Protein export in Escherichia coli Kit Johnson, Chris K. Murphy and Jon Beckwith Harvard Medical School, Boston, USA The export of proteins from Escherichia coil has been studied by genetic, biochemical and biophysical techniques. These studies have defined a number of steps in the export pathway and have identified the cellular components required for the translocation process. New information is presented on the function of some of these components. Current Opinion in Biotechnology 1992, 3:481-485 Introduction All living organisms must export proteins synthesized in the cytoplasm to other compartments of the cell. The export pathways of diverse organisms may share comm o n features as each of them face similar physical obstacles to protein export. For instance, the translocation of periplasmic and outer m e m b r a n e proteins across the inner m e m b r a n e of bacteria is analogous to translocation of proteins across the m e m b r a n e of the rough endoplasmic reticulum (RER) of eukaryotic ceils. Initially, protein export in Escherichia coli was characterized genetically. This analysis led to the demonstration of the requirement for an amino-terminal signal sequence and to the discovery of the sec genes, the products of which are required for export across the inner membrane. More recent studies using an in vitro secretion system and in vivo physiological studies have s h o w n that this pathway requires energy from both ATP and the proton motive force (PMF). These approaches have led to impressive progress towards an understanding of the export pathway. Substantial progress has also b e e n m a d e in understanding the process w h e r e b y bacteria such as E. coli secrete proteins into the medium [1] although this area is, for the most part, b e y o n d the scope of this review.

Signal sequences Amino-terminal signal sequences are the single most striking feature that distinguishes exported and cytoplasmic proteins. A signal sequence attached to a normally cytoplasmic protein will direct it to the export pathway, although, in some cases, such hybrid proteins cannot be exported because of folding in portions of the cytoplasmic protein. A signal sequence has three domains: a positively-charged amino-terminal region, a hydrophobic core consisting of 5 to

15 hydrophobic amino acids, and a leader peptidase cleavage site. Biophysical studies of synthetic signal peptides have demonstrated that they insert spontaneously into membrane-mimetic environments, w h e r e they adopt an ~x-helical conformation [2]. Using mutant signal peptides that reduce export in vivo, Gierasch and co-workers have s h o w n that both m e m b r a n e insertion and 0~-helical structure are correlated with signalpeptide function [3"]. One particularly revealing mutant left the conformation of the signal peptide largely unaltered, yet greatly reduced its ability to insert into m e m branes [4"]. This find implies that the ability of a signal peptide to interact with m e m b r a n e s is crucial for its in vivo function. It is suggested that the signal peptide directs secretory proteins to the inner membrane, w h e r e interaction with the m e m b r a n e - b o u n d Sec proteins can occur b y diffusion within the plane of the membrane. Several studies have emphasized the importance of the mature region adjoining the signal sequence. Yet, this region has b e e n s h o w n to have no effect u p o n the structure and membrane-binding activity of its preceding signal peptide, raising the possibility that the early portion of the mature region is a passive spacer selected not to interfere with the signal sequence [5"q. The presence of positively charged amino acids within a sharply defined area early in the mature region can interfere with export [6]. These observations d e m o n strate the importance of considering the potential effect that the early mature region m a y have u p o n the export of chimeric proteins.

Lipids m friend or foe? The lipid bilayer constitutes the major barrier to protein export. Crossing this bilayer requires an elaborate cellular machinery and a substantial supply of energy. Lipids are usually considered a passive impediment to the export process but the negatively-charged lipids,

Abbreviations MBP--maltose-bindingprotein; PG--phosphatidylglycerol;PMF--proton motive force; RBP--ribose-bindingprotein; RER---roughendoplasmicreticulum; SRP--signal recognitionparticle. © Current Biology Ltd ISSN 0958-1669

481

482 Expressionsystems however, specifically phosphatidylglycerol (PG) in E. coli, play an active role in the export pathway in vivo. The insertion of SecA [7",8"] and signal sequences [9,10-] into m e m b r a n e s is d e p e n d e n t on PG in vitro. De Kruijff and co-workers [7"] have suggested that the signal peptide interacts specifically with PG in the membrane, and that this interaction causes the signal peptide to adopt an a-helical conformation and insert into the bilayer.

