The interaction between the chaperone SecB and its ligands: evidence for multiple subsites for binding.

The chaperone protein SecB is dedicated to the facilitation of export of proteins from the cytoplasm to the periplasm and outer membrane of Escherichia coli. It functions to bind and deliver precursors of exported proteins to the membrane-associated translocation apparatus before the precursors fold into their native stable structures. The binding to SecB is characterized by a high selectivity for ligands having nonnative structure but a low specificity for consensus in sequence among the ligands. A model previously presented (Randall LL, Hardy SJS, 1995, Trends Biochem Sci 20:65-69) to rationalize the ability of SecB to distinguish between the native and nonnative states of a polypeptide proposes that the SecB tetramer contains two types of subsites for ligand binding: one kind that would interact with extended flexible stretches of polypeptides and the other with hydrophobic regions. Here we have used titration calorimetry, analytical ultracentrifugation, and electrospray ionization Fourier transform ion cyclotron resonance mass spectrometry to obtain evidence that such distinguishable subsites exist.     

Substrate specificity of the SecB chaperone

The bacterial chaperone SecB assists translocation of proteins across the inner membrane. The mechanism by which it differentiates between secretory and cytosolic proteins is poorly understood. To identify its binding motif, we screened 2688 peptides covering sequences of 23 proteins for SecB binding. The motif is approximately 9 residues long and is enriched in aromatic and basic residues, whereas acidic residues are disfavored. Its identification allows the prediction of binding regions within protein sequences with up to 87% accuracy. SecB-binding regions occur statistically every 20-30 residues. The occurrence and affinity of binding regions are similar in SecB-dependent and -independent secretory proteins and in cytosolic proteins, and SecB lacks specificity toward signal sequences. SecB cannot thus differentiate between secretory and non-secretory proteins via its binding specificity. This conclusion is supported by the finding that SecB binds denatured luciferase, thereby allowing subsequent refolding by the DnaK system. SecB may rather be a general chaperone whose involvement in translocation is mediated by interactions of SecB and signal sequences of SecB-bound preproteins with the translocation apparatus.     

The binding cascade of SecB to SecA to SecY/E mediates preprotein targeting to the E. coli plasma membrane

The export of many E. coli proteins such as proOmpA requires the cytosolic chaperone SecB and the membrane-bound preprotein translocase. Translocase is a multisubunit enzyme with the SecA protein as its peripheral membrane domain and the SecY/E protein as its integral domain. SecB, by binding to proOmpA in the cytosol, prevents its aggregation or association with membranes at nonproductive sites. The SecA receptor binds the proOmpA-SecB complex (Kd approximately 6 x 10(-8) M) through direct recognition of both the SecB (Kd approximately 2 x 10(-7) M) as well as the leader and mature domains of the precursor protein. SecB has a dual function in stabilizing the precursor and in passing it on to membrane-bound SecA, the next step in the pathway. SecA itself is bound to the membrane by its affinity (Kd approximately 4 x 10(-8) M) for SecY/E and for acidic lipids. The functions of SecB and SecA as a two-stage receptor system are linked by their affinity for each other.     

Preprotein transfer to the Escherichia coli translocase requires the co-operative binding of SecB and the signal sequence to SecA

In Escherichia coli , precursor proteins are targeted to the membrane-bound translocase by the cytosolic chaperone SecB. SecB binds to the extreme carboxy-terminus of the SecA ATPase translocase subunit, and this interaction is promoted by preproteins. The mutant SecB proteins, L75Q and E77K, which interfere with preprotein translocation in vivo , are unable to stimulate in vitro translocation. Both mutants bind proOmpA but fail to support the SecA-dependent membrane binding of proOmpA because of a marked reduction in their binding affinities for SecA. The stimulatory effect of preproteins on the interaction between SecB and SecA exclusively involves the signal sequence domain of the preprotein, as it can be mimicked by a synthetic signal peptide and is not observed with a mutant preprotein (Δ8proOmpA) bearing a non-functional signal sequence. Δ8proOmpA is not translocated across wild-type membranes, but the translocation defect is suppressed in inner membrane vesicles derived from a prlA4 strain. SecB reduces the translocation of Δ8proOmpA into these vesicles and almost completely prevents translocation when, in addition, the SecB binding site on SecA is removed. These data demonstrate that efficient targeting of preproteins by SecB requires both a functional signal sequence and a SecB binding domain on SecA. It is concluded that the SecB–SecA interaction is needed to dissociate the mature preprotein domain from SecB and that binding of the signal sequence domain to SecA is required to ensure efficient transfer of the preprotein to the translocase.     

Involvement of SecB, a chaperone, in the export of ribose-binding protein.

