Protein secretion in bacteria

Protein secretion in bacteria

Protein secretion in bacteria Joseph M. Gennity and Masayori Inouye University of Medicine and Dentistry of New Jersey, Piscataway, New Jersey, USA Mo...

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Protein secretion in bacteria Joseph M. Gennity and Masayori Inouye University of Medicine and Dentistry of New Jersey, Piscataway, New Jersey, USA Most secretory proteins are synthesized as precursors with an amino-terminal signal peptide. Genetic identification of proteins essential for signal peptide dependent translocation to the Escherichia coil periplasm has led to the biochemical dissection of the secretion pathway. Additional mechanisms exist in Gram-negative bacteria for protein secretion to the extracellular environment. Current Opinion in Biotechnology 1991, 2:661-667

Introduction Living organisms are confronted by a basic problem of cell biology. In order to carry out certain essential functions, large hydrophilic protein molecules must in some manner be translocated across the rather impermeable hydrophobic barrier that is the lipid bilayer of the cell membrane. The subject of this review will be the two basic mechanisms developed by bacteria to accomplish protein secretion. The major, and consequently better studied pathway, uses an amino-terminal protein extension known variously as the signal peptide or leader peptide sequence to direct proteins into the secretion pathway. The secreted protein is synthesized as a cytoplasmic precursor and the signal peptide is proteolytically removed during or immediately following membrane transfer. Such signal peptide dependent translocation across the cytoplasmic membrane into the periplasm of Gramnegative bacteria requires a common set of proteins referred to here as the Sec system. The secretion of proteins across the outer membrane of Gram-negative bacteria to the extracellular environment occurs by a Sec-independent pathway. This review will cover significant recent additions to our understanding of this process. Other reviews of bacterial protein secretion have been published in the past year [1--4]. In addition, the entire June issue of The Journal of Bioenergetics and Biomembranes [5] and the February-March issue of Biochimie [6] have been devoted to this topic.

Signal peptide dependent secretion by the Sec system The proteins required for signal peptide dependent secretion were originally identified through the isolation of mutants in E. coli that were either conditionally defective in the secretion of proteins that exert toxic effects when channeled into the export pathway or were able to suppress the defective secretion of precursor proteins har-

boring a signal peptide mutation [1]. This seminal work has led to the identification of the sec genes (sec,4/prlD, secB, secD, secE/prlG, secF, and secY/prlA) essential for secretion and the purification of their protein products. Current endeavors are focused on the in vitro molecular dissection of the secretion process.

Genetic characterization Identified as cold-sensitive mutations which pleotropically inhibit protein secretion, the secD locus has been shown by complementation analysis to contain at least two genes (secD and secF) that comprise an operon and are thought to encode integral membrane proteins [7"]. Alkaline phosphatase fusion experiments support the predicted membrane localization. These are the only sec genes known to be organized into an operon. Mutations that confer resistance to azide (azi) have been shown to be in alleles of secA [8,]. Azide was shown to inhibit protein secretion in vivo, SecA-dependent in vitro translocation and SecA ATPase activity, while the azi mutant or its SecA was resistant. It is likely that azide directly inhibits SecA by binding to an ATP-binding site (see discussion below). A genetic analysis of existing prL4 (seclO alleles indicates that expression of mutant SecY from the prlA gene in a genetic background containing reduced quantities of SecE, but normal levels of wild-type SecY, can block secretion [9]. Interference from the prlA locus is believed to be caused by titration of SecE by the defective SecY protein, indicating a direct interaction between these proteins during secretion. This result is consistent with the observation that SecY and SecE copurify (see discussion below).

Biochemical characterization The recent in vitro reconstitution of secretion activity from detergent-solubilized inner membrane vesicles

Abbreviations MBP--maltose-binding protein; MDR--multiple drug resistance; pmf--proton motive force; SRP--signal recognition particle.

