J. Cell Sci. Suppl. II, 73-83 (1989)Printed in Great Britain © The Company of Biologists Limited 1989

73

Involvement of membrane lipids in protein export in Escherichia coli

J A N T O M M A S S E N 1-2* , T R U U S D E V R I J E 3, H A N S D E C O C K 2,D I R K B O S C H 1 a n d B E N D E K R U I J F F 2,3

1 Department of Molecular Cell Biology, in s titu te of Molecular Biology and Medical Biotechnology, and 3Centre of Biomembranes and Lipid Enzymology, University of Utrecht, Padualaan 8, 3584 CH Utrecht, The Netherlands

SummarySeveral models for the transport of proteins across membranes predict a role for lipids. If these models are correct, then alterations in lipid metabolism may affect protein export and vice versa. We are investigating this possibility by studying Escherichia coli K-12 mutants with defects in protein export or phospholipid metabolism. A temperature-sensitive secA mutant, which is defective in protein export at 42°C, exhibited severe pleiotropic effects on membrane biogenesis. Incubation of this strain at 42°C resulted in the appearance of intracytoplasmic membranes, in alterations in lipopolysaccharide structure and in decreased cardiolipin and C1 8 :1 fatty acid content. On the other hand, a pgsA mutant which is defective in the synthesis of acidic phospholipids, exhibited a protein export defect when studied in vivo or in vitro. These results are in agreement with a postulated role of membrane lipids in protein export.

IntroductionThe cell envelope of Gram-negative bacteria consists of two membranes, the inner and the outer membrane, which are separated by the peptidoglycan-containing periplasm. Since protein synthesis takes place in the cytoplasm, proteins destined for the periplasm or the outer membrane have to cross the inner membrane to reach their final location. Such proteins are synthesized in a precursor form with an N-terminal extension of approximately 20 amino acids which is called the signal sequence. Such signal sequences consist of three domains (Von Heijne, 1985; Trun & Silhavy, 1989; Hardy & Randall, 1989): the N terminus containing positively charged amino acids; a hydrophobic core, and a more polar C-terminal region, which contains the recognition site for signal peptidase, an enzyme which cleaves off the signal sequence from the precursor proteins. The signal sequences are essential for export, since mutations especially in the hydrophobic core can prevent export (Emr et al. 1980; Bedouelle et al. 1980). Although a signal sequence does not seem to be sufficient to mediate export (Moreno et al. 1980) the rest of a precursor does not seem to contain

* Author for correspondence.

Key words: protein export, Escherichia coli, secA, pgsA, lipid metabolism.

74 y. Tommassen and others

essential export information. Series of overlapping deletion mutations have been described in the structural genes of the outer membrane proteins PhoE (Bosch et al. 1986, 1988) and OmpA (Freudl et al. 1985, 1987) of Escherichia coli. These deletions together covered the complete phoE and ompA genes, except for the signal sequence-encoding parts. The mutant proteins were all exported across the inner membrane, showing that no parts of the mature proteins are essential for export. Probably they only play a passive role, i.e. they should be export compatible.

Not much is known about the mechanism of export across the membrane or the cellular components involved in this process. In eukaryotes, several proteinaceous components of the export apparatus have been identified, such as the signal recognition particle (Walter et al. 1981) and docking protein (Meyer et al. 1982). Equivalents of these factors have not yet been identified in prokaryotes. Crooke et al. (1988) demonstrated in an in vitro system that the precursor of OmpA can translocate across inner membrane vesicles in the absence of any other soluble protein. Nevertheless, soluble proteins have been purified from E. coli that stimulate the translocation of purified precursors in vitro. These proteins, designated trigger factor (Crooke & Wickner, 1987) or cytoplasmic translocation factor (Weng et al. 1988), probably maintain the translocation-competent state of the purified precur­sors (Crooke et al. 1988). Whether they play a role in protein export in vivo has not yet been demonstrated.

Also, genetic studies have been performed to identify components of the export apparatus in i', coli (for reviews, see Oliver, 1985; Trun & Silhavy, 1989). Mutations in the sec genes lead to the accumulation of precursors of exported proteins under non-permissive growth conditions. On the other hand, prl suppressor mutations restore the export of precursors with a defective signal sequence. The product of the secB gene appears to be a soluble protein, which maintains the export-competent, unfolded state of precursors (Collier et al. 1988). The function of the other sec and prl gene products is unknown. They might also be part of the export apparatus, but on the other hand they might also be only indirectly involved in protein export.

