Export and assembly of outer membrane proteins in E. coli

EXPORT AND ASSEMBLY OF OUTER MEMBRANE PROTEINS IN E. COLt

Jan Tommassen and Hans de Cock

I. II.

Ill.

IV. V.

VI.

......

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Outer Membrane Proteins . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. Function of Outer Membrane Proteins . . . . . . . . . . . . . . . . . . . B. Structure of Outer Membrane Proteins . . . . . . . . . . . . . . . . . . . Transport Across the Inner Membrane . . . . . . . . . . . . . . . . . . . . . . A. The Export Apparatus . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. Role of SecB in Protein Export . . . . . . . . . . . . . . . . . . . . . . . C. lntragenic Export Information . . . . . . . . . . . . . . . . . . . . . . . Pathway to the Outer Membrane . . . . . . . . . . . . . . . . . . . . . . . . . Insertion and Assembly in the Outer Membrane . . . . . . . . . . . . . . . . . A. Assembly Intermediates, Detected in vivo . . . . . . . . . . . . . . . . . B. Role of LPS and Lipid Biosynthesis . . . . . . . . . . . . . . . . . . . . C. In vitro Reconstitution of the Insertion and Assembly Process . . . . . . . D. Sorting Signals in Outer Membrane Proteins . . . . . . . . . . . . . . . . Conclusions and Future Prospects . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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Advances in Cell and Molecular Biology of Membranes and Organeiles Volume 4, pages 145-173. Copyright 9 1995 by JAI Prms Inc. All rights of reproduction in any form reserved. ISBN: 1-55938-924-9 145

146 146 146 148 150 150 150 152 154 155 155 158 159 162 165 166

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JAN TOMMASSEN and HANS DE COCK I.

INTRODUCTION

The outer membrane of Escherichia coil is an extremely asymmetric bilayer containing phospholipids and lipopolysaccharides (LPS) only in the inner and outer monolayer, respectively (77). It protects the cell by forming a barrier for harmful compounds, like bile salts and enzymes, and prevents periplasmic proteins leaking into the medium. The outer membrane also contains a number of proteins (OMPs). After their synthesis in the cytoplasm, OMPs have to pass through the inner membrane before reaching their destination. The fact that these proteins don't end up in the inner membrane suggests either that these proteins have a structure that is incompatible with stable insertion into this membrane or that they somehow bypass this membrane on their way out. Indeed, OMPs have a completely different structure as compared with inner membrane proteins. In this chapter, we will review the current knowledge on the biogenesis of OMPs. First, we will introduce the leading characters in this process and discuss their structure within the outer membrane, since this is important to understand the assembly process. For a more detailed description of the structure of outer membrane porins, we refer to the chapter of G. Schulz in this book. For the transport across the inner membrane, OMPs follow the same pathway as periplasmic proteins (59). This pathway will be discussed here only briefly, with only some special attention to the role of SecB protein, which is of special importance in the export of OMPs. For more details we refer to the chapters of K. Ito and of S. Mizushima. The focus of this chapter will be on the folding process of OMPs and on their insertion and assembly into the outer membrane. We will restrict the discussion to integral outer membrane proteins. For the biogenesis of lipoproteins, the reader is referred to a recent review (55).

II.

OUTER MEMBRANE PROTEINS

A. Function of Outer Membrane Proteins Most OMPs of E. coil are involved in the uptake of nutrients (Table 1). The porins, OmpF, OmpC and PhoE, form general diffusion pores in the outer membrane through which hydrophilic solutes with molecular masses up to about 600 Da can pass (10, 77, 90). These proteins, which exhibit extensive sequence similarity (85), form homo- or heterotrimers (46). Expression of the OmpF and OmpC proteins is regulated by the osmolarity of the growth medium (130), whereas the synthesis of PhoE is induced by phosphate-starvation (97). In contrast to the OmpF and OmpC pores, which are cation-selective, the PhoE pores are anion-selective (11). In addition to the general porins, the outer membrane contains proteins that form specific channels (90). These proteins have no sequence similarity to the porins. They also function as general diffusion pores, but in addition, they contain a specific

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Table 1. Survey of E. coli Outer Membrane Proteins and Their Functions a Protein

Function

OmpF

general pore, cation-selective

OmpC PhoE LamB

general pore, cation-selective general pore, anion-selective specific channel for maltoseand maltodextrins specific channel for nucleosides receptor for vitaminB 12 receptor for Fe3+-ferrichrome receptor for Fe3+-enterochelin pore; integrityof outer membrane phospholipase protease secretion of pill secretion of hemolysin

Tsx BtuB FhuA FepA OmpA PldA OmpT PapC TolC Note: ~

list is not intended to be complete, but only mentions the proteins described in this chapter.

binding site for a certain solute. An example of a protein that forms specific channels is LamB protein. LamB is a trimeric protein in the outer membrane, like the porins, and contains a binding site for maltodextrins (12). It facilitates the diffusion of maltose and maltodextrins and of some other sugars (64, 123). Another example is Tsx protein, which has a binding site for nucleosides (81). Another category of transport proteins in the outer membrane consists of receptors that bind their ligands with high affinity. The subsequent transport of the ligands across the outer membrane requires cytoplasmic membrane energy, which is coupled to the transport process via TonB protein (98). Except for BtuB, which is required for the transport of vitamin B 12 (61), all other known TonB-dependent receptors of E. coli are induced under iron-limitation, and they are involved in the uptake of iron-siderophore complexes (98). Examples are FhuA and FepA, the receptors for ferrichrome and enterochelin, respectively. The physiological function of OmpA protein, a major monomeric outer membrane protein, has remained an enigma for a long time. Together with Braun's lipoprotein, it is involved in maintaining the structural integrity of the outer membrane (115). It was recently demonstrated that this protein has pore properties as well (106, 120). Finally, the outer membrane of E. coli contains a few enzymes. The PldA protein has phospholipase A1 and A2 activities (94, 107), and OmpT is a protease (48). The exact physiological function of these enzymes is not known.