(~ Current BiologyLtd ISSN0958-1669

661

662 Expressionsystems [10-12] has enabled the molecular characterization of the proteins involved in translocation to be characterized (see [13]). Wickner and collaborators [14..] have isolated a complex of three proteins, SecY, SecE and an unidentified third component, protein 1, that can reconstitute proOmpA secretion in a SecA- and ATP-dependent manner into phospholipid vesicles. SecA mediates binding of a SecB-proOmpA complex to the membrane SecY/E complex [15]. There are independent SecB and proOmpA binding sites on SecA. Both the signal peptide and mature region of the proOmpA participate in Seca recognition [16]. Proton motive force (pmf) is not required for SecA binding to the membrane, but both the ApH and A~ components stimulate translocation [17]. The ATP-dependent accumulation of a membrane bound proOmpA translocation intermediate occurs in the absence of membrane potential. Subsequent addition of membrane potential supports intermediate translocation. This step is inhibited by deuterium oxide (D20) suggesting that proton transfer is a rate limiting step.Many of these observations have been independently confirmed by Mizushima and coworkers [18,]. They have shown that a proOmpA translocation intermediate containing an intramolecular disulfide bridge accumulates in membrane vesicles in an ATP-dependent manner. Either the ApH or A~ component of the pmf is required for subsequent translocation. This interesting finding indicates that proteins do not have to be completely unfolded during translocation. The pmf (ApH or A~t) lowers the ATP requirement for translocation of a hybrid precursor [19]. The conformation of SecA was altered by interactions with ATP or its non-hydrolyzable analogues, and by proOmpA in a manner that includes the signal peptide, and by acidic phospholipids [20]. Cross-linking experiments have defined two regions of SecA required for ATP and precursor (but not mature) protein binding [21].

Chaperone proteins The SecB and GroEL proteins maintain different subsets of precursor proteins in a conformation capable of effective interaction with the translocation machinery. As such, they are recognized as belonging to a larger family of proteins, known as chaperones, which exert antifolding activity. The role of the signal peptide during recognition of precursor proteins by SecB is currently under debate. I n vitro translocation experiments using a truncated precursor of maltose-binding protein (MBP) have indicated that SecB recognizes the signal peptide of precursor proteins [22]. In contrast, studies of preMBP-SecB binding have indicated that the signal peptide is not directly involved [23]. It has now been suggested that SecB binds with high affinity, but low specificity, to a number of sites consisting of unfolded protein sequences [24]. In this model, the signal peptide acts primarily to slow down the folding of the precursor and so to permit SecB interaction with unfolded sequences in the mature region of the protein. This view is supported by evidence that SecB cannot normally form an immunoprecipitable corn-

plex with mature MBP in vitro, but is able to if there is a mutation in the MBP region that slows folding in the absence of the signal peptide [25]. Furthermore, experiments that exchange the signal peptides of MBP and of ribose binding protein, which is secreted independently of SecB, indicate that the mature region of MBP, and not the signal peptide, confers SecB secretion dependence [26]. New findings tend to reconcile these opposing views. Using a series of LacZ fusions to the amino terminus of the outer membrane protein LamB, a region of the mature lamB protein was identified that interfered with secretion in vivo by titrating SecB [27"]. Deletion of this region, however, was not required for efficient LamB secretion which remained SecB dependent. This apparently paradoxical result was explained by the following observations: firstly, both the signal peptide and the mature region of preLamB interacting with SecB were required for stable SecB interaction with preLamB in vitro, secondly, efficient in vivo translocation of a signal peptidedefective prelamB required the SecB-interacting mature region; and thirdly, the specific mature sequence was required for maintenance of preIamB in a translocation competent state in vivo [28.]. SecB-binding to both the signal peptide and a specific site within the mature region of precursor proteins may be required for efficient secretion.

Role of the signal peptide Recent findings stress the importance for optimal secretion rates of the compatibility of the signal peptide and mature region of precursor proteins. The translocation rate of heterologous nuclease A is improved when it is fused to a mutated OmpA signal peptide [29], but there is no effect on homologous OmpA secretion [30]. Thus, the interaction between the OmpA signal peptide and its mature region appears to have evolved to a state of maximal translocation efficiency. A synthetic signal peptidase I cleavage site incorporating a predicted cleavage site structure, was functional in vivo in secretion of downstream sequences when introduced immediately after a hydrophobic sequence of a truncated signal peptidase derivative [31].These studies illustrate the advances in protein engineering of secretory proteins made possible by extensive studies of signal peptide structure designed in accordance with the 'loop model' of signal peptide function [32]. In this model, interaction of the basic amino terminus of the signal peptide is thought to mediate association with acidic phospholipids of the cytoplasmic membrane. This is followed by insertion of the hydrophobic signal sequence core as a loop into the lipid bilayer. This insertion is coincident with conformational changes in the secondary structure of the signal peptide which aid in precursor translocation.