According to the signal hypothesis (Blobel & Dobberstein, 1975) protein export is mediated by a completely proteinaceous export apparatus. Thus, exported proteins pass the membrane via pores, formed by integral membrane proteins. However, other models suggest that lipids are directly or indirectly involved in protein export (DiRienzo et al. 1978; Engelman & Steitz, 1981; Nesmeyanova, 1982; Von Heijne & Blomberg, 1979; Wickner, 1980). For instance, the precursors of exported proteins might directly bind to phospholipids in the inner membrane, and the proteins might pass the hydrophobic environment of the membrane if the protein stays in contact with the hydrophilic part of the phospholipids by the formation of non-bilayer structures (De Kruijff et al. 1985). In this view, the co-transport of lipids may play an important role in facilitating protein export. If these models are correct, mutants defective in protein export may have pleiotropic defects in lipid metabolism, and on the other hand mutants defective in lipid metabolism may be pleiotropically defective in protein export. Both aspects are under investigation (De Cock et al. 1989; De Vrije et al. 1988).

Involvement of lipids in protein export IS

Fig. 1. Intracytoplasmic membranes as observed in ultra-thin cryosections of secA mutant strain MM52 after growth for 5 h at 42 °C.

Pleiotropic effects of a secA mutationMorphological alterations in a secA m utantE. coli strain M M 52 is a temperature-sensitive secA mutant, which accumulates precursors of exported proteins when the temperature is raised above the permissive temperature of 30°C (Oliver & Beckwith, 1981). T o detect any possible pleiotropic effects of this secA5l mutation on lipid metabolism, the membrane organisation of the mutant strain was first studied morphologically (De Cock et al. 1989). Examination of ultra-thin cryosections of MM52 cells, grown at 42°C, by electron microscopy revealed the appearance of new membrane-like structures within the cytoplasm of these cells (Fig. 1). Such membrane-like structures were hardly observed in MM52 cells growth at 30°C and not at all in the secA+ parental strain grown at either 30 °C or 42 °C. These intracytoplasmic structures were also observed in freeze-fracture experiments, with the fracture plane going through several membranes (De Cock et al. 1989). This confirms that the intracytoplasmic struc­tures observed in the ultra-thin cryosections of the secA mutant are actually membranes. Thus, these morphological studies show that the secA51 mutation has pleiotropic effects on membrane biogenesis.

Interestingly, morphologically similar intracytoplasmic membranes were observed in cells where protein export was blocked by the induction of the synthesis of Lam B- LacZ fusion proteins (Voorhout et al. 1988). Such fusion proteins consist of an N- terminal moiety, including the signal sequence, of outer membrane protein LamB and the cytoplasmic enzyme /3-galactosidase (Hall et al. 1982). After induction of the synthesis of such a fusion protein, export is initiated at the LamB moiety, but ¡3- galactosidase cannot efficiently be transported across the inner membrane, probably

76 J. Tommassen and others

because it rapidly folds into an export-incompatible form. As a result the hybrid protein becomes stuck in the membrane and this leads to a block in protein export. However, it should be noted that the appearance of intracytoplasmic membranes is not a general feature of cells where protein export is blocked. For instance, in a secY mutant, hardly any intracytoplasmic membranes were observed (De Cock et al. unpublished observation). In addition, the few intracytoplasmic membranes ob­served in this mutant strain were morphologically distinct from those observed in the secA mutant. In a secB mutant, no intracytoplasmic membranes were observed.

Alterations in lipid composition in a secA mutantIn freeze-etch experiments, the concave side of the outer fracture face of the outer membrane of wild-type cells is completely occupied by particles (Verkleij et al. 1976), which probably consist of protein-lipopolysaccharide complexes (Van Alphen et al. 1978). Similar experiments with the secA mutant revealed the appearance of particle-free areas in the outer membrane after growth at 42 °C (De Cock et al. 1989). Previously, it has been reported that reduction in the amount of major outer membrane proteins as well as alterations in the lipopolysaccharide (LPS) structure lead to a reduced content of these particles. Of course, a reduction in the amount of outer membrane proteins is to be expected in a secA mutant, but we also investigated whether an altered LPS structure could be the cause of the disappearance of particles.

