Hans de Cock and Jan Tommassen. Department of Molecular ...... de Cock,H., van Blokland,S. and Tommassen,J. (1996) In vitro insertion and assembly of outer ...
The EMBO Journal vol.15 no.20 pp.5567-5573, 1996
Lipopolysaccharides and divalent cations are involved in the formation of an assembly-competent intermediate of outer-membrane protein PhoE of E.coli Hans de Cock and Jan Tommassen Department of Molecular Cell Biology, Institute of Biomembranes, Utrecht University, Padualaan 8, 3584 CH Utrecht, The Netherlands
To identify the requirements for the biogenesis of outermembrane proteins in Gram-negative bacteria, the sorting and assembly of the trimeric, pore-forming protein PhoE was studied in vitro. Purified lipopolysaccharide (LPS) in combination with low amounts of Triton X-100 and divalent cations induced the formation of folded monomers. LPS of deep-rough strains was far less efficient in the formation of folded monomers than wild-type LPS was. These folded monomers could be converted into heat-stable trimers upon addition of outer membranes and higher amounts of Triton X-100. Trimerization could precede the insertion step. These in vitro data suggest that the assembly in vivo proceeds sequentially by (i) formation of a folded monomer by interaction with LPS; (ii) sorting of the folded monomers to assembly sites in the outer membrane; (iii) trimerization; and (iv) insertion. Keywords: assembly/lipopolysaccharide/outer membrane/ porin/protein folding
Introduction The cell envelope of Gram-negative bacteria, such as Escherichia coli, consists of a cytoplasmic or inner membrane and an outer membrane (Lugtenberg and van Alphen, 1983; Nikaido and Vaara, 1985). The periplasm in between these two membranes can be considered as a transshipment region carrying out traffic between the interior and exterior of the cell. The biogenesis of outer-membrane proteins is generally supposed to proceed in two steps (Tommassen, 1988; Nikaido and Reid, 1990), although one-step translocation models have also been proposed, implicating a role for the zones of adhesion between inner and outer membrane (Nikaido and Reid, 1990). The twostep model postulates a periplasmic intermediate that inserts into the outer membrane. Outer membrane proteins, such as the pore-forming protein PhoE (Tommassen, 1988), are synthesized in the cytosol as precursors. After translocation of the precursor via the Sec machinery across the inner membrane (Wickner et al., 1991), the mature protein is assembled into the outer membrane. Porins are assembled in the membrane as trimers, their functional units. Insertion into the outer membrane has been proposed to be determined by the total conformation of the protein, rather than by a unique sorting signal (Freudl et al., 1985; Bosch et al., 1986). Nevertheless, sequence comparisons of unrelated outer-membrane proteins have indicated some C-terminal sequence similarity (Struyve et al., 1991), K Oxford University Press
which could possibly represent a sorting signal (Klose et al., 1988; Tommassen and de Cock, 1995). The development of in vitro systems is expected to be very useful for the identification of the requirements for the assembly of outer-membrane proteins. Previously, we have demonstrated that in vitro synthesized PhoE protein was assembled into heat-stable trimers in the presence of high amounts of Triton X-100 [2% (mass/vol)], albeit with slow kinetics (Van Gelder et al., 1994). The kinetics of the trimerization process were strongly increased in the presence of outer membranes. However, the active outermembrane components were not identified. In addition to trimers, folded monomers with a higher electrophoretic mobility than the completely denatured PhoE monomer were formed in the presence of TX- 100. It is not known whether this form represents an intermediate in the assembly pathway. Alternatively, it might be an artificial offpathway folding product. When in vitro synthesized PhoE protein was incubated with crude cell envelopes or purified outer membranes in the absence of detergents, only very low amounts were converted into a trypsin-resistant conformation, characteristic of a correctly folded PhoE protein. These results indicate that the in vitro synthesized protein is not able to insert and assemble efficiently into these membranes. Interestingly, low amounts of TX-100 [optimal 0.08% (mass/vol)] strongly stimulated the correct insertion and assembly into the cell envelopes (H.de Cock, unpublished observation) or the purified membranes (de Cock et al., 1996). A monomeric form of OmpF protein, secreted by spheroplasts, was also shown to be assembled into outer membranes in vitro in the presence of TX-100 (Sen and Nikaido, 1990). The detergent was shown to be required for the formation of an assembly-competent intermediate of PhoE protein with a very short half-life (de Cock et al., 1996), possibly a loosely folded non-native state of the protein. The goal of the present study was to identify outermembrane components involved in the folding of PhoE protein. We demonstrate that folded monomers are intermediates of the assembly process and that LPS and divalent cations are involved in the formation of these intermediates.