Ribose-binding protein (RBP) is an exported protein of Escherichia coli that functions in the periplasm. The export of RBP involves the secretion machinery of the cell, consisting of a cytoplasmic protein, SecA, and the integral membrane translocation complex, including SecE and SecY. SecB protein, a chaperone known to mediate the export of some periplasmic and outer membrane proteins, was previously reported not to be involved in RBP translocation even though small amounts of in vitro complexes between SecB and RBP have been detected. In our investigation, it was shown that a dependence on SecB could be demonstrated under conditions in which export was compromised. Species of RBP which carry two mutations, one in the leader that blocks export and a second in the mature protein which partially suppresses the export defect, were shown to be affected by SecB for efficient translocation. Five different changes which suppress the effect of the signal sequence mutation -17LP are all located in the N domain of the tertiary structure of RBP. All species of RBP show similar interaction with SecB. Furthermore, a leaky mutation, -14AE, generated by site-specific mutagenesis causes reduced export in the absence of SecB. These results indicate that SecB can interact with RBP during secretion, although it is not absolutely required under normal circumstances.     

SecB, a molecular chaperone with two faces

SecB is a molecular chaperone unique to the phylum Proteobacteria, which includes the majority of known Gram-negative bacteria of medical, industrial and agricultural significance. SecB is involved in the translocation of secretory proteins across the cytoplasmic membrane. The crystal structure of the Haemophilus influenzae SecB provides new insights into how SecB simultaneously recognizes its two ligands: unfolded preproteins and SecA, the ATPase subunit of the translocase. SecB uses its entire molecular surface for these two functions, but for preprotein release and its own membrane release, SecB relies on the catalytic activity of SecA. This defines SecB as a translocation-specific molecular chaperone.     

The molecular chaperone SecB is released from the carboxy-terminus of SecA during initiation of precursor protein translocation.

The chaperone SecB keeps precursor proteins in a translocation-competent state and targets them to SecA at the translocation sites in the cytoplasmic membrane of Escherichia coli. SecA is thought to recognize SecB via its carboxy-terminus. To determine the minimal requirement for a SecB-binding site, fusion proteins were created between glutathione-S-transferase and different parts of the carboxy-terminus of SecA and analysed for SecB binding. A strikingly short amino acid sequence corresponding to only the most distal 22 aminoacyl residues of SecA suffices for the authentic binding of SecB or the SecB-precursor protein complex. SecAN880, a deletion mutant that lacks this highly conserved domain, still supports precursor protein translocation but is unable to bind SecB. Heterodimers of wild-type SecA and SecAN880 are defective in SecB binding, demonstrating that both carboxy-termini of the SecA dimer are needed to form a genuine SecB-binding site. SecB is released from the translocase at a very early stage in protein translocation when the membrane-bound SecA binds ATP to initiate translocation. It is concluded that the SecB-binding site on SecA is confined to the extreme carboxy-terminus of the SecA dimer, and that SecB is released from this site at the onset of translocation.     

GTPase domain of the 54-kD subunit of the mammalian signal recognition particle is required for protein translocation but not for signal sequence binding

The 54-kD subunit of the signal recognition particle (SRP54) binds to signal sequences of nascent secretory and transmembrane proteins. SRP54 consists of two separable domains, a 33-kD amino-terminal domain that contains a GTP-binding site (SRP54G) and a 22-kD carboxy-terminal domain (SRP54M) containing binding sites for both the signal sequence and SRP RNA. To examine the function of the two domains in more detail, we have purified SRP54M and used it to assemble a partial SRP that lacks the amino-terminal domain of SRP54 [SRP(-54G)]. This particle recognized signal sequences in two independent assays, albeit less efficiently than intact SRP. Analysis of the signal sequence binding activity of free SRP54 and SRP54M supports the conclusion that SRP54M binds signal sequences with lower affinity than the intact protein. In contrast, when SRP(-54G) was assayed for its ability to promote the translocation of preprolactin across microsomal membranes, it was completely inactive, apparently because it was unable to interact normally with the SRP receptor. These results imply that SRP54G plays an essential role in SRP-mediated targeting of nascent chain-ribosome complexes to the ER membrane and also influences signal sequence recognition, possibly by promoting a tighter association between signal sequences and SRP54M.     