Bacteriophage C21 can infect only E. colt cells with galactose-deficient LPS (Lindberg, 1973). In contrast to its parental strain, secA strain MM52 was sensitive to this phage when grown at 37 °C (De Cock et al. 1989), which is consistent with an altered LPS structure in the secA strain. The altered structure of the LPS was also detected by SDS—polyacrylamide gel electrophoresis (Fig. 2). Apparently, the activity of one of the glycosyl transferases which substitute LPS is affected in a secA mutant.

Analysis of the phospholipid composition of MM52 cells, grown for 6h at 42°C, revealed that the cardiolipin (C L) content was slightly but significantly reduced as compared to the control strain grown under identical conditions (3-5 % and 5-0 % of the total phospholipids, respectively). Interestingly, in aprlA4 mutant strain which is able to export proteins with a defective signal sequence the C L content was found to be increased 2-fold as compared to its prlA+ parental strain (De Cock et al. unpublished observation). These results suggest that C L could play an important role in protein export, which hypothesis is particularly important because this lipid has a strong tendency to form type II non-bilayer structures (De Kruijff et al. 1985), the induction of which upon interaction with amphipatic polypeptides (Batenburg & De Kruijff, 1988) is of special interest.

Finally, fatty acid analysis revealed that strain MM52 contained a reduced amount of cis-vaccenic acid after 6 h at 42 °C as compared to the control strain (11 % and 18 % of the total fatty acids, respectively). A role for this fatty acid in the export of alkaline phosphatase has previously been suggested (Nesmeyanova, 1982). In conclusion, in

Involvement of lipids in protein export 77

1 2 3 4 5

Fig. 2. Silver stained SD S—polyacrylamide gel containing proteinase K-treated cell envelopes, showing the LPS patterns of secA mutant MM52 grown at 30 °C (lane 1) or at 42°C (lane 2), secA+ parental strain MC4100 grown at 30°C (lane 3) or at 42°C (lane 4) and a galE mutant strain MC1000 grown at 37°C (lane 5).

a secA mutant in which protein export is disturbed, important pleiotropic effects on membrane composition and lipid content are observed.

Pleiotropic effects of a pgsA mutationMorphological alterations in a pgsA m utant

Phosphatidylglycerol (PG ) phosphate synthetase is a key enzyme in the biosynthesis of the major acidic phospholipids, PG and C L, in E. coli. Strains carrying a null allele in the structural gene, pgsA, for this enzyme are not viable (Haecock & Dowhan, 1987). However, apgsA3 allele has been isolated which does not influence the growth rate when present in derivatives of strain SD 12 (Miyazaki et al. 1985). In these strains, the PG and C L contents are dramatically decreased. Since thep^sA3 allele could not be transferred to other strains (Haecock & Dowhan, 1987), it appears that SD 12 contains a suppressor mutation which allows the growth of cells with reduced capability to synthesize acidic phospholipids.

Light microscopic examination of cells carrying the pgsA3 allele revealed that these cells tend to form filaments, especially at elevated growth temperatures (Fig. 3).

78 J. Tommassen and others

pgsA+

pgs A 3V. ( \ o

^ " 'y - c \ r P t- _ ' \ \

1, c _v . - rFig. 3. Fuchsin-staining of cells of wild-type strain SD 12 (pgsA+) and of its pgsA3 derivative HD3122 after growth at 42°C.

Interestingly, filamentation has also been observed in cells in which a protein export defect is imposed by a secA mutation (Oliver & Beckwith, 1981) or by induction of the synthesis of a Lam B-LacZ fusion protein (H allei al. 1982). Electron microscopic examination of ultra-thin cryosections of the pgsA3 mutant cells did not reveal any intracytoplasmic membranes (Overduin et al. unpublished observation).