Results LPS and divalent cations are required for folding of PhoE protein To identify outer-membrane components required for folding of in vitro synthesized PhoE protein into a trypsinresistant conformation, outer membranes were extracted with various amounts of the non-ionic detergent TX-100 (Table I; see Figure LA for an example). Two bands with similar apparent molecular weights as two of the three 5567
H.de Cock and J.Tommassen L.
Table I. Composition and folding capacity of outer-membrane detergent extracts
Extractiona
Foldingb
TX-100 (%)
LPS in SUP (%)
LPS (nmol/ml)
TX-100 (%)
Tryp.R (%)
0 0.02 0.06 0.08 0.1 0.5 1 2
7 12 27 28 30 49 49 49
0.7 1.3 2.9 3 3.2 5.2 5.2 5.2
0 0.003 0.010 0.013 0.016 0.080 0.16 0.32
0 0.2 15 34 35 13 12 9
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aExtractions of outer membranes were performed as described in Materials and methods. The final amount of TX-100 [% (mass/vol)] present during the extraction and the amount of LPS (%) solubilized are indicated. bFolding of in vitro synthesized PhoE protein was induced by the addition of detergent extracts of outer membranes as in Figure lB. The final amount of LPS (nmol/ml) and TX-100 (%) during the folding reactions and the amount of trypsin-resistant PhoE formed [Tryp. (%)] are indicated. Hardly any protein (94 kDa), which might be derived from contaminating inner-membrane vesicles in the outer-membrane fraction, were detected in the extracts. However, the total amount of protein extracted with TX-100 was very low (Figure IA). In contrast, ~50% of the total amount of lipopolysaccharide (LPS) present in the outermembrane preparations could be extracted (Figure IA and Table I). These TX-100 extracts were able to induce folding of in vitro synthesized PhoE protein into a trypsinresistant conformation, indicating folding of the protein into the native state (Table I). The efficiency of folding was strongly dependent on the final amount of TX-100 and the amount of outer-membrane components in the extract. TX-100 around the critical micelle concentration [0.015% (mass/vol)] was found to be optimal for folding. The folding efficiency decreased 3- to 4-fold when the final amount of TX-100 during folding was raised from 0.016% to 0.08% at a constant LPS concentration (3.2 nmollml; data not shown). Strikingly, in all cases only folded monomers were formed (see Figure lB for an example), and no trimers were detected even after prolonged incubations (up to 18 h) of PhoE protein with the detergent extracts (data not shown). These results suggest that certain outer-membrane components required for trimerization are not present in the detergent extracts. Since LPS is a major outer-membrane component in the detergent extracts (Table I), we investigated whether mixed micelles of purified LPS and 0.015% TX-100 were able to induce the folding of in vitro synthesized PhoE protein into a trypsin-resistant conformation, which indeed appeared to be the case (Figure IC). The efficiency of folding increased linearly with the amount of LPS, and virtually all full-length PhoE protein synthesized could be converted into a trypsin-resistant form (Figure IC). Again, only folded monomers but no trimers were formed (results not shown). However, on an LPS basis the outer-membrane extracts were 10-20 times more efficient than the mixed 5568