Secretion model In the current model of protein secretion, as illustrated in Fig. 1, proteins containing signal peptides are targeted

Protein secretion in bacteria Gennity and Inouye to the secretion pathway. SecB interacts with certain precursor proteins to retain them in a translocation competent conformation. As SecB is not essential, this step is not required for all precursors. The SecB-precursor complex interacts specifically with membrane associated Sec.A. The membrane association of SecB-independent precursors has not been extensively studied. The precursor is channeled through SecA to the integral membrane protein SecY. At some point in this process ATP is obligately hydrolyzed by an ATPase activity residing in SecA. Translocation of the precursor across the membrane is dependent upon the integral membrane proteins SecY and SecE as well as the membrane potential. The integral membrane proteins SecD and SecF are thought to be required late in the translocation. Cleavage by a signal peptidase removes the signal peptide, probably late in the process, to permit release of the translocated protein to the cell periplasm. Future research will be directed at defining whether secretion occurs through the lipid bilayer, or via a proteinaceous channel. The role of ATP hydrolysis and coupling of translocation to membrane energization must be better defined. The function of the Sec integral membrane

Cytoplasm

f

Is there a prokaryotic particle?

signal recognition

The observation that E. coli harbors two essential proteins and an essential RNA species that are similar to components of the eukaryotic signal recognition particle (SRP) and its membrane docking protein, has led to considerable controversy concerning the possible existence of a prokaryotic SRP and its relevance to protein secretion [33,34]. Thefts gene of E. coli encodes a 4.5S RNA molecule with homology to the 7S RNA component of eukaryotic SRP. This 4.5S RNA is believed to interact with the ribosome and nascent polypeptide chains during translation. The ffh gene product encodes a protein (P48 or Ffh homologous to eukaryotic SRP54. A protein

mRN DDDQDDD~

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proteins and the molecular details of how signal peptide loop formation is integrated with the Sec translocation proteins need to be established. This should be greatly facilitated by the large number of structurally and functionally well defined signal peptide mutations presently available.

C)

C)

z/ (b)

Fig. 1. Signal peptide dependent inner membrane protein translocation. The current model of the Sec-dependent secretion pathway is shown by solid arrows. (a) SecB interacts with certain precursor proteins to retain them in a translocation competent conformation (this step is not required of all precursors). (b) The SecB-precursor complex interacts specifically with membrane associated SecA. (c) Translocation of the precursor across the membrane is dependent upon the integral membrarne protein SecY and SecE, Sec D and Sec F, as well as the membrane potential. A hypothetical signal recognition particle (SRP) dependent pathway is shown by dashed arrows. The hatched rectangle represents the signal peptide of a precursor protein. This model is modified from [33].

663

664 Expressionsystems (FtsY) homologous to the SRP docking protein subunit SR~, is coded by flY, a gene in an operon involved in cell division [35]. Two groups have independently demonstrated a remarkable structural and functional similarity between these prokaryotic gene products and eukaryotic SRP. Ribes et aL [36"] showed that human 7S RNA is able to replace E. coli 4.5S RNA and both RNAs are associated in a nucleoprotein complex with P48 in vivo. Both P48 and SRP54 bind to 4.5S RNA in vitro. Poritz et al. [37"] showed that 4.5S RNA can replace 7S RNA in activation of SRP-SRP receptor GTPase activity in vitro. Deletion of 4.5S RNA [36"'], or production of a dominant mutant 4.5S [37°-], halts cell growth and interferes with prefl-lactamase secretion. This effect on secretion, however, occurred hours after a shift to non-permissive conditions and may not represent a direct effect on secretion. Furthermore, the secretion of several other precursor proteins was unaffected. At present, therefore, there is no compelling evidence to indicate that the 4.5S complex is a prokaryotic SRP participating in secretion. In addition, the secretory proteins thus far examined are all capable of post-translational translocation, indicating that SRP may not be required for their secretion. Nevertheless, the similarity between eukaryotic SRP and the bacterial nucleoprotein complex leaves open the possibility that a prokaryotic SRP participates in the obligate cotranslational secretion of unidentified proteins.

Extracellular protein secretion in Gram-negative bacteria Klebsiella p n e u m o n i a e can secrete the lipid-modified protein pullulanase across the outer membrane into the extracellular environment. The first step in this pathway is the translocation of prepullulanase across the inner membrane in a Sec-dependent manner, as also occurs in E. coli [38]. Once translocated, processed and lipidmodified, exposure of pullulanase on the cell surface of either Klebsiella or E. coli, and subsequent release of the lipid-modified protein, is dependent upon 14 additional Klebsiella gene products. When an 834 residue amino-terminal segment of pullulanase is fused to 13-1actamase, the hybrid pullulanase-13-1actamase molecule can also be secreted to the growth media of E. coli, made secretion competent by the addition of the required Kleb siella genes [39"]. The structural information permitting pullulanase release is therefore located somewhere in a large amino-terminal portion of the molecule. In contrast, the poor secretion of a pullulanase-alkaline phosphatase chimera points to the existence of conformational constraints on effective export.