Protein export in a pgsA m utant

T o determine whether a reduced content of acidic phospholipids has a pleiotropic effect on protein export, pulse-label and pulse-chase experiments were performed (De Vrije et al. 1988). When cells containing a wild-type pgsA allele were pulse-

Pre O m p A - ______ ^ 'OmpA

chase 0 30" 0 30" 1 2 3 4

- 4 5

cat — — —

5 6 7i- 2 5

Fig. 4. Effect of PG and C L content on in vivo synthesis and processing of OmpA (A) and PhoE (B). A: cells of apgsA+ (lanes 1 and 2) and apgsA3 mutant strain (lanes 3 and 4) were pulse-labelled for 30 s with [35S] methionine and subsequently chased with unlabelled methionine for 0 or 30 s. Mature OmpA and its precursor were immuno- precipitated and analyzed by SD S—polyacrylamide gel electrophoresis, followed by fluorography. B : cells of a wild-type strain (lane 5), a pgsA mutant (lane 6) and a els mutant (lane 7) were pulse-labelled with [3oS]methionine for 10 s and labelled proteins were analyzed by SDS-polyacrylamide gel electrophoresis and fluorography. cat: chloramphenicol transacetylase.

Involvement o f lipids in protein export 79

labelled for 30 s, the majority of OmpA protein was in the mature form and hardly any precursor could be detected (Fig. 4A, lane 1). In contrast, substantial amounts of OmpA precursor were found in similar experiments with a pgsA3 mutant strain (Fig. 4A, lane 3). During a 30s chase period, this precursor was processed into mature OmpA (Fig. 4A, lane 4). Apparently, the processing and/or export of OmpA are retarded in the mutant strain. In similar pulse-label experiments, the ratio of precursor to mature PhoE protein was found to be substantially higher in the pgsA3 mutant as compared to the wild-type strain (Fig. 4B). In addition, it appeared that the total amount of PhoE protein produced during a 10 s pulse period was lower in the mutant strain. In contrast, cytoplasmic proteins such as chloramphenicol transacetylase were produced in normal amounts (Fig. 4B). It should be noted that reduced synthesis of certain envelope proteins in export-defective mutants has been observed previously (e.g. Liss & Oliver, 1986). Pulse-label experiments in a els mutant, which contains normal levels of PG and reduced levels of C L , did not reveal any precursor accumulation (Fig. 4B, lane 7), suggesting that the reduced PG content and not the reduced CL content is responsible for the export defect observed in the pgsA3 mutant.

The influence of low acidic phospholipid levels on protein translocation was also studied in an in vitro translation-translocation system (De Vrije et al. 1988). The PhoE precursor was synthesized in vitro and incubated with inverted inner membrane vesicles isolated from a wild-type strain or from the pgsA3 mutant. Translocation of the precursor was monitored by its protection against added protease. The results of a post-translational translocation assay are shown in Fig. 5. The rate of translocation appeared to be reduced in vesicles with a low PG content. Thus, these in vitro experiments also suggested that acidic phospholipids are required for efficient protein export. In addition, in these experiments it appeared that the efficiency of signal peptidase was severely reduced in the vesicles with reduced PG content (De Vrije et al. 1988).

What is the function o f acidic phospholipids in protein export?Signal sequences contain at their N termini a number of basic amino acid residues. Pulse-chase experiments revealed that the replacement of these basic residues by acidic residues in the PhoE precursor results in delayed processing and/or export (Fig. 6). Localization studies showed that the mutant precursor accumulated in the cytoplasm (Bosch et al. 1989). Also, in in vitro experiments the translocation of the mutant precursor appeared to be disturbed. In similar replacement studies, the positively charged residues at the N terminus of the major outer membrane lipoprotein precursor turned out to be essential for efficient export (Inouye et al. 1982; Vlasuk et al. 1983). A possible explanation for the results described is that the positively charged residues at the N termini of signal sequences interact with the polar head groups of acidic phospholipids in the membrane prior to translocation. This assumption is supported by studies with model membranes. A synthetic PhoE signal peptide was shown to be able to penetrate lipid monolayers and this penetration was particularly efficient when the monolayers consisted of negatively

80 J . Tommassen et al.

Fig. 5. Kinetics of post-translational translocation of PhoE protein across inner mem­brane vesicles of wild-type strain SD12 (• ------ • ) and its pgsA3 derivative HD3122(O------ O ). During the incubation of in ■wiro-synthesized PhoE precursor with thevesicles, translocation was stopped at the indicated time points by the addition of proteinase K and the percentage of translocated PhoE was determined.

p.PhoE m .PhoE

chase(min)o 0.5

charge

f0.5

pPHCI-201 ( + 2 )

pPHO .202( - 2 )