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Fig. 1. Extraction of purified outer membranes with TX-100 and folding of PhoE protein with these extracts or with TX-100 and purified LPS. (A) Outer membranes from Ecoli strain U20 (S chemotype) were extracted with 2% TX-100 as described in Materials and methods. Extracted membranes (P) and supematant (S) were analysed by SDS-PAGE (15% acrylamide gels) and proteins were visualized with Coomassie Blue R-250. Relatively four times more supernatant than pellet was loaded. LPS analysis by SDS-PAGE was performed on gels containing 15% acrylamide as described (van der Ley et al., 1986). MS, molecular weight standard proteins. (B) Folding of PhoE protein with TX-100 outer-membrane extract. An in vitro translation mixture containing synthesized PhoE protein was incubated with a 2% TX-100 outer-membrane extract and subsequently treated with trypsin. Prior to electrophoresis, protein aliquots were incubated for 10 min at room temperature (lane a), 56°C (lane b) or 100°C (lane c). Even though gels (containing 11% acrylamide) were run at 20 mA in a temperature-controlled room at 4°C, some denaturation of folded monomers did occur during electrophoresis, resulting in a smear of labelled proteins running between the 31 kDa form (the completely folded monomer, m*) and the 38 kDa form (the completely denatured monomer, m). Chloramphenicol transacetylase (Cat) is the product of the cat gene, which is also present on the plasmid used to direct PhoE synthesis, and is visible in the lane containing the total in vitro translation products (TL). (C) Folding of PhoE protein with purified LPS and TX-100. Twenty microlitres of a solution containing 0.0338 % TX-100 and varying amounts of either LPS from strain U20 (0) or equimolar amounts of LPS and phospholipids (-) were incubated for 30 min at 37°C with 25 ,ul of a translation mixture. Subsequently, samples were treated with trypsin. Samples were incubated for 10 min at 100°C in sample buffer and analysed by SDS-PAGE. The amount of trypsin-resistant PhoE [TrypR PhoE (%)] formed versus the amount of LPS (nmollml) in the reaction mixture is plotted.
micelles of TX-100 and purified LPS, indicating the of additional outer-membrane components in the detergent extracts that stimulate folding. This component is not phospholipid, since inclusion of E.coli phospholipids into the mixed micelles of TX-100 and LPS did not enhance their protein-folding capacity (Figure IC). In addition, since incubation of the TX-100 extracts with presence
Role of LPS in the assembly of a bacterial porin
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Fig. 2. Folding of PhoE requires intact LPS and divalent cations. (A) Folding of PhoE protein with LPS from wild-type strain MC4100 (chemotype Ra) and from its deep-rough derivative CE1229 (chemotype Re). In vitro synthesized PhoE protein was incubated with subsaturating amounts of LPS (18 nmol/ml) and TX-100 (0.015%) for 30 min at 37°C and trypsin-resistant PhoE formed was analysed as described in Figure 1. (B) Aliquots of 10 ,ul translation mixture were incubated for 2 min at 37°C in the presence or absence of EDTA (2 PI EDTA pH 8.0; the final concentration is indicated). Subsequently. 8 PI of mixed micelles of LPS and TX-100 (0.015%. end concentration) were added and trypsin-resistant PhoE formed was analysed as described in Figure IC. TL, in vitro translation products; m. denatured monomers; m*, folded monomers; MS, molecular weight standard proteins (in kDa); and Cat, chloramphenicol acetyltransferase.