Protein folding, secretion and cytoplasmic chaperones Proteins that are not rapidly exported from the cytoplasm can leave the export pathway b y being degraded, b y folding, or b y precipitating into an exportincompetent state. In 1986 Randall and Hardy s h o w e d that w h e n maltose-binding protein (MBP) is retained in the cytoplasm, its loss of export c o m p e t e n c e is correlated with folding into a protease-resistant conformation. Recently, Teschke et al. [11-'] used this finding to design a selection technique for mutants of ribosebinding protein (RBP) that fold slowly. Starting with a signal-sequence mutant that resulted in slow export of RBP, they selected mature-region mutants that allowed more efficient export of RBP. Using tryptophan fluorescence, they showed that these mutations slowed folding of RBP up to 13-fold. This system, and that previously described for MBP, m a y be superb subjects for genetic approaches to protein folding. While MBP and RBP clearly cannot be exported if they fold into a protease-resistant conformation, the question of what constitutes an e x p o r t q n c o m p e t e n t state remains. Specifically, h o w folded can a protein be and still be exported? Reed and Cronan [12] s h o w e d that a biotinated polypeptide chain can be translocated across the cytoplasmic m e m b r a n e by the Sec system. The biotination domain consists of 89 amino acids that must fold before the post-translational addition of biotin. The secretory apparatus can export this domain in an u n k n o w n manner, possibly by causing it to unfold during the process. Certain exported proteins require cytoplasmic chaperones for their export. One such chaperone is SecB, which interacts with the mature region of a protein such as MBP to maintain its export c o m p e t e n c e and to p r o m o t e its efficient targeting to the secretory a p p a ratus. The relative importance of these two functions of SecB m a y vary for each e x p o r t e d protein. It has b e e n s h o w n recently by de Cock and Tommassen [13"] that SecB binding to pre-PhoE does not maintain its export competence, and is thus required primarily for rapid entry into the secretion pathway. The secretion defect in secB mutants can be c o m p e n sated for b y induction of the heat shock r e s p o n s e [14"']. It is p r e s u m e d that other chaperones induced b y heat shock can replace the missing SecB chaperone. Whether heat shock proteins prevent the folding

of normally SecB-dependent proteins in this case or promote their targeting to the export pathway, is not known.

The secretion machinery Genetic studies have revealed six proteins required for secretion in vivo: SecA, SecB, SecD, SecE, SecF and PrlA/SecY. Studies with i n vitro translocation systems have confirmed the importance of SecA and SecB. In addition, two laboratories have demonstrated the complete d e p e n d e n c e of such systems u p o n SecY and SecE [15-18], despite a previous report of a SecY-independent system [19]. In addition, a third protein, Band 1, which w a s not identified b y genetic approaches, is found in the SecY-SecE c o m p l e x [18]. No role for SecD or SecF has yet b e e n demonstrated in vitro. This inconsistency b e t w e e n the in vivo and in vitro results could mean either that these proteins play a less direct role in the export process or that the in vitro systems are not entirely replicating the i n vivo process. For instance, it is not clear whether such in vitro systems can catalyze multiple rounds of translocation, w h e t h e r they are as efficient at export as in vivo systems, or whether they measure the entire export process. The two best characterized c o m p o n e n t s of the secretion machinery are SecB (described above) and SecA. SecA is the translocation ATPase, and undergoes a cycle of m e m b r a n e binding and dissociation during prorein export. The high affinity m e m b r a n e binding of SecA requires PG, SecY and SecE [8"], and is inhibited b y ATP [7"]. According to a model for the SecA binding cycle based u p o n the effects of analogs of ATP that cannot be hydrolyzed, a SecA-precursor-ATP complex binds the membrane, and ATP hydrolysis facilitates release of the precursor proteins from SecA and partial translocation into the m e m b r a n e [7",8°]. The membrane-binding region of SecA has b e e n m a p p e d in vivo to its amino-terminal quarter [20"q. This region is highly conserved (70% identical) in the Bacillus subtilis secA homologue, divA, suggesting that it is crucial for the function of SecA [21,22]. Given the central role of SecA in protein export, it is not surprising that its expression is regulated according to the secretion needs of the cell. Oliver and co-workers [23] have provided evidence that regulation occurs at the level of translational initiation of the secA gene. Recent in vitro studies suggest that SecA itself m a y bind directly to its mRNA [23]. On the basis of sequence inspection, Koonin and Gorbalenya [24] p r o p o s e that SecA may b e an RNA helicase. SecE is a small integral m e m b r a n e protein containing three transmembrane stretches. Although this protein is essential for protein export in E. coli (CK Murphy, unpublished data), only a single transmembrane stretch and a small cytoplasmic segment are necessary for its function [25"]. In contrast to SecA, SecE is not regulated b y the secretion needs of the cell.