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JAN TOMMASSEN and HANS DE COCK

B. Structure of Outer Membrane Proteins

Integral inner membrane proteins contain hydrophobic stretches of approximately 20 amino acid residues that span the bilayer in an o~-helical configuration. Inspection of the primary sequences of OMPs does not reveal the presence of such hydrophobic stretches and suggests that these proteins are very hydrophilic. This is probably related to the fact that OMPs have to be transported across the inner membrane before they reach their destination, since hydrophobic stretches would act as stop-transfer sequences (26). Indeed, it has been shown that the introduction of hydrophobic sequences in OMPs interferes with their transport across the inner membrane (2, 5, 80). Much effort has been invested in the understanding of how these apparently hydrophilic OMPs can be accommodated in the hydrophobic environment of the outer membrane. A variety of biophysical measurements, including infrared absorption, high-angle X-ray diffraction, circular dichroism measurements, and Raman spectroscopy, have revealed that the porins, OmpA and LamB, have a predominant 13-structure with many antiparallel ~strands in the membrane (63, 134). Further information on the topology of OMPs has been derived from genetic investigations. These experiments were directed to identify cell-surface-exposed amino acid residues, involved in the binding of bacteriophages (69, 87) or monoclonal antibodies (mAbs) (33, 132). Alternative approaches consisted of insertion of foreign antigenic determinants (2, 22) or protease-sensitive sites (67) into OMPs and determining if these epitopes/sites became accessible in intact cells, which would prove insertion in an exposed loop of the OMPs. After the identification of a few cell-surface-exposed amino acids in PhoE, we have proposed a model for the topology of this protein in the membrane, postulating 16 amphipathic, antiparallel [3-strands spanning the membrane, thereby exposing eight regions at the cell surface (126, 132). This model was largely confirmed by the resolution of the crystal structures of OmpF and PhoE (25), which showed that each monomer of these porins forms a 16-stranded, antipamllel 13-barrel (Figure 1A), with hydrophobic amino acids exposed to the lipids and to the subunit interface. Therefore, it is possible to postulate topology models for OMPs with a fair amount of accuracy on the basis of only a few genetic experiments or from sequence data only. A model, similar to the PhoE model, has been proposed for LamB (23). For the much larger FhuA protein, a 32-stranded [3-barrel has been predicted (67). OmpA consists of two domains, an N-terminal membrane-embedded domain and a C-terminal periplasmic tail (Figure IB) (18). The N-terminal domain is proposed to contain eight antiparallel amphipathic ~3-strands (87, 134). In conclusion, it seems that the majority of OMPs have a similar type of organization within the membrane, with multiple membrane-spanning amphipathic ~-strands. The hydrophobic side of these [3-strands is exposed to the lipidic environment of the membrane, and, in the case of multimeric proteins, to the subunit interface.

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TRANSPORT ACROSS THE INNER MEMBRANE A. The Export Apparatus

At least the initial steps in the export of OMPs and periplasmic proteins appear to be identical. OMPs as well as periplasmic proteins are synthesized as precursors with N-terminal signal sequences, which are essential for export. The signal sequences of OMPs and periplasmic proteins are functionally exchangeable (129). Furthermore, it has been observed that export of hybrid proteins consisting of an N-terminal part of the periplasmic maltose-binding protein (MBP) and the cytoplasmic enzyme [3-galactosidase was initiated but not completed, presumably because the ~-galactosidase moiety of the hybrid protein interferes with the passage of the polypeptide through the inner membrane (9). Expression of such a hybrid protein blocked the export apparatus, resulting in the accumulation of the precursors of both OMPs and periplasmic proteins. Also, the mutational distortion of components of the export apparatus, e.g., by secA mutations (96), simultaneously affected the export of OMPs and periplasmic proteins. Genetic approaches have been used to identify components of the export apparatus. A variety of selection procedures were developed to select for export mutants (for a review, see ref. 108). In this way, six genes, designated secA (prlD), secB, secD, secE secE (priG), and sec Y (prlA ), were identified that encode components of the export apparatus. A multisubunit enzyme designated translocase is central in the export apparatus. Protein purification and reconstitution of the export process showed that SecE and SecY are essential integral inner membrane components of the translocase (20, 124). SecA is a peripheral component with an intrinsic ATPase activity that is stimulated by the binding of a precursor and by membrane vesicles (75). ATP hydrolysis as well as the proton motive force are required for efficient transport (35, 47). SecD and SecF are also integral inner membrane proteins; they contain, however, a large periplasmic domain and were postulated to have a role late in the export process (45). Such a role could not be established in the in vitro reconstitution of the export process (75, 124). Antibodies directed against SecD inhibited the secretion of processed envelope proteins from spheroplasts into the medium (82), suggesting that SecD is required for the release of translocated proteins from the inner membrane.

B. Role of SecB in Protein Export In contrast to the central components of the export apparatus, SecB is required for the export of only a subset of envelope proteins (71). Very interestingly, all OMPs examined thus far are dependent on SecB for efficient export, whereas periplasmic proteins, with the exception of MBP, are not. SecB is a cytoplasmic protein that binds to the mature domains of precursor proteins (24, 30, 70,101). Its exact role in the export process is not yet resolved. Two different but not mutually