proteinase K or trypsin did not significantly change their capacity to stimulate folding, no protease-sensitive component required for folding seems to be present in these extracts (data not shown). Therefore, the observed difference in the folding capacity might be due to structural differences between mixed micelles in the outer-membrane extracts and those made of purified LPS. Interestingly, LPS purified from a deep-rough mutant (chemotype Re) was far less efficient in inducing the folding of PhoE than LPS from an isogenic rough strain (chemotype Ra) was (Figure 2A). This observation might reflect the defect in the biogenesis of outer-membrane proteins in deep-rough mutants observed in vivo (Ames et al., 1974; Koplow and Goldfine, 1974; Tommassen and Lugtenberg, 1981; Ried et al., 1990). Together, the results show that LPS is of special importance for the folding of PhoE into a nativelike folded monomeric form. Interestingly, folding of in vitro synthesized PhoE protein into a trypsin-resistant conformation was strongly inhibited in the presence of EDTA (Figure 2B). Folded monomers, once formed with LPS and 0.015% TX-100, were not denatured by the presence of 10 mM EDTA in the sample buffer. These results indicate a requirement for divalent cations during the folding process.
Folded monomers are assembly-competent intermediates Previously, we have demonstrated that in vitro synthesized PhoE protein assembled into folded monomers and heatstable trimers in the presence of 2% TX-100 at 4°C (Van Gelder et al., 1994). However, when trypsin-resistant folded monomers were formed with purified LPS and 0.015% TX-100, they did not assemble into trimers when subsequently incubated in 2% TX-lO0 (results not shown), suggesting that binding of LPS to folded monomers
LPS(nmol/mD) Tryp.R (o/cb) Tim (%,,) Lane:
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b c a b c a b c +
63.3 15.8 33 0.7 10 0.6 18 41 3 45 6 7 8 9 1 2 0
Fig. 3. LPS inhibits trimerization. Aliquots of 25 p1 of a translation mixture containing in vitro-synthesized PhoE protein were incubated for 30 min at 37°C with 2% TX-100 and the indicated amounts of LPS from U20 (S chemotype), and subsequently treated with trypsin (45 pg/ml). Protein samples were analysed by SDS-PAGE as in Figure 1. The protein band migrating with an apparent molecular weight of approximately 48 kDa visible in lanes 1, 4 and 7 is the protease-resistant, oligomeric form of the cat gene product (Cat). The total amount of trypsin resistant (Tryp.R) PhoE protein formed is expressed as the percentage of the total amount synthesized. T, trimers: m, denatured monomers; and m*, folded monomers. The ratio of trimers over the total amount of trypsin-resistant PhoE (T/m X 100%) is indicated.
inhibits trimerization of the protein and/or that folded intermediates for trimerization. We therefore investigated the effect of LPS on folding of PhoE protein in 2% TX- 100 in more detail. The efficiency of folding of PhoE protein into a trypsin-resistant conformation in the presence of 2% TX-100 at 37°C was strongly increased in the presence of LPS (Figure 3). However, the ratio of heat-stable trimers over monomers (T/m ratio) decreased with increasing amounts of LPS (Figure 3A). These data demonstrate that LPS is required for the efficient folding of PhoE protein into a trypsinresistant folded monomer but that it inhibits the subsequent trimerization of the monomers, possibly by interacting with them at the subunit interface. To determine whether the folded monomers are indeed associated with LPS, flotation gradient centrifugation was carried out. Trypsinresistant folded monomers, formed with mixed micelles of 0.015% TX-100 and 31 nmol/ml LPS, were quantitatively recovered in the top fraction of the gradient, showing that they are indeed associated with LPS (Figure 4). Previously, we have demonstrated that in vitro synthesized PhoE protein was inserted and assembled into trimers when directly incubated with outer membranes and 0.08% TX-100 (de Cock et al., 1996). In this study, we investigated whether pre-folded, trypsin-resistant monomers were assembly-competent intermediates under these conditions. Indeed, trimerization of folded monomers proceeded very efficiently when they were incubated with outer membranes and 0.08% TX-100 (Figure 5A). Up to 80% of the folded monomers assembled into heat-stable trimers, which were associated with the outer membranes. Also, small amounts of folded monomers and dimers were detected in the membranes (hardly visible in Figure 5A; see Figures SB or 6). The proteins were inserted into, and monomers are no