Fig. 6. Effect of the replacement of the two lysine residues in the PhoE signal sequence by aspartic acid residues on the kinetics of processing in vivo. Cells expressing wild-type PhoE protein (lanes a-d) or the mutant PhoE (lanes e-h ) were pulse-labelled for 30 s with [35S]methionine and chased with unlabelled methionine for the indicated time period. Mature PhoE and its precursor were immunoprecipitated and analyzed by SD S-poly- acrylamide gel electrophoresis and autoradiography.

charged lipids (Batenburgei al. 1988). Also the binding of in uzfro-synthesized PhoE precursor to phospholipid liposomes has been studied (De Vrije et al. 1989). It appeared that the precursor was only efficiently bound by negatively charged liposomes (Fig. 7). Thus, acidic phospholipids may play a role in (translocation- competent) binding of precursors to the membrane, but other roles in the protein export pathway are also possible.

Involvement of lipids in protein export 81

40 K - — « m » PELLET

40 K - SUPERNATANT

- PC PC PG E . coli

DDA LIPID

Fig. 7. Binding of in vitro synthesized PhoE precursor to various phosphilipid lipo­somes. After incubation of PhoE precursor with the liposomes, the liposomes were pelleted by centrifugation. The pellet and supernatant fractions were collected and analyzed by fluorography of SDS-polyacrylamide gels. —, no liposomes; PC, phosphati­dylcholine liposomes; PC + DDA, phosphatidylcholine liposomes containing 10mol% dimethyl dioctadecyl ammonium bromide; PG, phosphatidylglycerol liposomes; E. coli lipid, liposomes composed by E. coli lipids. (K = 103 M J:) .

ConclusionsA secA mutation, which leads to protein export defects, has severe pleiotropic defects in membrane biogenesis. It is difficult to assess what the primary effect is of the secA mutation. A blockage of protein export does not always lead to the same pleiotropic effects. For instance, the appearance of intracytoplasmic membranes was hardly or not observed in secY or secB mutants. This could mean that the export defect in a secA mutant is a secondary effect. For instance, the precursors of exported proteins could interact with the intracytoplasmic membranes which would prevent their export across the inner membrane. On the other hand, one could argue that protein export involves multiple steps and that a blockage in each individual step leads to different pleiotropic effects. The appearance of intracytoplasmic membranes in the lamB-lacZ fusion strains could then be explained by assuming that the hybrid protein titrates out the SecA protein.

We also found that a defect in the synthesis of acidic phospholipids leads to retardation of protein export. Possibly, a role of these phospholipids is in the binding of precursors to the membranes in an export-competent fashion. Taken together, our results suggest a role for phospholipids in protein export and a coupling between protein export and lipid metabolism.

ReferencesB a t e n b u r g , A. M ., D e m e l , R. A., V e r k l e ij , A. J . & D e K r u ijf f , B . (1988). Penetration of the

signal sequence of Escherichia coli PhoE protein into phospholipid model membranes leads to lipid-specific changes in signal peptide structure and alterations in lipid organization. Biochemis­try 27, 5678-5685.

B a t e n b u r g , A. M. & D e K r u ijf f , B . (1988). Modulation of membrane surface curvature by peptide-lipid interactions. Biosci. Rep. 8, 299-307.

B e d o u e l l e , H ., B a s s f o r d , P . J . J r , F o w l e r , A. V ., Z a b in , I . , B e c k w it h , J . & H o fn u n g , M. (1980). Mutations which alter the function of the signal sequence of the maltose binding protein of Escherichia coli. Nature, Lond. 285, 78-81.

B l o b e l , G. & D o b b e r s t e in , B . (1975). Transfer of proteins across membranes. I. Presence of proteolytically processed and unprocessed nascent immunoglobulin light chains on membrane- bound ribosomes of murine myeloma. J. Cell Biol. 67, 835-851.

82 J . Tommassen et al.

B o s c h , D ., D e B o e r , P., B i t t e r , W. & T o m m assen , J . (1989). Role of the positively charged N- terminus of the signal sequence of E. coli outer membrane protein PhoE in export. Biochim. biophys. Acta, 979, 69-76 .

B o s c h , D . , L e u n is s e n , J . , V e r b a k e l , J . , D e Jo n g , M ., V a n E rp , H. & T o m m assen , J . (1986). Periplasmic accumulation of truncated forms of outer membrane PhoE protein of Escherichia coli K 12 . J . molec. Biol. 189, 449-455.

