precursor forms o f the ch~merm proteins. Unless otherwise indicated, bacterial cells were c u l t u r e d in L - b r o t h . F o r cultivation o f strains possessing plasmids, 50 m g o f ampicillin/litre was a d d e d .
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FrO. I. m Si~s of recombination in individual cJ~imericproteins. The amino acid sequences of OmpF (upper sequence) and OmpC (lower sequence) are shown. Homologous _aminoacid sequences are boxed with thin lines, ilomotogous sequer,ces found to be sites of recombination for individual chimeric proteins are boxed with thick tines with chimer;.c protein numbers above and below, Recombination site 1501 was newly determined in thls study. Numbers above boxes are those of OmpF-OmpC chimeric proteins and numbers below boxes arc those of OmpC-OmpF chimeric proteins. The sh~,rt and long arrow with dotted lines indicate ~-strands.that typicalI~¢app~r~n OmpF ~nd OmpC. respectivew. ~- representz a long amino acia stretclz typical o i o m p t ; .
180
C. H I K I T A
ET AL.
P]asmids.
All of the plasmids carrying ompF-ompC or omt;C-om.~F chimeric genes used, ~xcept plasmid ]501 were described in the previous papers (Mizuno et al., 1987; .~.~logami et al., 1985), a n d are s u m m a r i z e d in figure 1. Piss.mid 1501, which also carries an .~mp.F-ompC chimeric gene, was newly chaxacterizcd in this : t u d y , a n d ',flso s h o w n in figure I. T h e sites o f h o m o l o g o u s r e c o m b i n a t i o n in these genes arc b o x e d with thick lines, with the chimeric gene n u m b e r s a b o v e or below. H e r e a f t e r , these chimeric genes, plasmids carrying these genes a n d chimeric proteins c o d e d for by these 7,enes will be referred to by these n u m b e r s .
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FIG. 2. --. Construction o f ompF-ompC-ompF chimeric genes in which homologous recombination between ompF and oft;pC genes had taken place twice.
Two homologous genes, ompF-om~C and ompC-ompFcifimeric genes, were cloned in tandem in a single ~lasmid. The arnplcillin-resistant gene (b/a) is also l ~ t e z i in the plasmid. Homologous recombination between the two chimeric g~nes was then carried out in vivo as depicted. Restriction endonuclease used are shown in pvrenthe~es, The open and solid at~'ows ind!cat¢ the ompC and ompF genes, respectively. The arrowheads represent the promoter distal ends of the genes.
P O R I N ASSEA4BL Y I N E. C O L I
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Constrnetion of ompg'-ompC-ompF chimerie genes in v i w . Piasmids carrying one of the ompF-ompC chimeric gen~s followed by one of the ompC-ompFchimeric genes were constructed as shown in f~gure 2. They were then linearized and then transferred into MH 1! 6~ for recA-dependent in ~,ivo recombination, which ~elded plasmids carrying ompF-ompC-ompF chimeric genes (fig. 2). Hereafter, such two-fold chimeric genes and the encoded proteins are referred to by combinations o f the numbers of the chimeric genes and those of the chimeric proteins from which they were derived, respectively (for example, gene 1354-17 is an ompF-ompC-ompF gene derived from ompF~oml~ getl6 1354 and om.pC.-ompF gene 17). Structures of alltwo-fold chimeric genes were confirmed by rcstri~mn analysis with relevant restriction enzymes.
Preparation ap~~ characterization of ceil envelopes. The outer membrane protein fraction was prepared by extraction of cell envelcme~ wi,h ~odjurn N-lauryi sarcosinate (Filip et aL, 1973). Ueptidoglycan fractions complexed with the ~:~,_V . . . . . m~ C proteins were prepared as described previously (Rosenbuseh, 1974). Envelope proteins were analysed by urea-SDSpolyacrylamide g¢1 electrophoresis (PAGE) as described previously (Mizuno et aL, 1984),
Pulse-labelling experiments. Cells grown in M9 medium (1 ml) were pulse-labelled at 37°C for 2 min with 20 aCi o f 35S-methionine and then chased in the presence of 0 . 1 % methionJne. Labelled c~lls were solubiliTed by boiling in 1 % SDS. The soluble fraction was treated with the rabbit anti-OmpF antiserum in 250 ~ of 0.2 % SDS as described (Halegoua et aL, 1974), and tLen the precipitate was analysed by SDS-polvac,-w!amide gel electrophoresis and subsequent fluorography (Mizuno et a£, 1984).
immunoblot analysis. The cell envelope fraction from a strain harbouring a chimeric gene was subjected to SDS-PAG~ (Mizuno et al., 1984). Proteins on the gel were then transferred to nitrocellulase filters. T~.-~cfilters were treat¢~t with ehe rabbit anti-OmpF antiserum and then further treated with alkaline phosphatas,..coupled goat anti-rabbit igG to detect cross-reactive proteins.
O:~¢; ~echniques. Piasmids were prepared by the method of Bimboim and, Dely (1979), and tr~n.~-fcrmation was carried out by tile method o f Dagert ~ i d Ehrlich ~1979). Nucleotide scquence~ were determined by the di-deoxy chain termination method o f Sanger et a.L (1977). Other D N A techniques used were described Previously (Matsuyama et aL, I984; Mizuno et at., 1987; :Nogami et al., 1985).
182
C. H I K I T A E T A L .
RESULTS AND DISCUSSION
Characterization of lrauslati,~ products o[ chimeric genes. o m p F - o m p C chimeri,, genes did not produce chimeric proteins in the outer membrane, when homologous recombination between the two gene~ took place at a central region (Nogami ¢t aL, 1985). In order to determine whether these chimeric genes can be expressed or not, a pulse-labelling experiment with aSS-methionine was carried out as shown in figure 3. Cells possessing thes~ chimeric genes expressed proteins that were immunoprecipitable with the a n t i - O m p F antiserum. With m a n y o f these chimeric genes, the level o f expression appeared to be as high as t h a t wita the native o m p F gene, suggesting that, although these genes are expressed normaUy, their translation products are unstable. The instability o f these chimeric proteins was proved by pulse-chase experiments (fig. 4). No degradetion was observed with the native O m p F protein for 18 rain, whereas chimeric proteins, such as the above, were (!0~graded quite rapidly, the half-life time being shorter than 5 rain. Essentially the same result was obtained with o m p C . o m p F c h i m e r i c gene 17, which dit not produce a detectable amount of a chimeric protein (Mizuno et aL, 1987).