Protein export in Escherichia cofilohnson, Murphy and Beckwith 483 In the case of SecY, two of its ten transmembrane stretches are highly conserved in SecY homologs from Cryptomonas plastid DNA, C h l a m i d i a t r a c h o m a tis, Lactococcus lactis, M e t h a n o c o c c u s vanniellii, and Bacillus subtilis, and m a y constitute important functional regions of SecY [26-29]. The M. vannielli and B. subtilis homologs can c o m p l e m e n t E. coli secYts mutants. Future analysis of SecD, SecE and SecF homologues m a y shed light on the role and the important features of these proteins. It has b e e n p r o p o s e d that there is a bacterial signal recognition particle (SRP), comprising at least the 4.5S RNA and a h o m o l o g u e of the 54 kd subunit of m a m malian SRPs. Brown [30"] has reviewed the evidence suggesting that the primary role of 4.5S RNA is in protein synthesis and has raised questions about a possible role in protein export.

The role of the proton motive force Recent in vitro experiments suggest that ATP hydrolysis b y m e m b r a n e - b o u n d SecA allows the initial translocation into the membrane, and that translocation may be completed either by the PMF, or, in its absence, b y continued ATP hydrolysis by SecA. A reversal of the polarity of the PMF [31"] or elimination of the PMF and SecA [32 °] causes reversed translocation of m e m b r a n e - s p a n n i n g translocation intermediates. These results suggest that the PMF provides b o t h energy and polarity to m e m b r a n e translocation.

The other side: folding in the periplasm The newly exported protein faces a harsh environment in the periplasm. If it folds slowly, it m a y be degraded by periplasmic proteases, or it may aggregate and precipitate. Therefore, it is reasonable to expect that there may be periplasmic chaperones that protect these proteins from proteases, catalyze their folding, and prevent non-specific associations. One such protein has b e e n described in the yeast S a c c h a r o m y c e s cerevisiae, the hsp70 homologue, BiP. BiP is located on the lumen of the RER and plays an important part in translocation across the RER m e m b r a n e [33]. The most intensively studied periplasmic chaperones in bacteria are those involved in the assembly of pili. The PapD pilin chaperone of E. coli is the prototype of this growing family, and has been purified and crystallized. PapD contains a binding cleft that is structurally similar to the antigen-binding fold of immunoglobulins. Conserved amino acids in this family of proteins are located within this binding cleft and in structurally important internal regions. This binding cleft is believed to recognize the conserved carboxyl terminus of pilin subunits. Variable residues are found in loops analogous to the immunoglobulin hypervariable