B o s c h , D ., V o o r h o u t , W. & T o m m assen , J . (1988). Export and localization of N-terminally truncated derivatives of Escherichia coli K12 outer membrane protein PhoE. J . biol. Chem. 263, 9952-9957.

C o l l i e r , D . N ., B a n k a it is , V . A., W e iss , J . B . & B a s s f o r d , P. J . J r (1988). The antifolding activity of SecB promotes the export of the E. coli maltose-binding protein. Cell 53 , 273-283.

C r o o k e , E . & W ic k n e r , W . (1987). Trigger factor: a soluble protein that folds pro-OmpA into a membrane assembly competent form. Proc. natn. Acad. Sci. U.S.A. 84, 5216-5220.

C r o o k e , E ., B r u n d a g e , L ., R ic e , M. & W ic k n e r , W. (1988). ProOmpA spontaneously folds in a membrane assembly competent state which trigger factor stabilizes. EM BO J. 7 , 1831-1835.

D e C o c k , H ., M e e l d ijk , J . , O v e r d u in , P., V e r k l e ij , A. & T o m m a ssen , J . (1989). Membrane biogenesis in E. coli: effects of a secA mutation. (Manuscript submitted).

D e K r u ijf f , B ., C u l l is , P. R ., V e r k l e ij , A. J . , H o p e , M. J . , V an E c h t e l d , C . J . A., T a r a sc h i, T . F . , V an H o o g e v e st , P., K il l ia n , J . A., R ie t v e l d , A. & V an d e r S t e e n , A. T . M. (1985). Modulation of lipid polymorphism by lipid-protein interactions. In Progress in Protein-Lipid Interactions (ed. A. Watts and J . J . H. H. M. D e Pont), pp. 89-142. Amsterdam: Elsevier.

D e V r i je , T ., D e L e e u w , C. & D e K r u u f f , B. (1989). Characterization of the binding of precursor protein PhoE to Escherichia coli inner membrane vesicles and phospholipid liposomes. (Manuscript submitted).

D e V r i je , T . , D e S w a r t , R. L ., D o w h a n , W ., T o m m a ssen , J . & D e K r u ijf f , B. (1988). Phosphatidylglycerol is involved in protein translocation across Escherichia coli inner mem­branes. Nature, Land. 334, 173-175.

D iR ie n z o , J . M ., N a k a m u ro , K . & In o u y ë , M. (1978). The outer membrane proteins of Gram­negative bacteria: biosynthesis, assembly and functions. A. Rev. Biochem. 47 , 481-532.

E m r , S. O ., H e d g p e t h , J . , C l é m e n t , J.-M ., S il h a v y , T . J . & H o fn u n g , M. (1980). Sequence analysis of mutations that prevent export of receptor, an Escherichia coli outer membrane protein. Nature, Lond. 285, 82-85.

E n g e lm a n , D. M. & S t e i t z , T . A. (1981). The spontaneous insertion of proteins into and across membranes: the helical hairpin hypothesis. Cell 23 , 411-422.

F r e u d l , R ., S c h w a r z , H ., D e g e n , M. & H e n n in g , U. (1987). The signal sequence suffices to direct export of outer membrane protein OmpA of Escherichia coli K12. J. Bact. 169, 66-71.

F r e u d l , R . , S c h w a r z , H ., K l o s e , M ., R oa M o vva , N. & H e n n in g , U. (1985). The nature of information, required for export and sorting, present within the outer membrane protein OmpA of Escherichia coli K12. EM BO J. 4, 3593-3598.

H a e c o c k , P. N. & D o w h a n , W. (1987). Construction of a lethal mutation in the synthesis of the major acidic phospholipids of Escherichia coli.J. biol. Chem. 262, 13044-13 049.

H a l l , M. N., S c h w a r t z , M. & S ilh a v y , T . J . (1982). Sequence information within the lamB gene is required for proper routing of the bacteriophage receptor protein to the outer membrane of Escherichia coli K12. J. molec. Biol. 156, 93-112.

H a r d y , S. J . S. & R a n d a l l , L . L . (1989). Biochemical investigation of protein export in Escherichia coli.J. Cell Sci. Suppl. 11, 29-43.