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carries a m a J E - ~ : Z fusion gent, *.h~ expression of which is induced by maltose (Bassford et aL, 1979). The M a l E - L a c Z fusion protein thus induced interferes with the export o f m a n y proteins, resulting in the a¢MMI8ompF-
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Ceils possessing one of the indicated chimeric gen¢~ were pulse-~ubeliexi with 3~-S-methionine for 2 mi~ and then the total cell lysate was immunoprec~pitated with anti-OmpF antiserum. The irnmuno.~tecipitatTe.was ana.JysedbySDS:polyacrylan~'degel ele.ctrophoresis and subsequent tluorograpny, , ne position~ ox .JmV~" ana ~rap~ are inuicatea oy arrowheaas.
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cumulation of precursor proteins including proOmpF (lto et al., 1981). In order to determine whether the unstable chimeric proteins are mature or precursor forms, plasmids carrying these chimeric genes were transferred into M M l 8 o r n p F - and the chimeric genes were expressed in the presence of maltose (fig. 5). Upon maltose induction, a slower migrating band, in addition to the one at the position for these chimeric proteins, appeared. This indicates that the new band is that of a precursor and hence the originally existing band is that of the mature form. The unstably accumulating chimeric proteins were localized in the envelope fraction in N-lauryl-sarcosinate-insoluble forms, suggesting that these proteins had interacted to some extert with the outer membrane (data not shown). Taking all these facts together, we conclude that these ch~:meric proteins were synthesized and then exported across the ~,toplasmic membrane, and that they were degraded rapidly due to the failure caused by improper assembly into the outer membrane. The rate of degradation of the chimeric proteins shown in figure 4, was much slower than that of porin assembly into the outer membrane (lto et al., 1977). We further conclude, therefore, that the rapid degradation was not the cause but the result of the lack of proper assembly. Construction of ompF-ompC..ompF chimeric genes and characterization of their products. In order to study the relationship between the submolecular structures of the porin proteins and their assembly into the cell envelope or their functions, a variety of o m p F - o m p C - o m p F chimeric genes, each of which possessed a different portion of the o m p C gene in the middle, was constructed as shown in figure 2. We then examined the following three characteristics: (l) appearance as a stable product in the envelope fraction, (2) assembly into tile outer membrane as a peptidoglycan-associated form (Hasegawa et al., 1976, Rosenbusch, 1974), and (3) functioning as a receptor for phage TuIa or TuIb, which specifically recognize OmpF or OmpC, respectively (Datta et al., 1977). The l esults axe summarized in figure 6 together with those for the relevant ompF-ompC chimeric genes previously described (Mizuno et al., 1987; Nogami et al., 1985). The results will be discussed in the following three sections. Regions determiuiug receptor specificity for phages Tula and Turn. OmpF and OmpC constitute receptors for TuIa and Tulb, respectively, in E. coli KI2 (Datta et al., 1977). As summarized in figure 6, most of the chimeric proteins that stably accumulated in the envelope as peptidoglycanassociated forms were active as receptors for either TuIa or TuIb. Among them, all of the chimeric proteins, in which the OmpC-OmpF recombination took place at site 7 (c~timeric proteins 7, 403-7 and 462-7~, were Tula R TuIb s,
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FtG. 6. - - Characterization o f chimeric proteins. O m p F - O m p C - O m p F chimeric proteins constructed in the present work were characterized. Phage sensitivity represents that o f ceils in which the genes coding for these chimeric protei.'_n_swereyxl~essed:lProl~e,tries o f rel eva2i..t OmpF- .Om.i)C and OmpC-OmpF chimeric pro.~.u~ ~tumcu pre-ac~.~y t lvtt~mo e t a~: l ys/., NogaLm et at., 1985) are also presented together w~t, muse ox u m p , , ana umpt~. ~slaCK oa=s = u m p F domain and white bars ~- OmpC domain.
whereas those with the recombination at site 16 (chimeric proteins 403-I0, 462-10 and 1354-10) were TuIa s TuIb It. The results strongly suggest that the region between sites 10 and 7 plays an important role in determining the receptor specificity for these phages. It should be ttoted in this respect that the sequence homology between OmpF and OmpC is quite low in this region
186
C. H I K I T A E T A L .
(Inokuchi et at.: 1982: Mizuno et al., 1983; Yamada et aL, 1987, see also fig. 1). Cells possessing OmpC-OmpF chimeric protein 10 were Tula R TuIb R despite the fact that the region in question of this protein is derived solely from OmpF (fig. 6). This may be due to improper assembly of this chimeric protein into the outer membrane, since it was not peptidoglycan-associated. Recently, Misra and Benson isolated three OmpC mutants that render cells resistant to OmpC-speeific phages (Misra and Benson, 1988). Two of the mutations were mapped at OmpC-specific amino acid residues in this region, whereas the other mutation, which was mapped outside the region, occurred in ~ region that was common to both OmpC and OmpF. Using OmpC-PhoE and PhoE-OmpC chimeric proteins, Tomm~tssen et al. (1985 ; Van der Ley et aL, 1987) assigned the region in OmpC, which determines the receptor specificity for TuIb, to the 74th-278th residues. The region we assigned (150th-261st residues in the OmpC sequence) for TuIb covers about ~he half of the latter region.