regions, and are implicated in the binding specificity of these chaperones. It is possible that these proteins m a y be descendants of the primordial immunoglobulin ancestor [34"']. The ability to purify these chaperones w h e n complexed with their pilin subunits has facilitated biochemical studies on their function and mechanism of action. PapD and FaeE have b e e n s h o w n to bind to their pilin subunits in a 1:1 ratio, to prevent both subunit aggregation and degradation. Interestingly, studies on the pilin-chaperone complex have demonstrated that the pilin subunit is in an almost native conformation. The pilin subunit is released from the chaperone in an ATP-independent manner prior to incorporation into the pilus, probably u p o n interaction with an outerm e m b r a n e protein [35",36"]. A second recently discovered class of periplasmic chaperones is the DsbA family of disulfide oxidases [37"-39"]. DsbA facilitates disulfide-bond formation in the periplasm of E. coli, so that in its absence, disulfide b o n d s only form very slowly. Disulfide b o n d formation is crucial for the stability of alkaline phosphatase and in the dsbA mutant, alkaline phosphatase is degraded b y periplasmic proteases. In dsbA mutants, however, despite the fact that alkaline phosphatase is not properly folded in the periplasm, its export into that compartm e n t is not affected.

Implications for biotechnology The field of chaperones, both cytoplasmic and periplasmic, may have a significant impact on biotechnology. These proteins m a y aid the folding of proteins in the cytoplasm or periplasm, promote their entry into the general export pathway, facilitate their export from the cytoplasm, and alleviate lethality caused by overexpression. It seems likely that more such proteins will b e discovered. Finally, two systems h a v e b e e n d e v e l o p e d for the export of heterologous proteins to the cell surface or extracellular milieu. O n e such system is based u p o n the hemolysin export system, and allows excretion of ~-galactosidase, chloramphenicol acetyltransferase and prochymosin to the extracellular medium [40]. Another system allows the quantitative localization of the normally periplasmic ]t-lactamase to the bacterial cell surface [41"]. These systems may not w o r k for every protein, but do increase the n u m b e r of alternatives available.

Acnowledgments This work was supported by a grant from the National Institute of General Medical Sciences and an American Cancer Society Research Professorship to Jon Beckwith, and by a training grant to Kit Johnson, and a postdoctoral fellowship (Chris K Murphy) from the National Institute of General Medical Sciences.

484

Expression systems

References and recommended reading Papers of particular interest, published with the annual period of review, have been highlighted as: of special interest "" of outstanding interest 1.

LORY S: D e t e r m i n a n t s o f E x t r a c e l l u l a r P r o t e i n Sec r e t i o n i n G r a m - n e g a t i v e Bacteria. J Bacteriol 1992, 174:3423-3428.

2.

MCKNIGHTCJ, RAFALSKI M, GIERASCH LM: F l u o r e s c e n c e A n a l y s i s o f T r y p t o p h a n - c o n t a l n i n g v a r i a n t s o f the L a m B Signal S e q u e n c e u p o n I n s e r t i o n i n t o a Lipid Bilayer. Biochemistry 1991, 30:6241-6246.

3.

HOYT DW, GIERASCHLM: H y d r o p h o b i c C o n t e n t a n d Lipid Interactions of Wt and Mutant OmpA Signal Peptides C o r r e l a t e W i t h T h e i r I n Vtvo F u n c t i o n . Biochemistry 1991, 30:10155-10163. The Gierasch group has demonstrated that t h e biophysical properties of mutant signal peptides correlate with their in vivo function. Such studies increase our understanding of the role of the signal peptide in protein export. HOYT DW, GIERASCH LM: A P e p t i d e C o r r e s p o n d i n g to a n E x p o r t - d e f e c t i v e M u t a n t O m p A S i g n a l Sequence w i t h A s p a r a g i n e i n t h e H y d r o p h o b i c Core is Unable to I n s e r t i n t o M o d e l M e m b r a n e s . J Biol Chem 1991, 266:14406-14412. The substitution of Ash in the hydrophobic core of the O m p A signal s e q u e n c e causes complete loss of export in vivo. This mutation leaves t h e conformational properties of the signal s e q u e n c e largely unaltered, yet renders it unable to interact with membranes, suggesting that this interaction is essential in vivo.