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151

exclusive functions have been proposed, for instance, (1) it might stabilize the precursors in an export-competent conformation (23, 141), and (2) it might be required for proper targeting of the precursors to the membrane components of the export apparatus (122, 140). It is essential that a precursor is maintained in an export-competent (i.e., an unfolded or loosely folded conformation), since folding into a stable tertiary structure prevents its translocation from the cytoplasm (100). Indeed, it has been demonstrated that SecB retards the folding of the MBPprecursor into the native structure of the protein (24, 51). However, it should be noted that MBP might be an exceptional case in being the only periplasmic protein known so far that is dependent on SecB for efficient export. The situation might be different in the case of OMPs, which are unlikely to fold into their native structure in the cytoplasm in the absence of other outer membrane components. Initially, some evidence was published that the precursors of OMPs are also stabilized in an export-competent conformation by SecB. In in vitro translocation experiments, the purified precursor of PhoE protein (prePhoE) remained translocation-competent in the presence of SecB with a functional half-life of several hours, whereas it rapidly lost its translocation competency in the absence of SecB (72). SecB had little, if any, effect on the adoption of secondary and tertiary structure of prePhoE as measured by circular dichroism and fluorescence spectroscopy (! 9). However, it prevented the purified precursor from aggregating after dilution from urea (19), which might fully explain its effect on maintaining the translocation competent state of the precursor. Similar effects of SecB on the folding of the precursor of OmpA have been reported (73). It seems extremely unlikely that in vivo the concentration of the precursors of OMPs in a cell will ever increase to levels that support aggregation, unless in genetically manipulated overproducing systems. Furthermore, the in vitro translocation systems that employ purified precursors diluted from urea are somewhat suspicious, since proteins that are not involved in protein export in vivo have been reported to stabilize the export-competent state of a precursor protein (49). Therefore, the data referenced so far do not allow the conclusion that the role of SecB in the translocation of the precursors of OMPs is to stabilize their export-competent state. Recently, we employed a different in vitro system to study the role of SecB in prePhoE translocation (32). Radioactively labeled prePhoE was synthesized in vitro in an E. coli cell extract, and its posttranslational translocation into inverted inner membrane vesicles was studied in the presence or absence of SecB. The binding of SecB to prePhoE was studied by coimmunoprecipitation. The translocation of prePhoE into the membrane vesicles was much more efficient in the presence of SecB, confirming that SecB is required for efficient transiocation of this OMP. However, the translocation-competency ofprePhoE was rapidly lost in the presence of SecB with a functional half-life of only 14 min, even though the coimmunoprecipitation experiments did not reveal the dissociation of the SecB-prePhoE complexes during the incubations. Apparently, SecB-binding did not stabilize the export-competent state of prePhoE, prePhoE was translocated in the absence of

JAN TOMMASSENand HANSDE COCK

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SecB, although less efficiently. Under these conditions, the export competency was lost with the same kinetics (i.e., with a functional half-life of 14 min) as occurred in the presence of SecB (32). From these experiments it can be concluded that SecB does not stabilize the export-competent state of prePhoE, but is probably required for efficient targeting to the membrane components of the export apparatus. This conclusion might be extrapolated to the precursors of other OMPs. It is not yet clear how this targeting function operates. However, direct recognition of SecB by SecA has been demonstrated (52). Thus, it seems likely that a SecB-precursor complex will be more readily bound by SecA than the precursor alone. It should be noted that in vivo experiments underscore the conclusions described above. If the role of SecB is to stabilize the export-competent state of the precursors of outer membrane proteins, one would expect that export-incompetent precursors accumulate in a secB mutant. This is not the case (except under conditions of overproduction). Pulso-chase experiments showed that the processing (and therefore probably export) of the precursor of an outer membrane protein is retarded in a secB mutant, but eventually all the precursors are processed (Figure 2).

C. Intragenic Export Information OMPs and periplasmic proteins are synthesized as precursors with an N-terminal signal sequence. Signal sequences are in general 20-25 amino acid residues long and consist of three domains: (1) an amino-terminal positively charged region, (2) a central hydrophobic part, and (3) a more polar carboxy-terminal region containing the signal peptidase recognition site (136). Signal sequences probably interact via their positively charged N-terminal region with the polar headgroups of anionic

Outer Membrane Proteins in E. coil

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phospholipids in the membrane, whereas the hydrophobic core inserts between the fatty acyl chains and thus initiates translocation (34). In addition, the signal sequence may interact with the proteinaceous components of the export apparatus, like SecA (5) or the prokaryotic analogue of the eukaryotic signal-recognition particle (78). Whereas a signal sequence is essential for efficient export, it is not always sufficient to mediate this process. For instance, [$-galactosidase was not exported out of the cytoplasm when fused to the signal sequence of LamB (86). It is now generally assumed that [3-galactosidase rapidly adopts its stable tertiary structure in its native environment, the cytoplasm, which prevents it from being exported. However, an alternative or additional interpretation of this result is that the mature domain of a precursor contains export information. The latter hypothesis was tested by creating a series of overlapping deletions coveting the complete mature domains ofprePhoE (14,16) or proOmpA (42). All mutant proteins were exported, showing that no essential export signals are present in the mature domains of these OMPs. However, the efficiency of the in vitro translocation of many mutant prePhoE molecules into inverted inner membrane vesicles appeared to be reduced (30). This result suggests that the mature domain contains information that contributes to the efficiency of the export process. The nature of this information is not yet completely clear, but it probably involves the binding sites for components of the export apparatus, like SecB. The mutational removal of such a binding site has no drastic effect on export, probably because there are multiple SecB-binding sites present in a precursor; for example, four SecB-binding sites have been mapped in the mature domain of prePhoE (30). Alternatively, such deletions prevent the folding of the precursors into a stable, export-incompetent configuration, thus alleviating the need for rapid targeting to the translocase in the membrane by SecB. It should be noted that SecA recognizes, in addition to the signal sequence, elements in the mature domain of a precursor (76). However, such a binding site has not yet been defined in any precursor protein. Possibly, it is located directly adjacent to the signal sequence in the N-terminal region of the mature protein. Mutations in this region had an effect on the efficiency of the in vitro translocation of PhoE without disturbing SecB-binding (30). Furthermore, deletions in the corresponding region of LamB had an effect on export kinetics in vivo (102). The charge of the N-terminal region of the mature domain of a precursor seems to be of importance. A database analysis showed that this region is in general negatively charged (135). Especially position +2 was found to be enriched in acidic residues, whereas a basic residue was never found in this position in wild-type precursors. The introduction of basic residues in this region had a drastic effect on export efficiency (7, 74, 79, 116, 143). How these positively charged residues interfere with translocation is not known. Possibly they prevent proper interaction of the precursor with SecA or the phospholipids in the membrane or they prevent the proper orientation of the signal sequence with respect to the electrochemical gradient across the membrane. The observation that the strength of the inhibitory

JAN TOMMASSEN and HANS DE COCK

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effect depended on the pKa of the inserted residues (116) suggests that basic residues in this region have to be deprotonated to become translocated.