Roles of the amino-tern~na! and carboxy-terminal regions in porin assembly. Studies on series of OmpF-OmpC and OmpC-OmpF chimeric proteins ~uggested that the amino-terminal region before site 462 can be derived either from OmpF or OmpC for stable assembly of chimeric proteins (Mizuno et al., 1987 ; Nogami et aL, 1985). The experiments involving two-fold chimeric proteins in the present work (fig. 6) further indicated that this is true irrespective of whether the carboxy-terminal reg';on is d~rived from OmuF or OmpC, although some of these proteins were not peptidoglyean-associated. These studies also suggested that the carboxy-terminal region after site 26 can be derived either from OmpF or OmpC, irrespective of the source of the aminoterminal region. S~nce the homology between OmpF and OmpC in their terminal regions is not significantly higher than that in the remaining regions, this interex.:hangeability cannot be accounted for solely by homology. The assembly of these terro~nal regions may take place rather independently of the central regions, which possibly play important roles in porin assembly as discussed below. Recently, Bosch et aL (1988) reported that deletions in the amino-terminal region of PhoE, a porin that ressembles OmpF and OmpC, affected the efficiency of assembly, however.
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The above discussion implies that the central regions of O_rvtoFand OmpC are important in the respective assembly. Why does the central region liave to be solely derived from either OmpF or OmpC? In order to answer this question, we constructed a large number of o m p F - o m p C - o m p F chimeric genes and characterized their products. Chimeric proteins possessing the fragment from sites 462 to 7 solely from OmpC can be stably assembled as
P O R I N A S S E M B L Y I N E. COLI
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peptidoglycan-ass~ciated forms, and those possessing the OmpC fragment from sites 462 to 26 can be stably synthesized but are not peptidoglycanassociated. When the site of OmpF-OmpC recombination was moved downward f¢om 462 to 1354, the resultant chimeric proteins became quite unstable, irrespective of the so~c~ of *,he~rboxy-~erminal region, These results support the view that the central rcgions of OmpF ~t~,.dOft,pC are critically different in tertiary stru~ure and behave as the core in the respective assembly into the outer membrane. It should be mentioned in this respect that a shilft of the recombination site from 462 to 1354 resulted in a drastic difference in por~n assembly. Due to the high OmpF/OmpC homology between sites 462 and 1354, only 5 c._mino acid residues differ in this region between the two porins (fig. 1). How does this minor change result in such a drastic change in the modes of assembly of these proteins? ~ approach for the structural prediction of OmpF and bacteriorhodopsin in membranes was propvsed (Paul and Rosenbusch, 1985). The method includes identification of segments causing polypeptides t¢ reserve their direction and ass~gment of ~-strands between the turns. The authors stressed the importance of the [3-strands rather than hydrophobic str~=hes as membrane-spanning domains (Paul and Rosenbusch, 1985). Although the folding profiles predicted using this method are simiIar between OmpF and OmpC (data not pre~ented), OmpC has a long [3-strand (15 amino acid residues) around sites 462 and 1354, whereas OmpF has a short [3-strand (6 amino acid residues) in the corresponding region (see fig. 1). Chimeric proteins with the C~mpF-OmpC junction at site 462 possess the OmpC-type-~-strand, whereas those with the junction at site 1354 possess the OmpF-type one. Since this was the only significant difference, that co~Id be observed between the two ~oroups of chimeric proteins, it is possible that this region, perhaps in combination with other parts of the central domain, plays an important role in the specific assembly of OmpC and Or,~pF, although this view is totally hypothetical, it should be mentioned in this respect that OmpF-OmpC chimeric protein 1354 was unstable, whereas OmpC-OmpF chinleric protein 14, possessing the crossover at essentially the same po~ition, was stable. It is probable that the requirement of the OmpC-type [3-strand for other parts of the central domain is less strict than that of the OmpF-type -strand. Another interesting fact is that the inseztion of a long OmpC-specific polypeptide chain in the central region of OmpC (see fig. 1) into OmpF prevented neither stable assembly nor peptidoglycan-association of the latter porin. According to the folding profile prediction (Paul and Rosenbusch, 1985), this long chain is located in a turn region between two 13-strands. Thus, it is likely that the folding profile of the central region, to which the array of ~-strands contributes appreciably, rather than the primary structure of the central region plays an important role in the assembly of porin proteins.
188
C. H I K I T A E T A L .
RI~SUMI~ STRUCTUR~ ET FONCTIONS DES PORINES O M P F ET O M P C DE LA MEMBRANE EXTERNE DE ESCHERICHIA COLI : IMPLICATION DI~S PROTI~INF.~ CHIMI~RES
Le r61e des r6gions sous-mol~culaires des prot6ines majeuxes OmpF and OmpC de la membrane ~ t e r n e de Escherichia coil, concernant leur biogen~se, leur structure et Ieur fonctlon, a ~6 6tudi6 it l'alde d'un grand hombre de g~nes chim6riques construits/t partir des g ~ e s ompF et 9rnpC it travers des r~ombinalsons homo logues in vivo simples ou doubles. Quand la recombinaison entre les cteux genes prena ptace /~certains endroits de louts r~gions centrales, aucune prot6ine chim~re n'est d~tect6e, ind~pendamment du f~*. qu¢ los r~gions N- ou COO-terminales soient d~riv~es de OmpF ou OmpC. Los 6tud~ biochimiques r*v~lent queces prot~ines sont synth~is6'es et export~s a travers la membrane cytoplasmique normalement mais qu'elles ne sont pas convenablement assembl6es dans la membrane externe et que, par cons6quent, olios sont rapidement d~grad6es. La caract6risation de cos prot~mes chim6riques, darts lesquelles la recombin~ison entre OmpF et OmpC prend place une ou deux lois, sugg~re que la r~gion centrale de chacune de ces prot~inea jouc un r61e important dans ~ur assemblage respectif, alors que le r61e des r~gions N ou COO-terminales peut etre marginal. La caract~nsaUon fonctionnelle de cos pro~6ines chim~riques r~v~le los r~gions importantes pour les fonctions r~cepteurs de OmpF et OmpC pour los bacteriophages TuIa et Tulb, respectivement. MOTS-CL~S: Eschericl~ia coti~ Membrane externe, Porine; OmpF, OmpC, Prot~ines chim~riques. #
ACKNOWLEDGEMENTS
We thank K. Ito for E. coli MMI8 and M. Yoshida mad I. Sugihara for secretarial assistance. This work was supported by gram from the Ministry of Education, Science of Culture of Japan (No. 61060001).