11. •"

TESCHKECM, KIM J, SONG T, PARK S, PARK C, RANDALLLL: Mutations that A f f e ~ t h e Folding o f Ribose-binding P r o t e i n Selected as S u p p r e s s o r s o f a Defect i n E x p o r t i n E s c b e r i e h i a c o i l J Biol Chem 1991, 266:11789-11796. The authors describe a selection w h i c h should provide a m e a n s of isolating protein folding mutations in m a n y proteins. 12.

13.

DE COCK H, TOMMASSENJ: See.B-binding D o e s N o t M a i n t a i n t h e T r a n s l o c a t i o n - c o m p e t e n t State of P r e P h o E . Mol Micro 1992, 5:599-604. A direct demonstration that not all proteins that require SecB for their export remain in an export-competent state w h e n b o u n d to SecB. Thus for pre-PhoE, the primary role of SecB is as a targeting factor. 14. •"

ALTMANE, KUMAMOTO C, EMR SD: Heat S h o c k P r o t e i n s C a n Substitute for SecB F u n c t i o n D u r i n g P r o t e i n Exp o r t i n E s c b e r i c h i a coll. EMBO J 1991, 10:239-245. A genetic demonstration that s o m e u n k n o w n heat shock proteins can substitute for SecB in vivo. Further analysis of these s u p p r e s s o r mutations should lead to the identification of these proteins. 15.

AKIMARUJ, MATSUYAMAS, TOKUDA H, MIZUSHIMA S: Reconstitution o f a P r o t e i n T r a n s l o c a t i o n S y s t e m C o n t a i n i n g P u r i f i e d SecY, SecE, a n d SecA f r o m E $ c h e r i c h i a c o i l Proc Nail Acad Sci USA 1991, 88:6545-6549.

16.

NISHIYAMA K, KABUYAMA Y, AKIMARU J, MATSUYAMA S, TOKUDA H, MIZUSHIMA S: SeeY is a n I n d i s p e n s a b l e C o m p o n e n t o f t h e Protein Secretory M a c h i n e r y o f E $ c h e r i c h i a coll. Biochim Btophys Acta 1991, 1065:89-97.

17.

TOKUDA H, AKIMARU J, MATSUYAMA S, NISHIYFAMA K, MIZUSHIMA S: P u r i f i c a t i o n o f SecE a n d R e e o n s t i t u t i o n o f SecE-dependent P r o t e i n T r a n s l o c a t i o n Activity. FEBS Lett 1991, 279:233-236.

18.

BRUNDAGE L, FIMMEL CJ, MIZUSHIMA S, WICKNER W: SecY, SecE , and Band 1 F o r m t h e M e m b r a n e - e m b e d d e d D o m a i n o f E s c h e r i c h i a coil P r e p r o t e i n Translocase. J Biol Chem 1992, 267:4166-4170.

19.

WATANABEM, NICCHITrA CV~ BLOBEL G: R e c o n s t i t u t i o n o f P r o t e i n T r a n s l o c a t i o n f r o m Detergent-solubilizes E s c b e r i c h i a coli I n v e r t e d Vesicles: PrlA P r o t e i n Deficient Vesicles Efficiently Translocate Precursor Proteins. Proc Nail Acad Sci USA 1990, 87:1960-1964.

4.

McKNIGHT CJ, STRADLEYSJ, JONES J n , GIERASCH LM: C o n f o r m a t i o n and M e m b r a n e B i n d i n g P r o p e r t i e s o f a S i g n a l Sequence are L a r g e l y U n a l t e r e d b y its Adjacent M a t u r e Region. Proc Nail Acad Sci USA 1991, 88:5799-5803. The conformation of a signal peptide is not changed w h e n adjacent to a 28-amino-acid s e g m e n t of its mature region. The authors suggest that the first part of the mature region of m o s t exported proteins has b e e n selected to be a passive spacer that will not interfere with the role of the signal sequence.