5569
H.de Cock and J.Tommassen
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not peripherially associated with, the membranes, since they remained membrane-associated after incubation of the membranes with 4 M urea (Figure 5A). The efficiency of insertion was dependent on the amount of outer membranes and was quantitative when sufficient membranes were present (Figure 5B). Apparently, the trypsin-resistant folded monomer is an intermediate that can be assembled into outer membranes in trimers. The assembly sites must be limiting since relatively high amounts of outer membranes (nmol range on an LPS basis) were required for insertion of radiochemical amounts of PhoE protein (fmol range). Folded monomers were not associated with or assembled into the outer membrane when the TX-100 concentration was not increased to 0.08% or when the incubation was performed at 0°C (results not shown). Interestingly, pre-folded monomers were assembled with similar efficiencies into outer membranes derived from a wild-type (Figure 5B) or mutant strain with deep-rough LPS (Figure 5C), and in both cases similar amounts of heat-stable trimers were formed (Figure 5D). Hence, folding of PhoE into trypsin-resistant folded monomers is inefficient with defective LPS, whereas assembly of the protein as heat-stable trimers, into the outer membranes derived from deep-rough mutants, proceeds normally, suggesting that defective LPS does not affect later steps of the assembly process. Typically, trimers formed with outer membranes from MC4100, containing Ra LPS, migrated as multiple bands in contrast to those formed with OMs from CE1229, containing Re LPS.
Trimerization can precede insertion Subsequently, we investigated whether trimerization could precede insertion. In vitro-synthesized PhoE protein was previously shown to be assembled in an insertion-independent manner into folded monomers and heat-labile and 5570
Fig. 5. Sorting and assembly of folded monomers into outer membranes. (A) Trypsin-resistant folded monomers (FM) were formed as in Figure lC, with 62 nmol/ml LPS from strain U20 (S chemotype) and 0.015% TX-100. Aliquots of 20 gl, containing the folded PhoE protein, were subsequently incubated with purified outer membranes (OM) derived from strain MC4100 (1.2 nmol LPS) and the TX-100 concentration was increased to 0.08% (total volume, 25 gl). After 30 min at 37°C, outer membranes were re-isolated by centrifugation. The supernatant (SUP) was directly analysed by SDS-PAGE and the membranes in the pellet were incubated with 4 M urea. Membranes were re-isolated by centrifugation and pellet (PEL, urea) and supematant (SUP, urea) were analysed. (B) The amounts of PhoE protein recovered in pellet (0) and supernatant (0) fractions at different amounts of outer membranes [nmol LPS (Ra)] are plotted. (C) Assembly of folded monomers into outer membranes of a deeprough strain. Trypsin-resistant folded monomers were formed with 62 nmollml LPS from strain U20 (S chemotype) and 0.015% TX-100. Aliquots of 20 gu were subsequently incubated with purified outer membranes from strain CE1229 (chemotype Re) and the TX-100 concentration was increased to 0.08%. After 30 min at 37°C the amount of PhoE protein in pellet (0) and supernatant (0) fractions at different amounts of OMs [nmol LPS (Re)] were determined and plotted. (D) Fluorogram of a gel containing assembled PhoE proteins into outer membranes of Ecoli K-12 strain MC4100 (chemotype Ra) or of its isogenic derivative CE1229 containing defective LPS (chemotype Re). Proteins were analysed by SDS-PAGE as in Figure lB. Positions shown in (A) and (D): T, heat-stable trimers; D, dimers; m*, folded monomers; and m, denatured monomers.
heat-stable trimers when incubated with 0.08% TX-100 and limiting amounts of outer membranes (Figure 6, SUP 1; de Cock et al., 1996). Interestingly, all these folded forms were assembly-competent intermediates, which were sorted and assembled efficiently into freshly added outer membranes (Figure 6, PEL 2). These proteins remained associated with the outer membranes after incubation with 4 M urea, indicating complete insertion into the outer membrane (Figure 6, PEL 3). Apparently, trimerization can precede insertion of the protein in this in vitro system.