REFERENCES BmSFORV, P. Jr, StLHAVY,T.J. & BECKWlTH,J.R. (1979), Use of gone fusion to study secretion of maltose-bindlng protein into Eaeheriehia coli periplasm. £ Baet., 139, 19-31. BmN~OIM, H.C. & DOLY, J. (1979), A rapid alkaline extraction p~ocedure for screening recombinant plasmid DNA. NucL Acids Res., 7, 1513-1524. BoscN, D., VOORHOUT,W. & TOMMmSEN, J. (1988), Export and localization of Nterminally trancated derivatives of Eseheriehia coil KI2 outer membrane protein PhoE. J. bioL Chem., 263, 9952-9957. DAO~:RT, M. & EHRLtCH, S.D. (1979), Prolonged incubation in calcium chloride improves the competence of Escherichia coli cells. Gene, 6, 23-28. D.~,TrA, D.B., Am0EN, B. & H•NNISO, U. (1977), Major proteim of the Escherichia coli outer cell envelope membrane as bacteriophage receptors. J. Bact., 131, 821-829.
PORIN ASSEMBLY
I N E. COLI
189
FILw, C., FLETCHER,G., WULFF,J.L. & EARHAI~T,CoF. (1973), Solubilization of the cytoplasmic membrane of F~cherlch:a coil by the ionic detergent sodium iaury.~lsarcosinate. J. Bact., 11$, 717-722. FURUKAWA,H., YAMADA:H. & MIZUSHIMA,S. (1979), Interaction of bacteriophage T4 with reconstituted cell envelopes of Escherichia coil KI2. J. Bact., 140, 1071-1080. HALEGOUA, S., HIRASHIMA,A. & INOUYE,M. (1974), Exsistence of a free from of a specific membrane lipoprotein in Gram-negative bacteria. J. Bact., 120, 1204-1208. HALL, M.N. & SILaAVY, T.J. (1981), The ompB locus and the regulation of the major outer membrane porin proteins of Escherichia coti KI2. J. tool Biol., 146, 23-43. ~ , K. (1978) Major outermembrane proteins of E. coli KI2 serve as receptors for the phages T2 tprotein Ia) and 434 (protein Ib). MoL gen. Genetics, 164, 131-135. HASEGAWA,Y., YAMADA,H. ~" MIZUSHIMA,S. (1976), Interaction of outer membrane proteins 0.8 and 0-9 with peptidogly¢an sacculus of Escherichia coli K12. J. Biochem. (Tokyo), ~0, 1401-1409. INOKUCH1, K., MUTOH, N., MATSUYAMA,S. & I~IiZUSH~tA,S. (1982), Primery structure of the o m p F gone that codes for a major outer membrane protein of E3cherichia coli KI2. Nuci. Acids Res., 10, 6957~6968. ITO, K., BASSt~ORD,P.J., Jr & BECKWXTH,J. (1981), Protein localization in E. coli: is there a common step in the setection of periplasmic and outer-membrane proteins? Cell, 24, 707-717. h-o, K., SATO,T. & Y u ~ , T. (1977), Synthesis and assembly of the membrane proteins in E. coll. Cell, 11, 551-559. MATSUVAMA,S., INOKUCHt,K. & MIZUSHIMA,S. (1984), Promoter exchange ~etween o m p F and ompC, genes for osmoregulated major outer membrane proteins of Escherichia coil KI2. J. Bact., 158, 1041-1047. MISRA, R. & BENSON, S.P. (1988), Genetic identificatie~l of t~e pore domain of the OmpC porin of Escherichia coli KI2. J. Bact., 170, 3611-3617. MLzuNo, T., CHOU, M.-Y. & INougw, M. (1983), A comparative study on the genes for three porins of th~ Escherichia coil outer membrane. DNA sequence of the osmoregulated o m p C gone. J. biol. Chem., 2 . ~ 6932-6940. Mxztnqo, T., CHOU, M.-Y. & INot~'~, M . . ~1984), A unique me¢hanism regulating gene expression: translation~ inhibition Dy a complementary KNA transcript (mic RNA)~ Proc. nat. Acad. Sci. (Wash.), 81, 1966-1970. MJzuNo, T., KAsAt, H. & MXZUStI~_A, S. (1987), Construction of a series of ompCompFclfimeric genes by in vivo homologous recombination in Escherichia coIi and characterization of their translational products. MoL gen. Genetics. 207, 217-223. N[~An3¢~, H , ROSF~,~ERO, E.Y. & FOULD, J. (1983), Porin chmanels in Escherich.;a coli: studies with ~lactams in intact cells. J. Bact., 153, 232-240. NmAn)O, H. & V A ~ , M. (1985), Molecular ~Jasis of bacterial outer membrane permeability. Microb~ol. Roy., 49, 1-32. NOC3AMI, T., MIZUNO, T. & M~2VSmMA, S. (1985), Construction of a series of ompF-ompC chimeric genes by in vivo hor~ologous recombination in Escherichia coil and characterization of the translational products. J. BacL~ 164, 797-801. PA, ~L, C. & ROSeNeUSCN, J.P. (1985), Folding patterns of porin and bacteriorhodopsin. EMBO J. 4, 1593-1597. RosENau~m, J.P. (1974). Chaxacterizatiog of-the major envelope proteins from Escherichia coli. regular arrangement, on the peptidoglycan and ~nusual dodecyl sulfate binding. J. biol. Chem., 249, 8019-8029. S~oeR, F., N1c~t~N, S. & Cota.so~ A.R. (1977), DNA sequencing with chainterminating inhibitors. Proc. nat. Acad. Sci. (Wash.), 74, 5463-5467.