5. •"

6.

ANDERSSONH, VON HEIJNE G: A 3 0 - R e s i d u e - l o n g E x p o r t I n i t i a t i o n D o m a i n Adjacent to t h e Signal S e q u e n c e is Critical f o r P r o t e i n T r a n s l o c a t i o n Across t h e I n n e r Membrane of E s c h e r i c h i a coli. Proc Nail A c a d Sci USA 1991, 88:9751-9754.

REED RE, CRONAN JE: E s c h e r i c h i a coil E x p o r t s Previo u s l y Folded and Biotinated Protein D o m a i n s . J Biol Chem 1991, 266:11425-11428.

BREUKINKE, DEMEL RA, DE KORTE-KOOL G, DE KRUIJFF B: SecA Insertion into Phosphoh'pids is S t i m u l a t e d b y Negatively C h a r g e d Lipids a n d Inhibited b y ATP: A M o n o l a y e r Study. Biochemistry 1992, 31:1119-1124. An investigation of the SecA membrane-binding cycle w h i c h d e m o n strates that this step requires PG a n d is inhibited by ATP. A model for the SecA cycle and ATP hydrolysis is presented.

CABELLIRJ, DOLAN KM, QIAN L, OLIVER, DB: Characterization o f Membrane-associated and Soluble States o f SecA Protein F r o m Wild-type a n d secASl(Ts) Mutant S t r a i n s o f Escherichia colt. J Biol Chem 1991, 266:24420-24427. The authors investigate the SecA membrane-binding cycle in vtvo, a n d m a p the membrane-binding d o m a i n to the amino-terminal quarter of SecA. Interestingly, this d o m a i n can c o m p l e m e n t the secA51 (Ts) mutationi in vivo, a n d is highly conserved in the B. subtilis SecA homologue.

8.

21.

SADAIEY, TAKAMATSUH, NAKAMURAK, YAMANEK: S e q u e n c i n g R e v e a l s S|m|l~rity o f the W i l d - t y p e div + G e n e o f Bacillus subtilis to the E s c b e r i c h i a coli secA Gene. Gene 1991, 98:101-105.

22.

OVERttOFFB, KLEIN M, SPIES M, FREUDL R: I d e n t i f i c a t i o n of a Gene Fragment W h i c h Codes for t h e 364 Amltxo-ter~ m | ~ o Acid Residues o f a SecA H o m o l o g u e f r o m Bacillus subtilis: Further Evidence for t h e Conservat i o n o f t h e Protein Export Apparatus in Gram-positive and Gram-negative Bacteria. Mol Gen Genet 1991, 228:417-423.

23.

DOLANKM, OLIVER DB: C h a r a c t e r i z a t i o n o f E s c h e r i c h i a coli SecA P r o t e i n B i n d i n g to a Site o n its m R N A Inv o l v e d i n A u t o r e g u l a t i o n . J Biol Chem 1991, 266:2332923333.

24.

KOONIN EV, GORBALENYA AE: A u t o g e n o n s T r a n s l a t i o n R e g u l a t i o n b y E s c b e r i c h i a coli ATPase SecA m a y b e

7.

HENDRICKJP, WICKNERW: SecA P r o t e i n Needs B o t h Acidic P h o p h o l i p l d s a n d SecY/E P r o t e i n for Functional Highaffinity B i n d i n g t o t h e E s c h e r i c b i a coil P l a s m a Membrane. J Biol Chem 1991, 266:24596-24600. The authors demonstrate that high affinity binding of SecA to vesicles requires Sec¥, SecE, a n d PG. 9.

KUSTERSR, DOWHAN W, DE KRUIJFF B: N e g a t i v e l y C h a r g e d P h o s p h o l i p i d s Restore PrePhoE Translocation Across Phosphatidylglycerol-depleted E s c h e r i c b i a coli I n n e r Membranes. J Btol Chem 1991, 266:86594662.