Discussion The development of an in vitro system to study the sorting and assembly of outer-membrane protein PhoE resulted in
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Fig. 6. Sorting and assembly of trimers into the outer membrane. In vitro-synthesized PhoE protein was incubated for 30 min at 37°C with 0.08% TX-100 and outer membranes of Ecoli U20 (0.7 nmol LPS). The mixture was subsequently incubated for 20 min with trypsin (30 Pg/ml) and 5 min with PMSF (1 mM) at O°C. and membranes containing inserted PhoE protein (PEL 1) were separated from soluble folded forms of PhoE protein (SUP 1). The supernatant (SUP 1), containing folded monomers and heat-labile (i.e. denaturing at 56°C) and heat-stable trimers, was incubated for 30 min at 37°C with outer membranes of MC4100 (1.2 nmol LPS). One half of the mixture was directly centrifuged to obtain a pellet (PEL 2). containing the membranes, and a supernatant (SUP 2). whereas the other half was first incubated with 4 M urea before fractionation to obtain PEL 3 and SUP 3. Membranes and supernatants were analysed by SDS-PAGE. Protein samples were incubated for 10 min at room temperature (a). 56°C (b) or 100°C (c) prior to electrophoresis. T. trimers: D, dimers; m, denatured monomers; mi, folded monomers; MS, molecular weight standard proteins (in kDa); TL, total translation products; and Cat.
chloramphenicol acetyltransferase.
the identification of LPS as an important outer-membrane component required for efficient folding of the protein. Previously, it has been demonstrated that outer-membrane protein OmpF, either secreted from spheroplasts (Sen and Nikaido, 1990) or synthesized in vitro (Sen and Nikaido, 1991a), could be trimerized with a mixture of LPS and detergents, including TX- 100. Furthermore, it was reported that the assembly of this porin was less efficient with LPS with a defective core region (Sen and Nikaido, 1991b). However, no intermediate stages in the assembly process of OmpF were detected. Our present work allows to dissect the assembly process into distinct stages. We demonstrate that a stable folded monomer of PhoE protein is an intermediate that can be converted into trimers and inserted into outer membranes. In addition, we show that trimerization can precede insertion of the protein in this in vitro system. Furthermore, we demonstrate that LPS and divalent cations are required early in the assembly process, i.e. for the folding into a folded monomer. The conditions for the formation of the assembly-competent folded monomer require very low amounts of the non-ionic detergent TX-100 (0.015%), LPS and divalent cations, conditions that were previously not met (de Cock et al., 1990a,b, 1996; Van Gelder et al., 1994). Previously, we demonstrated that the detergent is required for the formation of an assembly-competent intermediate of PhoE protein with a short half-life (de Cock et al., 1996). This could be due to dissociation of a chaperone and/
or detergent-induced folding of the protein. Here, we demonstrate the formation of a new, stable intermediate, i.e. a folded monomer, by interaction with LPS. Divalent cations were shown to be required for folding. Possibly, essential LPS-protein interactions during folding are mediated via salt bridges formed between divalent cations and the negatively charged macromolecules. Alternatively or in addition, the S 135 cell extract in which PhoE is synthesized might contain certain components that are involved in folding and that require divalent cations for their activity. LPS are glycolipids present in the outer monolayer of the outer membrane (Lugtenberg and van Alphen, 1983; Nikaido and Vaara, 1985). Hence, the observation that LPS is required for folding of PhoE could be considered an indication that folding takes place only after insertion into the outer membrane. However, the biogenesis of the porins depends on lipid synthesis, since inhibition of fatty acid synthesis with the antibiotic cerulenin inhibits the sorting and assembly of these proteins (Boquet-Pages et al., 1981; Pages et al., 1982; Bolla et al., 1988). Consequently, de novo-synthesized LPS seems to