IV. PATHWAY TO THE OUTER MEMBRANE At some stage, the export pathways of OMPs and periplasmic proteins must diverge. In the simplest model (125), OMPs are completely translocated across the inner membrane like periplasmic proteins. The subsequent insertion into the outer membrane would occur in a separate step (Figure 3). Other models have been proposed according to which OMPs do not actually pass all the way through the cytoplasmic membrane to reach their final location, but rather move by some form of membrane-membrane contact (1/3). The latter type of models is supported by electron microscopic studies (114), which revealed that newly synthesized porins appeared at the cell surface in patches located above fusion sites between the two membranes. However, the nature and even the existence of these fusion sites, the so-called Bayer bridges, has recently been challenged on the basis of improved electron microscopy methods (62). Furthermore, this type of model was initially supported by the results obtained with LamB/~galactosidase hybrid proteins.

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Figure 3. Model for the biogenesis of outer membrane proteins. (1) Precursor proteins are co- or post-translationally targeted to the inner membrane (IM)-Iocated translocation apparatus (See). Translocation across the IM requires ATP and the protonmotive force. The signal sequence is removed during or after translocation. (2) After release of the mature protein in the periplasm (PP), it will fold into an insertion-competent conformation. (3) Insertion requires hydrophobic interactions between protein and outer membrane components. After insertion into the outer membrane (OM), proteins fold into their final 3-D structure. Porins have to assemble into their trimeric configuration probably after insertion of their folded monomers into the OM.

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Whereas, as discussed in Section IIIA, MBP/~l-galactosidase fusions were not exported to the periplasm because the 13-galactosidase moiety cannot pass the inner membrane (9), similar fusions containing an N-terminal part of the OMP Lamb instead of MBP fractionated with the outer membranes, suggesting that they were exported (50). This result was interpreted to mean that the C-terminus of the fusion proteins, and consequently of OMPs, does not have to be translocated across the inner membrane in order to reach the outer membrane, and therefore favored transport models that implicated a role of membrane contact sites. However, immunoelectron microscopy suggested that the co-fractionation of the fusion proteins with the outer membranes was actually artificial; the fusion proteins were mostly found in the cytoplasm as aggregates and also associated with the inner membrane and to intracytoplasmic membranes, which appeared upon induction of the synthesis of these hybrid proteins (127, 128, 137). Clearly, both the aggregate formation and the appearance of intracytoplasmic membranes may have deceived the interpretation of standard cell fractionations. Consequently, there is no solid experimental evidence that supports the transport of OMPs via membrane contact sites, and therefore, we favor the two-step transport model depicted in Figure 3. Several lines of evidence support this periplasmic pathway: 1. mutations that prevented the assembly of PhoE (14) or OmpA (42) into the outer membrane, resulted in the pedplasmic accumulation of the mutant proteins 2. pulse-chase experiments suggested that FhuA protein passed through a soluble, membrane-free pool after cleavage of the signal sequence and before insertion into the outer membrane (60) 3. after the removal of the outer membrane by EDTA/lysozyme treatment, newly synthesized OmpF porin was secreted by the spheroplasts into the medium (83) 4. OmpE secreted by spheroplasts, could be inserted in vitro into isolated outer membranes (111), suggesting that they were true assembly intermediates 5. treatment of spheroplasts with antibodies directed against SecD prevented the release of processed MBP and OmpA (82) suggesting that the function of SecD is to release periplasmic proteins as well as OMPs from the inner membrane after their translocation.

V. INSERTION AND ASSEMBLY IN THE OUTER MEMBRANE A. Assembly Intermediates, Detected in vivo After their linear translocation across the inner membrane, OMPs have to adopt tertiary structure, insert into the outer membrane and, in many cases, oligomerize (Figure 3). The identification of assembly intermediates is very beneficial to gain

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insight in this process. Correctly assembled OMPs have several distinct biochemical characteristics and the successive acquirement of these characteristics can be used to monitor the kinetics of the assembly process. In contrast to inner membrane proteins, OMPs are not readily solubilized in detergents like Sarkosyl (39) or Triton X-100 (I 09). Many OMPs, including the porins and LamB, are trimeric proteins that are highly resistant to denaturation. Even in 2% SDS, temperatures above approximately 70 ~ are required to denature these trimers into monomers (see also Figure 5, p. 000). Monomeric OMPs like OmpA (57) and PldA (94) display a particular electrophoretic behavior, designated heat-modifiability. When cell envelope-containing samples are not heated above 60 ~ before electrophoresis, these proteins migrate much faster in SDS-polyacrylamide gels than the fully denatured proteins. Furthermore, OMPs are highly resistant to proteases when they are correctly assembled into the outer membrane, being either totally protected against digestion (88) or yielding few, well-defined degradation products like the membrane-embedded part of OmpA (1119). Finally, monocional antibodies (mAbs) raised against native OMPs recognize often conformational epitopes that are not present in the denatured proteins (131). Pulse-chase experiments have been performed to study the assembly of OmpA (43). An assembly intermediate was detected, designated imp-OmpA (immature processed), which had already been processed by signal peptidase, but had not yet acquired the characteristic heat-modifiability and protease-resistance of native OmpA. This imp-OmpA fractionated like an inner membrane protein. A protein form with properties similar to imp-OmpA accumulated in cells overproducing OmpA, probably because of the limited capacity of the outer membrane to take up the protein. Immuno-electron microscopy revealed that imp-OmpA was localized in the periplasm under these conditions. The imp-OmpAcould be converted in vitro into the heat-modifiable form and displayed phage receptor activity upon addition of LPS, suggesting that imp-OmpA is a true assembly intermediate. Similarly, the assembly of LamB has been studied in pulse-chase experiments (138). Newly synthesized Lamb monomers had a half-life of about 20 seconds and were converted into trimers. However, these trimers were denatured in SDS at temperatures between 60 and 70 ~ and were therefore designated "metastable trimers." The metastable trimers were converted slowly, with a half-life of about 5.7 minutes, into stable trimers, possibly by interaction with other outer membrane components like LPS. These results show that the assembly of LamB into trimers requires multiple steps. Newly synthesized LamB was detected at the cell surface with a delay of 30-50 seconds (I 38), indicating that the metastable trimer intermediate is already inserted into the outer membrane. A metastable trimer was also detected by Fourel et al. (44) as an intermediate in the assembly of OmpE These authors used a large panel of mAbs, all recognizing conformational epitopes, and studied the kinetics of the appearance of the corresponding epitopes in pulse-chase experiments. The different epitopes were exposed on the assembling protein in four distinct stages. The earliest epitopes were detected