KELLERRCA, KILLIANJA, DE KRUIJFFB: A n i o n i c P h o s p h o lipids are Essential For m-Helix Formation o f t h e Sign a l Peptide o f PrePhoE U p o n I n t e r a c t i o n w i t h PhosphoHpid Vesicles. Biochemistry 1992, 31:1672-1677. The s e c o n d role for PG in protein export is described - to facilitate alpha helix formation b y the signal peptide w h e n it interacts with the m e m b r a n e . 10.

20. •"

Protein export in Escherichia coli]ohnson, Murphy and Beckwith 485 M e d i a t e d b y a n I n t r i n s i c RNA H e l i c a s e Activity o f t h i s P r o t e i n . FEBS Lett 1992, 298:6-8. SCHATZPJ, BIEKER KL, OTFEMANNKM, SILHAVYTJ, BECKW1TH J: O n e o f T h r e e T r a n s m e m b r a n e S t r e t c h e s is S u f f i c i e n t f o r t h e F u n c t i o n i n g o f t h e SecE P r o t e i n , a M e m b r a n e Component o f t h e E. coli S e c r e t i o n M a c h i n e r y . EMBO J 1991, 10:1749-1757. A surprising observation that a single transmembrane stretch a n d a small cytoplasmic d o m a i n of SecE can function in vivo. 25.

26.

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27.

KAUL R, GRAY GJ, KOEHNCKE NR, GU L: C l o n i n g a n d Sequence Analysis of the Chlamydia trachomatis s p c R i b o s o m a l P r o t e i n G e n e Cluster. J Bact 1992, 174:1205-1212.

28.

KOIVULAT, PALVA I, HEMILIAE H: N u c l e o t i d e S e q u e n c e o f t h e S e c Y G e n e f r o m L a c t o c o c c u s lactis a n d Identific a t i o n o f C o n s e r v e d Regions b y C o m p a r i s o n o f F o u r SecY" P r o t e i n s . FEBS Lett 1991, 288:114-118.

29.

AUERJ, SPICKER G, BOECK A: P r e s e n c e o f a G e n e i n t h e A r c h a e b a c t e r i u m M e t h a n o c o c c u s vannielii Homologous to S e c Y o f Eubacteria. Btochtmie 1991, 73:683-688.

35.

KUEHN MJ, NORMARK S, HULTGREN SJ: I m x n u n o g l o b u l i n like P a p D C h a p e r o n e C a p s a n d U n c a p s I n t e r a c t i v e S u r f a c e s o f N a s c e n t l y T r a n s l o c a t e d P i l u s S u b u n i t s . Proc Natl Acad Sci USA 1991, 88:10586-10590. Pilin (PapG) b o u n d to PapD resembles native PapG, b a s e d u p o n the maintenance of its receptor-binding ability w h e n c o m p l e x e d to PapD. The PapD-PapG c o m p l e x is extremely stable u n d e r non-reducing conditions. BAKKERD, VADER CEM, ROOSENDAALB, MOOI FR, OUDEGA, B, DEGRAAF FK: S t r u c t u r e a n d F u n c t i o n o f P e r i p l a s m i c C h a p e r o n e - l i k e P r o t e i n s I n v o l v e d i n t h e Biosynthesis o f K88 a n d K 9 9 F i m b r i a e i n E n t e r o t o x i g e n i c E$cherichia c o i l Mol Micro 1991, 5:875-886. A convincing demonstration that the pilin subunit b o u n d to FaeE (a PapD homologue) is in a nearly native conformation, judged by its ability to be recognized by monoclonal antibodies specific for native pilin. 36.

37.

BARDWELLJCA, McGOVERN K, BECKWITH J: I d e n t i f i c a t i o n of a Protein Required for Disulfide Bond Formation I n Vivo. Cell 1991, 67:581-589. T h e first demonstration that disulfide-bond formation is catalyzed in vivo. Studies o n an E. coli system.

30.