17,0
C. H I K I T A E T A L .
TOMMASS~N, J., VANDF.RL~¥, P., VANZEIJL, M. & AGTERBERG, M. (1985), Localization of functional domains in E. eoli KI2 outer membrane Corms. E M B O jr., 4, 1583-1587. VAN ALPHEN, W., VAN SELI~t, N. & LUOTENBERG, B. (1978), Pores in the outer membrane of Escherichia coil K12: involvement o f proteins b and e in the functionning of pores for nucle~Jtides. MoL gen. ~enetics, 159, 75-83. V,MqDI/RLEY, P., BURM, P., AGTERBERG, M., VAr~MEERSBERGEN,J. • TOMMASSEN, J. (1987), Analysis of structure-function relationship in Escherichia coli KI2 outer membrane porins with the aid of ompC-phoE andphoE-ompC hybrid genes. MoL gen. Genetics, 209, 585-591. YAMADA. H., OSHIMA, N., MIZUNO, T., MAI"SU[, H., KAI, Y., NOGUCHI, H. & MIZUSmMA, S. (1987), Use of a series of QmpF-OmpC chimeric proteins for locating antigenic detertrdnants recogmzed by monocional antibodies against the OmpC and OmpF proteins of the Escherichia coil outer membrane. J. Biochem., 102, 455-464.
~) INSTITUTPP.STEU~,/ELS~'~[E.~ Paris I989
STRUCTURAL A N D ]~UNCTIONAL CI~dtACTERIZATION OF THE OmpF A N D OmpC PORINS OF THE ESCHERICHLA COLI O U T E R MEMBRANE: STUDIES INVOLVING CHIMERIC PROTEINS C. Hikita (1), y . Saeake (2), H. Yamada (2), T. Mizuno (2) and S. Mizushima (l)(,} (J) Institute o f Applied Microbiology, The University of Tokyo, Yayoi, Bunkyo.ku, Tokyo 113, and (2) Laboratory o f Microbiology, School of Agricuituw, Nagoya University, Chitusa-ku, Nagoya 464 (Japan)
SUMMARY The roles of submolecular regions of OmpF and OmpC, lnajor outer membrane proteins of Escherichia coli, as concerns their biogenesis, structure and function were studied using a large number of chimeric genes constructed from the o m p F and o m p C genes through single or double homologous in vivo recomb'nation. When recombination between the two genes took place at ~ tain regions of their central regions, no chimeric protein was detected, irrespective of whether the amino-terminal and carboxy-terminal regions were derived from OmpF or OmpC. Biochemical studies revealed that these proteins were synthesized and exported across the cytoplasmic membrane normally, but that they were not properly assembled into the outer membrane and hence were degra~,~d rapidIy. Characterization o f th~se chimeric proteins, in which recombination between OmpF and OmpC took piece once or twice, suggeste~ that the central region of each of these proteins plays an important role in the respective assembly, whereas the roles of the amin~'...t¢~;m~naland carboxyterminal regions may be marginal. Functional character~ation of these chimeric proteins revealed the regions important for the receptor functions of O m p F and OmpC for phages Tula and TuIb, respectively.
Received April 18, 1989. (*) Correspondingauthor.
178
C. H I K I T A E T A L .
I~Y-WORDS" Escherichia coli, Outer membrane, Porin; OmpF, OmpC, Chimeric proteins.
INTRODUCTION
OmpF and OmpC are major outer membrane proteins vf Escherichia coli KI2. Although they are homologous proteins (Inokuchi et al., 1982; Mizuno et al., 1983, see also fig. 1), they function differently in several respects (Datta etal., 1977; Furukawa etal., 1979; Hantke, 1978; Nikaido et ate, 1983; Nikaido aud Vaara, 1985; Van Alphen et al,, 1978). As a means of elu._:dating the submolecular structures that determine the difference, a method was developed for constructing series of ompF.ompC and ompC-ompF chimeric genes through in vivo homologous recombination between these two genes (Mizuno et al., 1987; Nogami et al., 1985). Many of the chimeric genes thus constructed produced chimeric proteins which were stably assembled into the cell envelope. When homologous recombination took place within certain parts of their central regions, ht~wever, no chimeric proteins were observed, suggesting that the central regions play an important role in the biogenesis and assembly of the individual porin proteins. In the present work, we found, first of all, that these chimeric proteins are not accumulated because they cannot be assembled prol:rerly into the outer membrane and are hence degraded rapidly. We then coixsttucted a large number of ompF-ompC-ompF chimeric genes in which homologous recombination between the ompF and ompC genes had taken place twice, and studied the roles of the submolecular regions of the porins in their assembly into the outer membrane and their functions. The, results are also discussed in relation to the structures of these porin proteins.
MATERIALS AND METHODS Enzymes. Restriction endonuclease, 1"4 DNA polymerase and T4 DNA iigase were purchased from Takara Shuzo Co. Bacterial strains.
E. coii MHII60 (F-AiocUl#9 araD139 rpsL flbB relA ompBlOl) (Hall and Silhavy, 1981) was used for the construction of plasmids carrying omT~F-ompC-ompF chimeric genes. E. coli SM1005 (F-AlacU169rpsL :~lA thiA flbB syrA ompC
SDS-PAGE = sodium dod~'yl sulpbalc-polyacrylamid¢gel electrophoresis.
P O R I N A S S E M ~ L Y I N E. COLI
179
ompFl4) ( M a t s u y a m a , 1984) was used for identification o f the p r o d u c t s o f t h e chimeric genes. E. coli M M I 8 opmF- (F-AtacU169araDt39rpsLre~4 thiA 0,p72-47) ompF::TnS) was constructed t h r o u g h P 1 t r a n s d u c t i o n o f ompF::Tn=; f r o m M H 4 5 0 (Hall a n d Siihavy, 1981)to M M I I ; (Ito el ai., 1981), a n d used f o r t h e detection or precursor forms o f the ch~merm proteins. Unless otherwise indicated, bacterial cells were c u l t u r e d in L - b r o t h . F o r cultivation o f strains possessing plasmids, 50 m g o f ampicillin/litre was a d d e d .