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immediately after processing and they are probably present on a folded monomeric assembly intermediate. This intermediate could be immunoprecipitated from the periplasmic fraction, shortly after the pulse, consistent with the periplasmic pathway for OMP biogenesis (Figure 3). This early intermediate was converted successively into metastable trimers, stable trimers, and finally to native trimers, with each step being defined by the appearance of additional epitopes. A metastable trimeric assembly intermediate of OmpF was not detected by Reid et al. (103). However, these authors detected a novel intermediate with an electrophoretic mobility in between those of denatured monomers and trimers. This assembly intermediate (see also Figure 5) was identified as a dimer. The data discussed above show that the assembly of OMPs into their native structures is a multistep process. Unfortunately, the subcellular localization of the intermediates may be prone to artefacts. Standard cell fractionation methods have been established for fully assembled native proteins. Fractionation artefacts have previously been reported for genetically manipulated proteins (125). Even bona fide E. coli proteins may be prone to fractionation artefacts; for instance, the A and B subunits of heat-labile enterotoxin are periplasmic proteins, but they form aggregates in the Tris-EDTA buffers normally used for membrane isolation, and consequently, they pellet with the membranes (58). It is conceivable that assembly intermediates become membrane-associated or aggregated during cell disruption, or that they pass through a membranous compartment that behaves differently during fractionations than the bulk of inner or outer membranes. Probably the most reliable method for the subcellular localization of proteins is immunoelectron microscopy. However, this method is not very sensitive and therefore inappropriate to detect low amounts of assembly intermediates. A possible solution to this problem is the use of mutants with thermosensitive assembly defects. Such a mutant has been described for LamB (84). However, the assembly intermediate was rapidly degraded at the restrictive temperature. Degradation was reduced in a degP mutant strain, lacking a periplasmic protease. After a short incubation at the restrictive temperature, the accumulated assembly intermediate could be chased into trimers by a shift-down to the permissive temperature. However, after a longer incubation at the restrictive temperature, which would be required to accumulate sufficient assembly intermediates for detection by immunoelectron microscopy, the protein lost its assembly competence and could therefore no longer be considered as a true intermediate. Presently, only those intermediates that are already inserted into the outer membrane can reliably be localized. Whereas most OMPs are resistant to proteases, a protease-sensitive site can be created by inserting a short stretch of additional amino acid residues in an external loop (e.g., refs. 2, 41). These insertions do not generally interfere with the correct assembly of the proteins into the outer membrane. Thus, the outer membrane localization of assembly intermediates can be assessed by determining their sensitivity to externally added proteases.

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B. Role of LPS and Lipid Biosynthesis An important question to answer is why OMPs selectively insert into the outer membrane, and not in the cytoplasmic membrane. LPS could be important in this respect, since it is a lipidic component, specific to the outer membrane. Indeed, a tight interaction between several OMPs and LPS has been reported (36, 105, 142). LPS consists of a complex phosphorylated heteropolysaccharide that is linked to a glucosamine-containing lipid, lipidA (104). The polysaccharide portion has been divided into two major regions, an internal core region and the peripheral O-antigen, which shows a high degree of variability among different strains. Because of this variability, and also because many commonly used laboratory strains, including E. coli K-12, are lacking the O-antigen, one would not expect this part of the LPS to be important in the assembly of OMPs. Nevertheless, it has been observed that OmpF preferentially interacts with O-antigen-containing LPS (36). Mutants affected in the core region of the LPS, most notably heptose-deficient strains, are markedly decreased in the proportion of protein recovered in the outer membrane fraction (6, 68), indicating a strong correlation between OMP biogenesis and LPS structure. Not all OMPs were affected to the same extent by the LPS mutations (e.g., whereas OmpF was barely detectable, OmpA was only moderately and OmpC hardly, if at all, affected in heptose-deficient mutants [53]). This result suggests that different OMPs depend to different degrees on LPS structure or that they interact with different regions of the LPS molecule. The exclusive location of LPS in the outer leaflet of the outer membrane may be difficult to reconcile with a role as recognition site for the selective insertion of OMPs into the outer membrane. However, one should realize that LPS also has to be transported from its site of synthesis in the inner membrane to the outer membrane. Thus, assembly intermediates of the OMPs might interact with nascent LPS, and consequently, the biogenesis of the molecules might be coupled. This supposition was underscored by the observation that inhibition of fatty acid synthesis, and consequently of LPS, by the drug cerulenin interfered with the assembly of OmpF into trimers (13). Later experiments suggested that the conversion of metastable into stable trimers was inhibited by the drug (44). The assembly intermediates that accumulated during treatment with the drug were degraded in the cell (13). Furthermore, a dramatic decrease in the level of synthesis of OmpF was observed when its assembly was inhibited by the drug. The synthesis of several other OMPs was also found to be affected by cerulenin treatment, and this inhibition probably takes place at the translational level (89). These results suggest that there is a feedback inhibition of OMP synthesis by assembly intermediates. The observation that expression of an altered OmpC protein, lacking two amino acid residues from a transmembrane segment inhibited the expression of several other major OMPs (21), might be explained by the same feedback inhibition mechanism. In conclusion, there appears to be a direct coupling between the synthesis of lipid (most likely LPS) and the assembly of OMPs.