A similar secretion system appears also to operate in Pseudomonas. Two xcp (extracellular protein) genes from Pseudomonas have recently been found to be highly homologous to Klebsiella genes required for pullulanase secretion [40]. Mutations in the xcp genes, however, cause the periplasmic accumulation of proteins that are normally extracellular, indicating that many are probably not lipid-modified. Indeed, the Pseudomonas

solanacearum extracellular polygalacturonase, although synthesized as a cytoplasmic precursor, is not lipid-modified, and its export across the outer membrane requires the 13 carboxy-terminal residues [41]. The presence of a carboxy-terminal secretion signal in this protein is in marked contrast to the situation with puUulanase. Pseudomonas can also export an endoglucanase lipoprotein in a manner similar to the Iaebsiella puUulanase system [42]. However, after the protein is assembled at the cell surface, 26 residues of the lipid-modified amino terminus are proteolysed to release a soluble protein.

Extracellular toxins can be exported by E. coli without requiring factors in addition to the Sec proteins. These heat-stable enterotoxins (STa and STp) are synthesized as cytoplasmic preproproteins, which are translocated to the periplasm in a Sec-dependent manner and lose their pro-sequence following export to the extracellular environrnent [43]. The pro-sequence of STp is not required for translocation across the outer membrane and when fused to the heterologous protein nuclease A cannot direct it to the growth medium [44]. Therefore, the information required for outer membrane permeation appears to be localized in the enterotoxin carboxyl terminus.

Sec-independent secretion in Gram-negative bacteria E. coli secretes the extracellular protein hemolysin by a signal peptide- and Sec-independent pathway. The outer membrane protein TolC [45], with two additional proteins, HlyB and HlyD, are required for the export, which occurs without processing of the hemolysin. Hemolysin contains a carboxy-terminal secretion signal. The HtyB and HlyD proteins are believed to form a channel in the inner membrane that, in conjunction with TolC, permits hemolysin secretion directly from the cytoplasm to the growth medium without periplasmic intermediate. The HlyB protein is a member of a large family of eukaryotic and prokaryotic proteins with an amino-terminal transmembrane sequence and a carboxy-terminal ATP-binding site, which are involved in membrane transport of a wide variety of molecules - - the multiple drug resistance (mDR)-like family. Erwinia chrysanthemi and a number of other bacteria secrete a variety of extracellular proteins by a pathway that is quite similar to the hemolysin system and also uses an MDR-like protein [46]. Like hemolysin, secretion of a metaUoprotease (PrtB) by this pathway requires a secretion signal present in the 40 carboxy-terminal residues of the protein and three additional p r t gene products, which are homologous to HlyB, HlyD and TolC [47"]. In E. coli expressing the p r t gene products, this carboxy-terminal sequence can direct the extracellular secretion of a fused 200 residue fragment of the normally cytoplasmic protein amylomaltase. Specific conformational requirements for secretion are, however, indicated by the failure of export of other amylomaltase fusions.

Colicin V secretion in E. coli has also been found to require an export system analogous to the hemolysin path-

Protein secretion in bacteria Gennity and Inouye

way [48]. TolC plus two other proteins, one MDR-like, are required for the signal peptide- and Sec-independent secretion of colicin. In this case, however, the secretion signal is localized to the 39 amino-terminal residues of colicin V.

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Implications for biotechnology

GARDELG, JOHNSON K, JACQ A, BECKWITHJ: The secD Locus of E. coil Codes for T w o Membrane Proteins Required for Protein Export. EMBO J 1990, 9:3209-3216. A convincing genetic analysis of the essential secD locus reveals the first identified secretion operon is composed of at least two genes (secD and secF). Sequencing and alkaline phosphatase fusions indicate the genes encode integral membrane proteins.

The molecular characterization of the Sec secretion pathwaywill greatly aid attempts to utilize E. colias an expression system for heterologous proteins. Of particular relevance will be the clarification of signal peptide function. SecB can recognize and bind to proteins lacking signal peptides [24,25] and deliver these proteins to the membrane (P Bassford, personal communication). This association does not, however, lead to secretion. Clearly, the signal peptide plays some additional critical role in secretion. ff this role could be understood, it might be possible to construct a bacterium harboring altered sec alleles capable of Sec-dependent secretion of proteins lacking a signal peptide. Expression vectors employing the OmpA signal peptide have been utilized for secretion of high levels of human growth hormone [49]. The overproduction of proteins secreted to the periplasm can, however, result in aggregation [50] and cell toxicity [30]. Recent observations associated with such overproduction have led to speculation that a periplasmic protein with chaperone-like activity may exist [51]. If such a protein can be identified, its overproduction in expression systems could improve the yidd and stability of secreted proteins. The extracellular export of heterologous proteins in E. coli by either the puUulanase or hemolysin-like pathways offers obvious advantages. Both pullulanase [39"] and PrtB [47] have been used to secrete heterologous proteins to the E. coli growth medium. Although the exported products are by necessity hybrid proteins, it may be possible to overcome this drawback by the inclusion of appropriate protease cleavage sites at the point of fusion.