PrlC, a suppressor of signal sequence mutations in Escherichia coli, can direct the insertion of the signal sequence into the membrane

The prlC gene product of Escherichia coli can be altered by mutation so that it restores export of proteins with defective signal sequences. The strongest suppressor, prlC8, restores processing of a mutant signal sequence to a rate indistinguishable from the wild-type. Data obtained by changing gene dosage of the dominant suppressor and its specificity for different signal sequence mutations suggest that PrlC8 interacts directly with the hydrophobic core of the signal sequence. Despite the fact that signal sequence processing appears to be mediated by leader peptidase, the processed mature protein is not translocated efficiently from the cytoplasm. Results obtained with various double mutants indicate that PrlC8-mediated processing of mutant signal sequences does not require components of the cellular export machinery such as SecA, SecB or PrlA (SecY) and that the block in translocation from the cytoplasm occurs because PrlA (SecY) fails to recognize the defective signal sequence. We suggest that PrlC8 directs insertion of the mutant signal sequence into the membrane bilayer to an extent that processing by leader peptidase can occur. This reaction is novel in that it has not been observed previously in vivo.     

Selective SecA association with signal sequences in ribosome-bound nascent chains: a potential role for SecA in ribosome targeting to the bacterial membrane

The role of SecA in selecting bacterial proteins for export was examined using a heterologous system that lacks endogenous SecA and other bacterial proteins. This approach allowed us to assess the interaction of SecA with ribosome-bound photoreactive nascent chains in the absence of trigger factor, SecB, Ffh (the bacterial protein component of the signal recognition particle), and the SecYEG translocon in the bacterial plasma membrane. In the absence of membranes, SecA photocross-linked efficiently to nascent translocation substrate OmpA in ribosome-nascent chain (RNC) complexes in an interaction that was independent of both ATP and SecB. However, no photocross-linking to a nascent membrane protein that is normally targeted by a signal recognition particle was observed. Modification of the signal sequence revealed that its affinity for SecA and Ffh varied inversely. Gel filtration showed that SecA binds tightly to both translating and non-translating ribosomes. When purified SecA.RNC complexes containing nascent OmpA were exposed to inner membrane vesicles lacking functional SecA, the nascent chains were successfully targeted to SecYEG translocons. However, purified RNCs lacking SecA were unable to target to the same membranes. Taken together, these data strongly suggest that cytosolic SecA participates in the selection of proteins for export by co-translationally binding to the signal sequences of non-membrane proteins and directing those nascent chains to the translocon.     

Identification of a Sequence Motif That Confers SecB Dependence on a SecB-Independent Secretory Protein In Vivo

SecB is a cytosolic chaperone which facilitates the transport of a subset of proteins, including membrane proteins such as PhoE and LamB and some periplasmic proteins such as maltose-binding protein, in Escherichia coli. However, not all proteins require SecB for transport, and proteins such as ribose-binding protein are exported efficiently even in SecB-null strains. The characteristics which confer SecB dependence on some proteins but not others have not been defined. To determine the sequence characteristics that are responsible for the SecB requirement, we have inserted a systematic series of short, polymeric sequences into the SecB-independent protein alkaline phosphatase (PhoA). The extent to which these simple sequences convert alkaline phosphatase into a SecB-requiring protein was evaluated in vivo. Using this approach we have examined the roles of the polarity and charge of the sequence, as well as its location within the mature region, in conferring SecB dependence. We find that an insert with as few as 10 residues, of which 3 are basic, confers SecB dependence and that the mutant protein is efficiently exported in the presence of SecB. Remarkably, the basic motifs caused the protein to be translocated in a strict membrane potential-dependent fashion, indicating that the membrane potential is not a barrier to, but rather a requirement for, translocation of the motif. The alkaline phosphatase mutants most sensitive to the loss of SecB are those most sensitive to inhibition of SecA via azide treatment, consistent with the necessity for formation of a preprotein-SecB-SecA complex. Furthermore, the impact of the basic motif depends on location within the mature protein and parallels the accessibility of the location to the secretion apparatus.     

Biogenesis of outer membrane protein PhoE of Escherichia coli. Evidence for multiple SecB-binding sites in the mature portion of the PhoE protein.

Efficient in vivo translocation of the precursor of Escherichia coli outer membrane protein PhoE across the inner membrane is shown to depend on SecB protein. A set of mutants, carrying internal deletions in the phoE gene, was used to locate a possible SecB-binding site and/or a site that makes the protein dependent on SecB for export. Except for two small mutant PhoE proteins, the in vivo and in vitro translocation of all mutant proteins was more efficient in the presence of SecB. The interaction of SecB protein with wild-type and mutant PhoE proteins, synthesized in vitro, was further studied in co-immunoprecipitation experiments with anti-SecB protein serum. The efficiencies of co-immunoprecipitation of precursor and mature PhoE were very similar, indicating the absence of a SecB-binding site in the signal sequence. Moreover, all mutant proteins with deletions in the mature moiety of the PhoE protein were co-immunoprecipitated in these assays, albeit mostly with reduced efficiency. Taken together, these results indicate the existence of multiple SecB-binding sites in the mature portion of the PhoE protein.     