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C. In vitro Reconstitution of the Insertion and Assembly Process

Several laboratories have reported on the development of cell-free in vitro systems to study the molecular details of OMP assembly As mentioned above (Section V.A), an assembly intermediate of OmpA, designated imp-OmpA, accumulates in cells overproducing this protein. Addition of LPS to imp-OmpA resulted in conversion to the heat-modifiable form and in the acquisition of phage receptor activity (43). Earlier, it was described that the denatured OmpA, isolated from outer membranes, could be renatured by the addition of LPS but not by E. coil phospholipids or dimyristoylphosphatidylcholine (DMPC) (110). The renatured protein was heat-modifiable, displayed phage receptor activity, and its N-terminal part, which is normally embedded in the outer membrane (see Figure I B), became protease-resistant. The lipid A moiety of LPS appeared to be as effective as complete LPS in the renaturation experiments (I10). These results underscore the postulated involvement of LPS in the assembly of OMPs. However, it was demonstrated more recently that SDS-denatured OmpA could be refolded by adding the nonionic detergent octyiglucoside (OG), and could subsequently be reconstituted into DMPC lipid membranes (37). Hence, LPS is neither required for proper folding of OmpA nor for incorporation into a lipid membrane. Furthermore, urea-denatured OmpA was shown to refold and insert in the absence of detergent when diluted in a dispersion of small preformed DMPC vesicles (121). Large vesicles were not effective, unless in the presence of low concentrations of OG. The precise function of OG in the latter case is not clear. It seems unlikely that it induced the refolding of OmpA and allowed the subsequent insertion into the large vesicles, since concentrations below the critical micelle concentration were effective. Possibly, the detergent-induced small distortions in the large vesicles might be required to allow the insertion of OmpA. In small vesicles, the lipid bilayer is highly curved, and consequently, the lipid packing is not optimal. This feature may allow insertion in the absence of detergent. Interestingly, freeze-fracturing electron microscopy has revealed that the outer fracture face of the outer membrane is densely occupied with particles (133). Consequently, the periplasmic face of the outer membrane is covered with highly-curved micelle-like structures (schematically depicted in Figure 4). We suggest that these particles enable the insertion of OMPs, similar to the small DMPC vesicles in the in vitro reconstituted system for OmpA assembly. Moreover, the number of particles observed was drastically reduced in mutants with defects in the core region of the LPS, for example, in heptose-deficient mutants (133). Hence, the effect of LPS mutations on the biogenesis of OMPs (see section V.B) might be explained by the reduced number of particles (i.e., the reduced number of insertion sites). Recently, we have developed a system to study the assembly of PhoE protein in vitro (27, 28, 29). Radioactively labeled PhoE protein was synthesized in vitro in an E. coil lysate and its folding was probed in immunoprecipitation experiments with mAbs that recognize conformational epitopes. Since the protein could be

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161

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JAN TOMMASSEN and HANS DE COCK

with a defect in the core region of the LPS were less efficient in inducing trimerization (27). Furthermore, the trimers seemed to be associated with LPS, since their mobility during electrophoresis was slightly different, depending on the chemotype of the LPS in the outer membranes used to induce trimerization. The trimers obtained were highly resistant to denaturation in SDS (Figure 5), like the native PhoE produced in vivo. However, they were not inserted into the membranes since they remained in the supernatant after pelleting of the membranes by centrifugation (28). Furthermore, they were not protected against proteases. Insertion in a protease-resistant configuration was observed when small amounts of a detergent (0.06% Triton X-100) were present during trimerization (27). This observation is reminiscent of the results obtained for the insertion of OmpA in large DMPC vesicles, discussed above. Similar to the PhoE porin, the assembly of the related OmpF porin, obtained either by synthesis in an E. coli lysate (112) or by secretion from spheroplasts (111), has been studied in vitro. The results obtained were largely consistent with those described for PhoE. The major discrepancy is that trimerization of the OmpF proteins could not only be induced by outer membranes, but also by LPS preparations, and even by a mixture of an anionic and a nonionic detergent. Similarly, the renaturation of OmpF, isolated from the outer membrane and denatured in guanidium hydrochloride, by a combination of SDS and octyl-pentaoxyethylene, has been reported (38). An explanation for this discrepancy is not obvious at the moment. Anyhow, these results suggest that LPS is neither required for the folding nor for the trimerization of the OmpF molecules. However, it is still possible that the LPS plays an important role in the in vivo situation and that SDS functions as the substitute for LPS in vitro. Furthermore, the OmpF secreted by spheroplasts was reported to trimerize more efficiently than the OmpF or PhoE proteins synthesized in vitro in an E. coli lysate (112). There are several possible explanations for this difference: (1.) the in vitro synthesized proteins contained short N-terminal extensions of a few amino acid residues that may have interfered with the trimerization process, (2.) trimerization may have been inhibited by binding of cytoplasmic chaperones like SecB (30), which were present in the lysates used to direct the in vitro synthesis of these proteins, and (3.) secretion across the cytoplasmic membrane might be accompanied by conformational changes favoring trimerization. Indeed, the spheroplast-secreted OmpF porin appeared to be different from the in vitro synthesized OmpF in its reactivity with antibodies recognizing conformational epitopes (112).

D.

Sorting Signals in Outer Membrane Proteins

In general, the membrane-spanning segments of a polytopic inner membrane protein are hydrophobic and can insert into the membrane, independent of the other membrane-spanning segments. In contrast, the membrane-spanning segments of OMPs are ~-strands with only one hydrophobic side (see Section II.B). Conse-

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quently, OMPs are compatible with the lipidic environment of the membrane only after the formation of the entire ~barrel structure containing a hydrophobic exterior. It is, therefore, not surprising that large deletions (removing > 30 amino acid residues) in the mature domain of PhoE prevented the normal incorporation of the mutant proteins into the outer membrane and led to their periplasmic accumulation (14). Such large deletions can be expected to interfere with the folding of the protein into the [3-barrel configuration. Similarly, large deletions in the membrane-embedded N-terminal part of OmpA prevented the normal incorporation of this protein in the membrane (66), whereas deletions in the C-terminal periplasmic tail of this protein (see Figure IB) were tolerated (18). Although the entire conformation of OMPs is apparently required for their correct localization, some portions of these proteins might be more important to the correct conformation than others, and in addition, specific sorting signals might still be present to allow interaction with the appropriate membrane. A priori, one could expect that the J-strands are more important for the assembly of the proteins than the surface exposed loops, since there are restrictions on both the [3-structure and on the hydrophobicity of residues in these segments. Comparison of the primary structures of the porins OmpF, OmpC, and PhoE (8.5) show that the sequences of the membrane-spanning segments are much better conserved than those of the exposed loops. A similar observation was made when the primary structures of the OmpA proteins of different Enterobacteriaceae were compared (I 7). Furthermore, insertions (2, 4) and deletions (/) were well tolerated in the exposed loops of PhoE, as well as of other OMPs (e.g., refs. 22, 41, 67). However, the [3-strands also tolerated mutations. In the case of PhoE, hydrophobic residues in the J-strands that are exposed to the fatty acyl chains of the lipids or to the subunit interface of the trimers could be replaced by hydrophilic or even charged residues without affecting the assembly of the protein (119). However, two of such substitutions in a single [3-strand dramatically affected the efficiency of outer membrane incorporation. Furthermore, the first membrane-spanning segment of PhoE could be completely deleted (e.g., mutant protein A3-30, in ref. 16) and still, the mutant protein was correctly inserted in the membrane as evidenced by the binding of PhoE-specific bacteriophage and mAbs to intact cells. However, part of the total amount of the mutant protein produced accumulated in the periplasm, showing that the efficiency of outer membrane insertion was reduced. The first membrane-spanning J-strand of PhoE does not face the lipids, but is located at the subunit interface within the trimers (2.5). Consistently, no trimers of this mutant protein were detected, indicating that either the stability of the trimers is drastically reduced or that the protein inserts into the membrane as a monomer. According to the model for the localization of OMPs, depicted in Figure 3, these proteins adopt their tertiary structure in the periplasm before inserting into the outer membrane. Consequently, a putative sorting signal might be a conformational one and therefore difficult to detect by comparison of the primary structures of diverse OMPs. Nevertheless, a few short stretches of amino acid residues with vague