be required for the assembly of porins in vivo. It is quite possible that newly made LPS molecules interact with porin before assembly in the outer membrane. Several results obtained in our in vitro assembly system underscore this notion. First, the folded monomers, formed with LPS, were shown to represent true assembly intermediates that could trimerize and insert upon addition of outer membranes. Secondly, deep-rough mutants, containing highly defective LPS molecules, have reduced amounts of outer membrane proteins in vivo (Koplow and Goldfine, 1974; Tommassen and Lugtenberg, 1981; Ried et al., 1990; Ames et al., 1994). Here, we demonstrated that the formation of folded monomers by the LPS from such mutants was inefficient, whereas the insertion of folded PhoE monomers in the outer membranes of such mutants was not affected. The inhibition of trimerization of PhoE protein by LPS suggests interaction of LPS with the subunit interface. Dissociation of LPS from this area of the monomer is likely to be required for efficient trimerization, since LPS was not detected between the subunits of the trimer (Cowan et al., 1992). The temporary interaction of LPS with the subunit interface might be crucial in the biogenesis of trimeric outer-membrane proteins. This highly hydrophobic area might be prone to aggregation, and binding of LPS, via its lipid A part, introducing the charged core region to this area, and might thus prevent aggregation. In this respect, the glycolipid LPS might be regarded as a molecular chaperone. Folded monomers can be sorted and assembled into the outer membrane, suggesting that the outer-membrane assembly sites are required for dissociation of LPS from the subunit interface. Apparently, only very limiting amounts of assembly sites, of which the nature remains to be determined, are present in purified outer membranes. These sites must be very special in that they bring together three individual folded monomers, present in very low amounts, for trimerization. LPS, bound to the subunit interface of the monomers, might actually be required for targeting to the assembly machinery at these sites to guarantee efficient trimerization, explaining why outer membranes can increase trimerization kinetics in vitro (de Cock et al., 1990b; Van Gelder et al., 1994).
5571
H.de Cock and J.Tommassen
The inhibition of the assembly process at low temperatures could be due to the co-existence of the solid and the fluid phases in the outer membrane at this temperature (Nakayama et al., 1980). Alternatively, since protein folding is temperature dependent, the assembly into the outer membrane might require additional folding steps prior to insertion. Two arguments can be given for the hypothesis that folded monomers first assemble into trimers at the assembly sites before insertion. First, stable trimers were shown to be assembly competent. In addition, the efficiency of trimerization was not reduced when excessive amounts of outer membranes were added (data not shown). If insertion of folded monomers preceded trimerization such an inhibition would be expected because of titration of individual monomers by different outermembrane vesicles. In this respect, it is interesting to notice that correct folding and assembly of the protein can be a 'quality control' for correct sorting to the outer membrane in vivo, explaining why mutations perturbing the structure of outer-membrane proteins are defective in sorting and assembly (Freudl et al., 1985; Bosch et al., 1986). Such a mechanism is reminiscent of the stringent 'quality control' to which proteins exported from the endoplasmic reticulum in eukaryotes are subjected (Pelham, 1989). Thus, partially folded or misfolded proteins are retained and degraded, whereas correctly folded and assembled proteins proceed in the secretory pathway.
Materials and methods Bacterial strains S135 cell extracts were isolated from the Ecoli K-12 strain MC4100 were isolated also from this strain, as well as from its derivative CE1229 containing heptose-deficient LPS (de Cock et al., 1989) and from Ecoli strain U20 containing smooth LPS (van der Ley et al., 1986).