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FrO. I. m Si~s of recombination in individual cJ~imericproteins. The amino acid sequences of OmpF (upper sequence) and OmpC (lower sequence) are shown. Homologous _aminoacid sequences are boxed with thin lines, ilomotogous sequer,ces found to be sites of recombination for individual chimeric proteins are boxed with thick tines with chimer;.c protein numbers above and below, Recombination site 1501 was newly determined in thls study. Numbers above boxes are those of OmpF-OmpC chimeric proteins and numbers below boxes arc those of OmpC-OmpF chimeric proteins. The sh~,rt and long arrow with dotted lines indicate ~-strands.that typicalI~¢app~r~n OmpF ~nd OmpC. respectivew. ~- representz a long amino acia stretclz typical o i o m p t ; .
180
C. H I K I T A
ET AL.
P]asmids.
All of the plasmids carrying ompF-ompC or omt;C-om.~F chimeric genes used, ~xcept plasmid ]501 were described in the previous papers (Mizuno et al., 1987; .~.~logami et al., 1985), a n d are s u m m a r i z e d in figure 1. Piss.mid 1501, which also carries an .~mp.F-ompC chimeric gene, was newly chaxacterizcd in this : t u d y , a n d ',flso s h o w n in figure I. T h e sites o f h o m o l o g o u s r e c o m b i n a t i o n in these genes arc b o x e d with thick lines, with the chimeric gene n u m b e r s a b o v e or below. H e r e a f t e r , these chimeric genes, plasmids carrying these genes a n d chimeric proteins c o d e d for by these 7,enes will be referred to by these n u m b e r s .
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[ Xbalh~ker blac~ Xbld omoC~
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~
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Two homologous genes, ompF-om~C and ompC-ompFcifimeric genes, were cloned in tandem in a single ~lasmid. The arnplcillin-resistant gene (b/a) is also l ~ t e z i in the plasmid. Homologous recombination between the two chimeric g~nes was then carried out in vivo as depicted. Restriction endonuclease used are shown in pvrenthe~es, The open and solid at~'ows ind!cat¢ the ompC and ompF genes, respectively. The arrowheads represent the promoter distal ends of the genes.
P O R I N ASSEA4BL Y I N E. C O L I
t31
Constrnetion of ompg'-ompC-ompF chimerie genes in v i w . Piasmids carrying one of the ompF-ompC chimeric gen~s followed by one of the ompC-ompFchimeric genes were constructed as shown in f~gure 2. They were then linearized and then transferred into MH 1! 6~ for recA-dependent in ~,ivo recombination, which ~elded plasmids carrying ompF-ompC-ompF chimeric genes (fig. 2). Hereafter, such two-fold chimeric genes and the encoded proteins are referred to by combinations o f the numbers of the chimeric genes and those of the chimeric proteins from which they were derived, respectively (for example, gene 1354-17 is an ompF-ompC-ompF gene derived from ompF~oml~ getl6 1354 and om.pC.-ompF gene 17). Structures of alltwo-fold chimeric genes were confirmed by rcstri~mn analysis with relevant restriction enzymes.
Preparation ap~~ characterization of ceil envelopes. The outer membrane protein fraction was prepared by extraction of cell envelcme~ wi,h ~odjurn N-lauryi sarcosinate (Filip et aL, 1973). Ueptidoglycan fractions complexed with the ~:~,_V . . . . . m~ C proteins were prepared as described previously (Rosenbuseh, 1974). Envelope proteins were analysed by urea-SDSpolyacrylamide g¢1 electrophoresis (PAGE) as described previously (Mizuno et aL, 1984),
Pulse-labelling experiments. Cells grown in M9 medium (1 ml) were pulse-labelled at 37°C for 2 min with 20 aCi o f 35S-methionine and then chased in the presence of 0 . 1 % methionJne. Labelled c~lls were solubiliTed by boiling in 1 % SDS. The soluble fraction was treated with the rabbit anti-OmpF antiserum in 250 ~ of 0.2 % SDS as described (Halegoua et aL, 1974), and tLen the precipitate was analysed by SDS-polvac,-w!amide gel electrophoresis and subsequent fluorography (Mizuno et a£, 1984).
immunoblot analysis. The cell envelope fraction from a strain harbouring a chimeric gene was subjected to SDS-PAG~ (Mizuno et al., 1984). Proteins on the gel were then transferred to nitrocellulase filters. T~.-~cfilters were treat¢~t with ehe rabbit anti-OmpF antiserum and then further treated with alkaline phosphatas,..coupled goat anti-rabbit igG to detect cross-reactive proteins.
O:~¢; ~echniques. Piasmids were prepared by the method of Bimboim and, Dely (1979), and tr~n.~-fcrmation was carried out by tile method o f Dagert ~ i d Ehrlich ~1979). Nucleotide scquence~ were determined by the di-deoxy chain termination method o f Sanger et a.L (1977). Other D N A techniques used were described Previously (Matsuyama et aL, I984; Mizuno et at., 1987; :Nogami et al., 1985).
182
C. H I K I T A E T A L .
RESULTS AND DISCUSSION
Characterization of lrauslati,~ products o[ chimeric genes. o m p F - o m p C chimeri,, genes did not produce chimeric proteins in the outer membrane, when homologous recombination between the two gene~ took place at a central region (Nogami ¢t aL, 1985). In order to determine whether these chimeric genes can be expressed or not, a pulse-labelling experiment with aSS-methionine was carried out as shown in figure 3. Cells possessing thes~ chimeric genes expressed proteins that were immunoprecipitable with the a n t i - O m p F antiserum. With m a n y o f these chimeric genes, the level o f expression appeared to be as high as t h a t wita the native o m p F gene, suggesting that, although these genes are expressed normaUy, their translation products are unstable. The instability o f these chimeric proteins was proved by pulse-chase experiments (fig. 4). No degradetion was observed with the native O m p F protein for 18 rain, whereas chimeric proteins, such as the above, were (!0~graded quite rapidly, the half-life time being shorter than 5 rain. Essentially the same result was obtained with o m p C . o m p F c h i m e r i c gene 17, which dit not produce a detectable amount of a chimeric protein (Mizuno et aL, 1987).