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sequence homology were detected in the primary structures of the porins, LamB and OmpA (92). Probably, most of these sequences do not correspond to sorting signals since the homologies became insignificant when more OMP sequences became available, or because deletion analysis in OmpA or PhoE showed that they were not involved in outer membrane localization. For the most pronounced similarity region, sequence comparisons have recently been updated (91), and it appeared that only a single glycine residue, corresponding to Gly- 144 in PhoE, was completely conserved in this segment. Gly-144 is located in a periplasmic turn in the PhoE structure (25). In the OmpA model (134), the corresponding residue is located in an entirely different position near the cell surface, which makes a common function for these residues unlikely. Gly-144 of PhoE was replaced by a leucine residue (31). Indeed, outer membrane localization was somewhat defective, especially at higher growth temperatures. However, in the in vitro assembly system for PhoE (described in Section V.C), the mutant protein appeared to be defective in folding (31). Therefore, we believe that Gly-144 of PhoE is not part of a sorting signal, but is important for protein folding. A more significant similarity was recently detected at the C-termini of OMPs (118). Comparison of the last ten amino acid residues of diverse OMPs revealed the presence of potential amphipathic ~strands with hydrophobic residues at positions 1 (Phe), 3 (preferentially Tyr), 5, 7, and 9 from the C-terminus. In the porins this segment corresponds to the last membrane-spanning segment (25), and its deletion in PhoE had a detrimental effect on assembly in the membrane (15). This consensus sequence was found in the vast majority of bacterial OMPs, including E. coli proteins with widely diverse functions and OMPs of other Gram-negative bacteria (118). In a few cases, including LamB, Trp instead of Phe was found at the C-terminal position. However, like Phe, Trp is a hydrophobic aromatic residue and is therefore expected to functionally replace the Phe. In OmpA and its analogs of other bacteria, the consensus sequence is not found at the ultimate C-terminus, which is extending into the periplasm (Figure 1B), but at the C-terminus of the membrane embedded part (i.e., residues 161-170). The consensus sequence was not detected at the C-termini of OMPs that are involved in the secretion of macromolecular compounds like PapC, which is involved in the secretion of pill subunits (95), or TolC (93) known to be involved in the secretion of o~-haemolysin (139). Interestingly, the consensus sequence is located in a region in OmpA that was earlier implicated in sorting to the outer membrane (66). As mentioned in the beginning of this section, large deletions in OmpA prevented the correct incorporation of the protein into the outer membrane. However, immunoelectron microscopy revealed that the mutant proteins were still associated with the outer membrane, unless the deletions covered an area between residues 154 and 180. When this region was deleted, the mutant proteins accumulated in the periplasm (66). These results suggested that the area between residues 154 and 180 (including the consensus sequence between residues 161-170) contains an outer membrane

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sorting signal. Moreover, of many missense mutations created in ompA, only a mutant protein containing two amino acid substitutions in the consensus sequence was totally blocked in outer membrane assembly (65). Since the C-terminal Phe is the most pronounced feature of the consensus sequence, this residue was used as a target for extensive site-directed mutagenesis in PhoE (118). High-level expression of all mutant proteins obtained was lethal to the cells, and the efficiency of outer membrane incorporation was dramatically affected. The mutational effect was the least severe when the Phe was replaced by another aromatic residue and was most severe when this residue was deleted. In the latter case, hardly any PhoE could be detected in the outer membrane, and the protein accumulated as aggregates in the periplasm (117). In vitro the folding and the trimerization of this mutant protein were virtually unaffected, but the insertion in isolated outer membranes was drastically reduced. When the expression level of the mutant proteins carrying substitutions for or deletion of the C-terminal Phe was reduced, the detrimental effects on growth were alleviated and more mutant protein was correctly assembled into the outer membrane (117). This result suggests that the C-terminal Phe is not absolutely required for the correct assembly of the protein, but does contribute to the efficiency of the process. Apparently, there is a kinetic partitioning between outer membrane incorporation and aggregation of periplasmic assembly intermediates. At high levels of expression, more protein will aggregate and become assembly-incompetent, whereas at low expression levels the tendency to aggregate will be reduced, resulting in increased outer membrane incorporation. The C-terminal Trp in Lamb may play a similar role as the Phe in PhoE, since deletion of the last two amino acid residues of LamB has been reported to drastically affect outer membrane incorporation (56).

VI.