Acknowledgements

7. •

OLIVERDB, CABELH RJ, DOLAN KM, JAROSIK GP: Azide-resistant Mutants of Escherichia coli Alter the SecA Protein, an Azide-sensitive C o m p o n e n t o f t h e Protein Export Machinery. Proc Natl Acad Sci USA 1990, 87:8227-8231. Azide resistant E. coli mutants originally isolated by J Lederberg have mutations in secA alleles. Secretion and SecA are azide-sensitive in vivo and in vitro, respectively. Azide and azide-resistant SecA can be used to study SecA function in vitro. 8. •

9.

BIEKERKL, SILHAVYTJ: PrlA (SecY) and PriG (SecE) Interact Directly and Function Sequentially During ProteIn Translocation In E. coll. Cell 1990, 61:833-842.

10.

WATANABEM, NICCHrI'rA CV, BLOBELG: Reconstitution of Protein Translocation from Detergent-solubilized Escherichia coli Inverted Vesicles: PrlA Protein-deficient Vesicles Efficiently Translocate Precursor Proteins. Proc Natl Acad Sci USA 1990, 87:1960-1964.

11.

DRIESSENAJM, WICKNERW: SolubiliTation and Functional Reconstitution of the Protein-translocation Enzymes of Escherichia coll. Proc Natl Acad Sci USA 1990, 87:3107-3111.

12.

TOKUDA H, SrnOZUKA K, MLZUSHIMA S: Reconstitution of Translocation Activity for Secretory Proteins from Solubilized C o m p o n e n t s of Escherichia coll. Eur J Biochem 1990, 192:583-589.

13.

HURTLEYSM: Protein Translocation Into Reconstituted Vesicles. Trend Biochem Sci 1990, 15:211-212.

14. *o

BRUNDAGEL, HENDRICKJP, SCHIEBELE, DRIESSENAJM, WICKNER W: The Purified E. coli Integral Membrane Protein SecY/E is Sufficient for Reconstitution of SecA-dependent Precursor ProteIn Translocation. Cell 1990, 62:649-657. Purification of an integral membrane translocation complex consisting of three proteins (SecY, SecE, protein 1) capable of reconstituting SecA and ATP-dependent, pmf-stimulated translocation activity in proteollposomes. Characterization of the function of purified components will be based upon this work. 15.

HARTLFU, LECKER S, SCHIEBEL E, HENDRICKJP, WICKNER W: T h e Binding Cascade of SecB and SecA to SecY/E Mediates Preprotein Targeting to the E. coli Plasma Membrane. Cell 1990, 63:269-279.

16.

LILL R, DOWHAN W, WICKNER W: T h e ATPase Activity of SecA is Regulated by Acidic PhosphoUpids, SecY, and the Leader and Mature Domains of Precursor Proteins. Cell 1990, 60:271-280.

17.

DRIESSENAJM, WICKNER W; Proton Transfer is Rate-limiting for Translocation of Precursor Proteins by t h e Escherichta coli Translocase. Proc Natl Acad Sci USA 1991, 88:2471-2475.

Work in the author's laboratory was supported by a grant from the American Cancer Society (MV65907).

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

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SHIOZUKAK, TANI K, MIZUSHLMAS, TOKUDA H: T h e Proton Motive Force Lowers t h e Level of ATP Required for the

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39. •

KORNACKERMG, PUGSLEYAP: T h e Normally Periplasmic Enzyme [3-1actamase is Specifically and Efficiently Translocated T h r o u g h the Eschertchia coli Outer Membrane W h e n it is Fused to the Cell-surface Enzyme PuUulanase. Mol Microbiol 1990, 4:1101-1109. Pullulanase can direct the extracellular secretion of heterologous proteins in E. coll. 40.

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HUANG J, SCHELL MA: Evidence that Extracellular Export of the Endoglucanase Encoded by egl of Pseu, d o m o n a s solanacearum O c c u r s by a Two-step Process Involving a Lip*protein Intermediate. J Biol Chem 1990, 265:11628-11632.

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