BROWNS: 4.5S RNA: D o e s F o r m P r e d i c t F u n c t i o n ? The New Biologist 1991, 3:430-438. Summary of information available o n the function of 4.5S RNA.

KAMITANIS, AKIYAMAY, ITO K: I d e n t i f i c a t i o n a n d C h a r a c t e r i z a t i o n o f a n E s c h e r i c h i a coli G e n e R e q u i r e d for t h e F o r m a t i o n o f C o r r e c t l y F o l d e d A l k a l i n e P h o s p h a t a s e , a P e r i p l a s m i c E n z y m e . E M B O J 1992, 11:57--62. Identification of the same system as in [36"].

31.

39.

DmESSENAJM: P r e c u r s o r P r o t e i n T r a n s l o c a t i o n b y t h e E s c h e r i c h i a coli T r a n s l o c a s e is D i r e c t e d b y t h e P r o t o n Motive Force. EMBO J 1992, 11:847-853. The author describes a method for controlling both the magnitude a n d the direction of the PMF, to further reveal its role in protein export. 32.

SCHIEBELE, DRIESSEN AJM, HARTL FU, WICKNER W: Ap,l t + a n d ATP F u n c t i o n at Different Steps o f t h e C a t a l y t i c Cycle o f P r e p r o t e i n T r a n s l o c a s e . Cell 1991, 64:927-939. The in vitro system devised by Wickner and co-workers h a s proved to be capable of a detailed analysis o f the steps in protein export. In this paper, the different roles of ATP a n d the PMF are convincingly elucidated.

38.

PEEKJA, TAYLOR RK: C h a r a c t e r i z a t i o n o f a P e r i p l a s m i c Thiol:Disulfide Interchange Protein Required for the F u n c t i o n a l M a t u r a t i o n o f S e c r e t e d V i r u l e n c e Factors o f Vibrio cholerae. Proc Natl A c a d Sci USA 1992, 89:6210-6214. Identification of a disulfide-bond forming system in Vibrio cholerae similar to that in E. coB. 40.

KENNY B, HAIGH R, HOLLAND IB: A n a l y s i s o f t h e Haemolysin T r a n s p o r t P r o c e s s T h r o u g h t h e S e c r e t i o n f r o m E s c h e r i c h t a coil o f PCM, CAT o r ~ - G a l a c t o s i d a s e F u s e d to t h e H l y C - t e r m i n a l S i g n a l D o m a i n . Mol Micro 1991, 5:2557-2568.

41.

33.

SANDERSSL, WHITFIELD KM, VOGEL JP, ROSE MD, SCHEKMAN RW: Sec61p a n d BiP D i r e c t l y Facilitate Polypeptide T r a n s l o c a t i o n i n t o t h e E n d o p l a s m i c R e t i c u l u m . Cell 1992,-69:353-365.

HOLMGRENA, KUEHN MJ, BRANDEN CI, I-IULTGREN SJ: Cons e r v e d I m m u n o g l o b u l i n - l i k e F e a t u r e s i n a Family o f P e r i p l a s m i c Pilus Chaperones i n Bacteria. E M B O J 1992, 11:1617-1622. This work demonstrates the p o w e r of combining a phylogenetic analysis with a crystal structure to d e d u c e important structural features of proteins, in this case the PapD family of periplasmic chaperones. 34. ""

FRANCISCOJA, EARHART CF, GEORGIOU G: T r a n s p o r t a n d Anchoring of ~-Lactamase to the External Surface o f E s c h e r i c h i a c o i l Proc Natl A c a d Sci USA 1992, 89:2713-2717. T h e authors were able to quantitatively translocate and anchor Blactamase to the cell surface of E. coli by constructing a tripartite fusion protein of Lpp, O m p A a n d B-lactamase.

K Johnson, CK Murphy a n d J Beckwith, Department of Microbiolo g y and Molecular Genetics, Harvard Medical School, 200 Longwood Ave, Boston, MA 02115, USA.