CONCLUSIONS A N D FUTURE PROSPECTS

Whereas the molecular details of the mechanism of the transport of proteins across the cytoplasmic membrane are beginning to emerge, even the basic concepts of the last steps in the biogenesis of OMPs are far from clear. The proteins probably fold into their tertiary structure after passage through the inner membrane (Figure 3). After folding into a [~-barrel, the proteins have a hydrophobic exterior, compatible with insertion in the lipidic environment of the membrane. It is not yet clear where folding takes place. The process probably does not occur free in the periplasm, but is associated at the periplasmic face of one of the membranes, since amphiphiles were usually observed to induce folding in vitro. Although refolding of denatured OMPs was observed in the absence of any other proteins, this does not exclude the possibility that chaperones or enzymes are involved in this Process in vivo. Several other proteins have been reported to renature spontaneously in vitro, whereas their folding is guided by other proteins in vivo (e.g., ref. 40). The possible involvement of periplasmic proteins in the folding of OMPs will be an important topic of future

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research. Many OMPs, including the porins, are devoid of cysteine residues, and in these cases the involvement of protein disulfide isomerases can be excluded. Nevertheless, the expression of OmpF was affected at the transcriptional level in a dsbA mutant lacking a periplasmic disulfide isomerase (99). On the other hand, native OmpA does contain a disulfide bond and its formation was apparently retarded in a dsbA mutant (8). However, since the two cysteines of OmpA are located in the C-terminal periplasmic extension, no effect on outer membrane localization would be expected. The cis-trans isomerization of peptidylpropyl bonds is frequently a rate-limiting step in protein folding. An enzyme catalyzing this reaction has been detected in the periplasm of E. coli (54), but the involvement of this enzyme and of putative chaperones in the folding of OMPs remains to be investigated. Another important aspect for future research will be to gain insight in the mechanism of selective insertion of OMPs in the outer membrane and not in the inner membrane. There are some indications that LPS is involved in this process, but the effect of LPS could be indirect. The role of the observed outer membrane particles and of lipid packing will have to be investigated. Furthermore, if the C-terminal consensus sequence (118) indeed represents a sorting signal, a receptor for this signal can be expected to exist and should be identified.

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10. Benz, R., & Bauer, K. (1988). Permeation of hydrophilic molecules through the outer membrane of gram-negative bacteria. Review on bacterial porins. Eur. J. Biochem. 176, 1-19. 11. Benz, R., Schmid, A., & Hancock, R. E. W. (1985). Ion selectivity of Gram-negative bacterial porins. J. Bocteriol. 162, 722-727. 12. Benz, R., Schmid, A., Nakae, T., & Vos-Scheperkeuter, G. H. (1986). Pore formation by LamB of Escherichia coil in lipid bilayer membranes. J. Bacteriol. 165, 978--986. 13. Boll& J.-M., Lazdunski, C., & Pages, J.-M. (1988). The assembly of the major outer membrane protein OmpF of Escherichia coli depends on lipid synthesis. EMBO J. 7, 3595-3599. 14. Bosch, D., Leunissen, J., Verbakel, J., de Jong, M., van Erp, H., & Tommassen, J. (1986). Periplasmic accumulation of truncated forms of outer-membrane PhoE protein of Escherichia coli K-12. J. Mol. Biol. 189, 449--455. 15. Bosch, D., Scholten, M., Verhagen, C., & Tommassen, J. (1989). The role of the carboxy-terminal membrane-spanning fragment in the biogenesis of Escherichia coil K 12 outer membrane protein PhoE. Mol. Gen. Genet. 216, 144-148. 16. Bosch, D., Voorhout, W., & Tommassen, J. (1988). Export and localization of N-terminally truncated derivatives of Escherichia coli K- 12 outer membrane protein PhoE. J. Biol. Chem. 263, 9952-9957. 17. Braun, G., & Cole, S. T. (1984). DNA sequence analysis of the Serrotia marcescens ompA gene: implications for the organization of an enterobacteriai outer membrane protein. Mol. Gen. Genet. 195, 321-328. 18. Bremer, E., Cole, S. T., Hindennach, I., Henning, U., Beck, E., Kurz, C., & Schaller, H. (1982). Export of a protein into the outer membrane of Escherichio coil K 12. Stable incorporation of the OmpA protein requires less than 193 amino-terminal amino-acid residues. Eur. J. Biochem. 122, 223-231. 19. Breukink, E., Kusters, R., & de Kruijff, B. (1992). In vitro studies on the folding characteristics of the Escherichia coil precursor protein prePhoE. Evidence that SecB prevents the precursor from aggregating by forming a functional complex. Eur. J. Biochem. 208, 419--425. 20. Brundage, L., Hendrick, J. P., Schiebel, E., Driessen, A. J. M., & Wickner, W. (1990). The purified E. coli integral membrane protein SecY/E is sufficient for reconstitution of SecA-dependent precursor protein translocation. Cell 62, 649-657. 21. Catron, K. M., & Schnaitman, C. A. (1987). Export of protein in Escherichia coli: a novel mutation in ompC affects expression of other major outer membrane proteins. J. Bacteriol. 169, 4327--4334. 22. Charbit, A., Boulain, J. C., Ryter, A., & Hofnung, M. (1986). Probing the topology of a bacterial membrane protein by genetic insertion of a foreign epitope; expression at the cell surface. EMBO J. 5, 3029-3037. 23. Charbit, A., Cl6ment, J.-M., & Hofnung, M. (1984). Further sequence analysis of the phage lambda receptor site. Possible implications for the organization of the LamB protein in E. coli KI2. J. Mol. Biol. 175, 395-401. 24. Collier, D. N., Bankaitis, V. A., Weiss, J. B., & Bassford, E J. Jr. (1988). The antifolding activity of SecB promotes the export of the E. coil maltose-binding protein. Cell 53, 273-283. 25. Cowan, S. W., Schirmer, T., Rummel G., Steiert, M., Ghosh, R., Paupfit, R. A., Jansonius, J. N., & Rosenbusch, J. P. (1992). Crystal structures explain functional properties of two E. coil porins. Nature 358, 727-733. 26. Davis, N. G., & Model, P. (1985). An artificial anchor domain: hydrophobicity suffices to stop transfer. Cell 41,607-614. 27. De Cock, H., Blokland, S., & Tommassen, J. (1995). In vitro assembly and insertion of PhoE protein of E. coil K-12 into the outer membrane. (Manuscript submitted for publication.) 28. De Cock' H., Hekstra, D., & Tommassen, J. (1990). In vitro trimerization of outer membrane protein PhoE. Biochimie 72, 177-182.

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