I n o u y e , S . , S o be r o n , X ., F r a n c e sc h in i, T ., N a k a m u ra , K ., It a k u r a , K. & I n o u y e , M . (1982). Role of positive charge on the amino terminal region of the signal peptide in protein secretion across the membrane. Proc. natn. Acad. Sci. U.S.A. 79, 3438-3441.

L in d b e r g , A. A. (1973). Bacteriophage receptors. A. Rev. Microbiol. 27, 205-241.Liss, L . R. & O liv e r , D . B. (1986). Effects of secA mutations on the synthesis and secretion of

proteins in Escherichia coli. Evidence for a major export system for cell envelope proteins. J. biol. Chem. 261, 2299-2303.

M e y e r , D . E ., K r a u s e , E. & D o b b e r s t e in , B. (1982). Secretory protein translocation across membranes. The role of the ‘docking protein’. Nature, Lond. 297, 503-508.

M iy a z a k i, C., K u r o d a , M ., O h b a , A. & S h ib u y a , I. (1985). Genetic manipulation of membrane

Involvement o f lipids in protein export 83

phospholipid composition in Escherichia coli: pgsA mutants defective in phosphatidylglycerol synthesis. Proc. natn. Acad. Sei. U.S.A. 82, 7530—7534.

M o r e n o , F . , F o w le r , A. V ., H a l l , M ., S i lh a v y , T . J., Z a b in , I. & S c h w a r t z , M . (1980). A signal sequence is not sufficient to lead /3-galactosidase out of the cytoplasm. Nature, Land. 286, 356-359.

N esm e y a n o v a , M. A. (1982). On the possible participation of acid phospholipids in the translocation of secreted proteins through the bacterial cytoplasmic membranes. FEBS Lett. 142, 189-193.

O l iv e r , D . (1985). Protein secretion in Escherichia coli. A. Rev. Microbiol. 39, 615-648.O liv e r , D . B . & B e c k w ith , J. (1981). E. coli mutant pleiotropically defective in the export of

secreted proteins. Cell 25, 765-772.T r u n , N . J . & S i lh a v y , T . J. (1989). T h e genetics of protein targetin g in Escherichia coli K12.

Jf. Cell Sei. Suppl. 11, 13-28.V an A l p h e n , L . , V e r k l e ij, A ., L e u n is s e n -B ijv e l t , J . & L u g t e n b e r g , B . (1978). Architecture of

the outer membrane of Escherichia coli. I I I . Protein-lipopolysaccharide complexes in intramem- branous particles, jf. B a d . 134, 1089-1098.

V e r k le i j , A. J. , L u g te n b e r g , E. J. J. & V e r v e r g a e r t , P. H . J. T h . (1976). Freeze-etch morphology of outer membrane mutants of Escherichia coli K12. Biochim. biophys. Acta 426, 581-586.

V l a s u k , G. P ., I n o u y e , S ., It o , H ., I t a k u r a , K . & I n o u y e , M. (1983). Effects of the complete removal of basic amino acid residues from the signal peptide of lipoprotein in Escherichia coli. J. biol. Chem. 258, 7141-7148.

V on H e ijn e , G. (1985). Signal sequences: the limits of variation. J". molec. Biol. 184, 99-105.V on H e ijn e , G. & B l o m b er g , C. (1979). Transmembrane translocation of proteins. The direct

transfer model. Eur.J. Biochem. 97, 175-181.V o o r h o u t , W ., D e K r o o n , T . , L e u n is s e n -B ijv e l t , J., V e r k l e ij , A . J. & T o m m a s s e n , J. (1988).

A c c u m u la tio n o f L a m B -L a c Z h y b r id p ro te in s in in tra c y to p la s m ic m e m b ra n e - l ik e s t r u c tu r e s in Escherichia coli K 1 2 . J. gen. Microbiol. 134, 5 9 9 -6 0 4 .

W a l t e r , P., Ib rah im i, I . & B lo b e l , G . (1981). Translocation of proteins across the endoplasmic reticulum. I. Signal recognition protein (SRP) binds to in vitro assembled polysomes synthesizing secretory proteins. J'. Cell Biol. 91, 545-550. .

W e n g , Q., C h e n , L . & T a i , P. C . (1988). Requirement of heat-labile cytoplasmic protein factors for posttranslational translocation of OmpA protein precursors into Escherichia coli membrane vesicles. J. Bact. 170, 126-131.

W ic k n e r , W . (1980). Assembly of proteins into membranes. Science 210, 861-868.