(Casadaban, 1976). Membranes
Extraction of purified outer membranes with TX-100 Outer membranes (containing 6.6 nmol LPS) from Ecoli strain U20 (S chemotype) were incubated for 30 min at room temperature in 100 tl buffer L (50 mM triethanolamine acetate pH 7.5, 250 mM sucrose, 1 mM DTT), supplemented with varying amounts of TX-100. Membranes were re-isolated by centrifugation at 40 000 r.p.m. for 30 min at 15°C in a TLA 100 rotor (Beckman, USA) and resuspended in 100 gl buffer L. Aliquots of the pellet and supematant fractions were frozen with liquid nitrogen and stored at -80°C. The LPS content was determined by KDO measurements (van Alphen et al., 1978) after precipitation of the membranes or fractions with acetone to remove the sucrose.
In vitro translation, folding and assembly into outer membranes Isolation of S135 cell extracts and the in vitro transcription and translation reactions were performed as described previously (de Vrije et al., 1987). Plasmid pJP370 (de Cock et al., 1990a) was used to direct the in vitro synthesis of [35S]methionine-labelled quasi-mature PhoE protein, i.e. the complete mature protein extended with a methionine and serine at the N-terminus. Routinely, puromycin (10 gM) was added 25 min after initiation of synthesis. Outer membranes were isolated as described (de Cock et al., 1996). Folding with outer-membrane extracts. Aliquots of 25 gl of the translation were mixed at room temperature with S gl of a TX- 100 outer-membrane extract and 1.25 gl water, and incubated for 30 min at 37°C. Subsequently, 1 gl trypsin (1400 jg/ml in 100 mM Tris-HCl pH 8.0, 50 mM MgCl2) was added. After 15 min incubation at 37°C, phenylmethyl sulfonylfluoride (PMSF; 1 mM) was added and the mixture was transferred to ice.
Folding with purified LPS and 0.015% TX-100. LPS (in water) was dried in a speedvac and resuspended in buffer L. After mixing with TX100 (0.0338%), 20 p1 of the solution was incubated for 30 min at 37'C
5572
with 25 gl of a translation mixture. Samples were treated with trypsin (45 gg/ml) and, after 15 min at 37°C, PMSF was added and the mixture was transferred to ice. Assembly offolded monomers into outer membranes. Aliquots of 20 pl, containing folded PhoE protein, were mixed with purified outer membranes in buffer L and, subsequently, 2.1 pl 0.8% (w/v) TX-100 was added (total volume, 25 p1). After 30 min at 37°C, outer membranes were re-isolated by centrifugation at 40 000 r.p.m., for 30 min at 15°C, in a TLA 100 rotor (Beckman, USA). The membranes were resuspended in buffer L and, if required, incubated for 30 min at 0'C with 4 M urea in buffer L and again re-isolated by centrifugation. SDS-PAGE. Prior to electrophoresis, proteins aliquots were incubated in sample buffer, supplemented with EDTA (10 mM) for 10 min at either room temperature, 560 or 100'C. SDS-polyacrylamide gels (Lugtenberg et al., 1975) were run at 20 mA in a temperature-controlled room at 4°C to prevent denaturation of various folded forms of the PhoE protein during electrophoresis. Data were quantified with a PhosphorImager
(Molecular Dynamics). Flotation gradient centrifugation Flotation gradient centrifugations in metrizamide (ICN, USA) in buffer L were performed as described (Thom and Randall, 1988) using gradients of 500 p1 and centrifugation at 100 000 r.p.m., for exactly 5 h at 4°C, in a TLA 100.1 rotor (Beckman, USA).
Lipid purification LPS (chemotype S) was purified with warm phenol (Westphal and Jann, 1965). LPS of chemotype Ra and Re was purified from strains MC4100 and CE1229, respectively (Galanos et al., 1969) and phospholipids were removed by extraction of the LPS with chloroform/methanol (Bligh and Dyer, 1959). Ecoli phospholipids were isolated (Bligh and Dyer, 1959) from strain MC4100 and purified by chromatography (de Cock et
al., 1989).
Acknowledgements The research of Dr H.de Cock has been made possible by a fellowship of the Royal Netherlands Academy of Arts and Sciences.
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