U n s t a b l e c h i m e r i c p r o t e i n s erJs! as m a t u r e f o r m s .
carries a m a J E - ~ : Z fusion gent, *.h~ expression of which is induced by maltose (Bassford et aL, 1979). The M a l E - L a c Z fusion protein thus induced interferes with the export o f m a n y proteins, resulting in the a¢MMI8ompF-
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FIo. 3. -- Pulse-labelling detection of translational products of chimeric genes with recombination site in cemTa, '¢g" ...
Ceils possessing one of the indicated chimeric gen¢~ were pulse-~ubeliexi with 3~-S-methionine for 2 mi~ and then the total cell lysate was immunoprec~pitated with anti-OmpF antiserum. The irnmuno.~tecipitatTe.was ana.JysedbySDS:polyacrylan~'degel ele.ctrophoresis and subsequent tluorograpny, , ne position~ ox .JmV~" ana ~rap~ are inuicatea oy arrowheaas.
PORIN ASSEMBLY
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I~4
C. I t l K [ T A E T A L .
cumulation of precursor proteins including proOmpF (lto et al., 1981). In order to determine whether the unstable chimeric proteins are mature or precursor forms, plasmids carrying these chimeric genes were transferred into M M l 8 o r n p F - and the chimeric genes were expressed in the presence of maltose (fig. 5). Upon maltose induction, a slower migrating band, in addition to the one at the position for these chimeric proteins, appeared. This indicates that the new band is that of a precursor and hence the originally existing band is that of the mature form. The unstably accumulating chimeric proteins were localized in the envelope fraction in N-lauryl-sarcosinate-insoluble forms, suggesting that these proteins had interacted to some extert with the outer membrane (data not shown). Taking all these facts together, we conclude that these ch~:meric proteins were synthesized and then exported across the ~,toplasmic membrane, and that they were degraded rapidly due to the failure caused by improper assembly into the outer membrane. The rate of degradation of the chimeric proteins shown in figure 4, was much slower than that of porin assembly into the outer membrane (lto et al., 1977). We further conclude, therefore, that the rapid degradation was not the cause but the result of the lack of proper assembly. Construction of ompF-ompC..ompF chimeric genes and characterization of their products. In order to study the relationship between the submolecular structures of the porin proteins and their assembly into the cell envelope or their functions, a variety of o m p F - o m p C - o m p F chimeric genes, each of which possessed a different portion of the o m p C gene in the middle, was constructed as shown in figure 2. We then examined the following three characteristics: (l) appearance as a stable product in the envelope fraction, (2) assembly into tile outer membrane as a peptidoglycan-associated form (Hasegawa et al., 1976, Rosenbusch, 1974), and (3) functioning as a receptor for phage TuIa or TuIb, which specifically recognize OmpF or OmpC, respectively (Datta et al., 1977). The l esults axe summarized in figure 6 together with those for the relevant ompF-ompC chimeric genes previously described (Mizuno et al., 1987; Nogami et al., 1985). The results will be discussed in the following three sections. Regions determiuiug receptor specificity for phages Tula and Turn. OmpF and OmpC constitute receptors for TuIa and Tulb, respectively, in E. coli KI2 (Datta et al., 1977). As summarized in figure 6, most of the chimeric proteins that stably accumulated in the envelope as peptidoglycanassociated forms were active as receptors for either TuIa or TuIb. Among them, all of the chimeric proteins, in which the OmpC-OmpF recombination took place at site 7 (c~timeric proteins 7, 403-7 and 462-7~, were Tula R TuIb s,
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FtG. 6. - - Characterization o f chimeric proteins. O m p F - O m p C - O m p F chimeric proteins constructed in the present work were characterized. Phage sensitivity represents that o f ceils in which the genes coding for these chimeric protei.'_n_swereyxl~essed:lProl~e,tries o f rel eva2i..t OmpF- .Om.i)C and OmpC-OmpF chimeric pro.~.u~ ~tumcu pre-ac~.~y t lvtt~mo e t a~: l ys/., NogaLm et at., 1985) are also presented together w~t, muse ox u m p , , ana umpt~. ~slaCK oa=s = u m p F domain and white bars ~- OmpC domain.
whereas those with the recombination at site 16 (chimeric proteins 403-I0, 462-10 and 1354-10) were TuIa s TuIb It. The results strongly suggest that the region between sites 10 and 7 plays an important role in determining the receptor specificity for these phages. It should be ttoted in this respect that the sequence homology between OmpF and OmpC is quite low in this region
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(Inokuchi et at.: 1982: Mizuno et al., 1983; Yamada et aL, 1987, see also fig. 1). Cells possessing OmpC-OmpF chimeric protein 10 were Tula R TuIb R despite the fact that the region in question of this protein is derived solely from OmpF (fig. 6). This may be due to improper assembly of this chimeric protein into the outer membrane, since it was not peptidoglycan-associated. Recently, Misra and Benson isolated three OmpC mutants that render cells resistant to OmpC-speeific phages (Misra and Benson, 1988). Two of the mutations were mapped at OmpC-specific amino acid residues in this region, whereas the other mutation, which was mapped outside the region, occurred in ~ region that was common to both OmpC and OmpF. Using OmpC-PhoE and PhoE-OmpC chimeric proteins, Tomm~tssen et al. (1985 ; Van der Ley et aL, 1987) assigned the region in OmpC, which determines the receptor specificity for TuIb, to the 74th-278th residues. The region we assigned (150th-261st residues in the OmpC sequence) for TuIb covers about ~he half of the latter region.
Roles of the amino-tern~na! and carboxy-terminal regions in porin assembly. Studies on series of OmpF-OmpC and OmpC-OmpF chimeric proteins ~uggested that the amino-terminal region before site 462 can be derived either from OmpF or OmpC for stable assembly of chimeric proteins (Mizuno et al., 1987 ; Nogami et aL, 1985). The experiments involving two-fold chimeric proteins in the present work (fig. 6) further indicated that this is true irrespective of whether the carboxy-terminal reg';on is d~rived from OmuF or OmpC, although some of these proteins were not peptidoglyean-associated. These studies also suggested that the carboxy-terminal region after site 26 can be derived either from OmpF or OmpC, irrespective of the source of the aminoterminal region. S~nce the homology between OmpF and OmpC in their terminal regions is not significantly higher than that in the remaining regions, this interex.:hangeability cannot be accounted for solely by homology. The assembly of these terro~nal regions may take place rather independently of the central regions, which possibly play important roles in porin assembly as discussed below. Recently, Bosch et aL (1988) reported that deletions in the amino-terminal region of PhoE, a porin that ressembles OmpF and OmpC, affected the efficiency of assembly, however.
Roleof
the ccnt.~-! r e , o n in porin assembly.
The above discussion implies that the central regions of O_rvtoFand OmpC are important in the respective assembly. Why does the central region liave to be solely derived from either OmpF or OmpC? In order to answer this question, we constructed a large number of o m p F - o m p C - o m p F chimeric genes and characterized their products. Chimeric proteins possessing the fragment from sites 462 to 7 solely from OmpC can be stably assembled as
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187
peptidoglycan-ass~ciated forms, and those possessing the OmpC fragment from sites 462 to 26 can be stably synthesized but are not peptidoglycanassociated. When the site of OmpF-OmpC recombination was moved downward f¢om 462 to 1354, the resultant chimeric proteins became quite unstable, irrespective of the so~c~ of *,he~rboxy-~erminal region, These results support the view that the central rcgions of OmpF ~t~,.dOft,pC are critically different in tertiary stru~ure and behave as the core in the respective assembly into the outer membrane. It should be mentioned in this respect that a shilft of the recombination site from 462 to 1354 resulted in a drastic difference in por~n assembly. Due to the high OmpF/OmpC homology between sites 462 and 1354, only 5 c._mino acid residues differ in this region between the two porins (fig. 1). How does this minor change result in such a drastic change in the modes of assembly of these proteins? ~ approach for the structural prediction of OmpF and bacteriorhodopsin in membranes was propvsed (Paul and Rosenbusch, 1985). The method includes identification of segments causing polypeptides t¢ reserve their direction and ass~gment of ~-strands between the turns. The authors stressed the importance of the [3-strands rather than hydrophobic str~=hes as membrane-spanning domains (Paul and Rosenbusch, 1985). Although the folding profiles predicted using this method are simiIar between OmpF and OmpC (data not pre~ented), OmpC has a long [3-strand (15 amino acid residues) around sites 462 and 1354, whereas OmpF has a short [3-strand (6 amino acid residues) in the corresponding region (see fig. 1). Chimeric proteins with the C~mpF-OmpC junction at site 462 possess the OmpC-type-~-strand, whereas those with the junction at site 1354 possess the OmpF-type one. Since this was the only significant difference, that co~Id be observed between the two ~oroups of chimeric proteins, it is possible that this region, perhaps in combination with other parts of the central domain, plays an important role in the specific assembly of OmpC and Or,~pF, although this view is totally hypothetical, it should be mentioned in this respect that OmpF-OmpC chimeric protein 1354 was unstable, whereas OmpC-OmpF chinleric protein 14, possessing the crossover at essentially the same po~ition, was stable. It is probable that the requirement of the OmpC-type [3-strand for other parts of the central domain is less strict than that of the OmpF-type -strand. Another interesting fact is that the inseztion of a long OmpC-specific polypeptide chain in the central region of OmpC (see fig. 1) into OmpF prevented neither stable assembly nor peptidoglycan-association of the latter porin. According to the folding profile prediction (Paul and Rosenbusch, 1985), this long chain is located in a turn region between two 13-strands. Thus, it is likely that the folding profile of the central region, to which the array of ~-strands contributes appreciably, rather than the primary structure of the central region plays an important role in the assembly of porin proteins.
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RI~SUMI~ STRUCTUR~ ET FONCTIONS DES PORINES O M P F ET O M P C DE LA MEMBRANE EXTERNE DE ESCHERICHIA COLI : IMPLICATION DI~S PROTI~INF.~ CHIMI~RES
Le r61e des r6gions sous-mol~culaires des prot6ines majeuxes OmpF and OmpC de la membrane ~ t e r n e de Escherichia coil, concernant leur biogen~se, leur structure et Ieur fonctlon, a ~6 6tudi6 it l'alde d'un grand hombre de g~nes chim6riques construits/t partir des g ~ e s ompF et 9rnpC it travers des r~ombinalsons homo logues in vivo simples ou doubles. Quand la recombinaison entre les cteux genes prena ptace /~certains endroits de louts r~gions centrales, aucune prot6ine chim~re n'est d~tect6e, ind~pendamment du f~*. qu¢ los r~gions N- ou COO-terminales soient d~riv~es de OmpF ou OmpC. Los 6tud~ biochimiques r*v~lent queces prot~ines sont synth~is6'es et export~s a travers la membrane cytoplasmique normalement mais qu'elles ne sont pas convenablement assembl6es dans la membrane externe et que, par cons6quent, olios sont rapidement d~grad6es. La caract6risation de cos prot~mes chim6riques, darts lesquelles la recombin~ison entre OmpF et OmpC prend place une ou deux lois, sugg~re que la r~gion centrale de chacune de ces prot~inea jouc un r61e important dans ~ur assemblage respectif, alors que le r61e des r~gions N ou COO-terminales peut etre marginal. La caract~nsaUon fonctionnelle de cos pro~6ines chim~riques r~v~le los r~gions importantes pour les fonctions r~cepteurs de OmpF et OmpC pour los bacteriophages TuIa et Tulb, respectivement. MOTS-CL~S: Eschericl~ia coti~ Membrane externe, Porine; OmpF, OmpC, Prot~ines chim~riques. #
ACKNOWLEDGEMENTS
We thank K. Ito for E. coli MMI8 and M. Yoshida mad I. Sugihara for secretarial assistance. This work was supported by gram from the Ministry of Education, Science of Culture of Japan (No. 61060001).
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