The protein translocation machinery of the endoplasmic reticulum

The rough endoplasmic reticulum (r.e.r.) has been postulated to possess a single translation-coupled translocation system (in multiple copies) that effects signal sequence-mediated translocation of all secretory and lysosomal proteins and integration of all integral membrane proteins whose port of entry is the rough endoplasmic reticulum (G. Blobel 1980 Proc. natn. Acad. Sci. U.S.A. 77, 1496-1500). Two proteins have been isolated that are components of the r.e.r. translocation system. Their properties and function in protein translocation across and integration into membranes are discussed.     

Sec-dependent preprotein translocation in bacteria

Translocation of precursor proteins across the cytoplasmic membrane in bacteria is mediated by a multi-subunit protein complex termed translocase, which consists of the integral membrane heterotrimer SecYEG and the peripheral homodimeric ATPase SecA. Preproteins are bound by the cytosolic molecular chaperone SecB and targeted in a complex with SecA to the translocation site at the cytoplasmic membrane. This interaction with SecYEG allows the SecA/preprotein complex to insert into the membrane by binding of ATP to the high affinity nucleotide binding site of SecA. At that stage, presumably recognition and proofreading of the signal sequence occurs. Hydrolysis of ATP causes the release of the preprotein in the translocation channel and drives the withdrawal of SecA from the membrane-integrated state. Hydrolysis of ATP at the low-affinity nucleotide binding site of SecA converts the protein into a compact conformational state and releases it from the membrane. In the absence of the proton motive force, SecA is able to complete the translocation stepwise by multiple nucleotide modulated cycles. Received: 4 August 1995 / Accepted: 9 October 1995     

SecB protein: A cytosolic export factor that associates with nascent exported proteins

Soluble factors participate in protein translocation across a variety of biological membranes. TheEscherichia coli soluble protein SecB (the product of thesecB gene) is involved in the export of periplasmic and outer membrane proteins. The isolation ofsecB mutations permitted the demonstration that SecB is required for rapid and efficient export of certain proteins. Consistent with the results of these genetic studies, purified SecB has been shown to stimulate protein translocation acrossE. coli inner membrane vesiclesin vitro. This article presents a review of these past studies of SecB, speculation on the role of SecB in protein translocation, and a comparison of SecB and other factors, trigger factor and GroEL.     

In Vitro Studies with Purified Components Reveal Signal Recognition Particle (SRP) and SecA/SecB as Constituents of Two Independent Protein-targeting Pathways of Escherichia coli

The molecular requirements for the translocation of secretory proteins across, and the integration of membrane proteins into, the plasma membrane of Escherichia coli were compared. This was achieved in a novel cell-free system from E. coli which, by extensive subfractionation, was simultaneously rendered deficient in SecA/SecB and the signal recognition particle (SRP) components, Ffh (P48), 4. 5S RNA, and FtsY. The integration of two membrane proteins into inside-out plasma membrane vesicles of E. coli required all three SRP components and could not be driven by SecA, SecB, and DeltamicroH+. In contrast, these were the only components required for the translocation of secretory proteins into membrane vesicles, a process in which the SRP components were completely inactive. Our results, while confirming previous in vivo studies, provide the first in vitro evidence for the dependence of the integration of polytopic inner membrane proteins on SRP in E. coli. Furthermore, they suggest that SRP and SecA/SecB have different substrate specificities resulting in two separate targeting mechanisms for membrane and secretory proteins in E. coli. Both targeting pathways intersect at the translocation pore because they are equally affected by a blocked translocation channel.     

A family of bacteriocin ABC transporters carry out proteolytic processing of their substrates concomitant with export

Lantibiotic and non-lantibiotic bacteriocins are synthesized as precursor peptides containing N-terminal extensions (leader peptides) which are cleaved off during maturation. Most non-lantibiotics and also some lantibiotics have leader peptides of the so- called double-glycine type. These leader peptides share consensus sequences and also a common processing site with two conserved glycine residues In positions -1 and 2. The double-glycine-type leader peptides are unrelated to the N-terminal signal sequences which direct proteins across the cytoplasmic membrane via the sec pathway. Their processing sites are also different from typical signal peptidase cleavage sites, suggesting that a different processing enzyme is involved. Peptide bacteriocins are exported across the cytoplasmic membrane by a dedicated ATP-binding cassette (ABC) transporter. Here we show that the ABC transporter is the maturation protease and that its proteolytic domain resides in the N-terminal part of the protein. This result demonstrates that the ABC transporter has a dual function: (i) removal of the leader peptide from its substrate, and (ii) translocation of its substrate across the cytoplasmic membrane. This represents a novel strategy for secretion of bacterial proteins.     

SecB functions as a cytosolic signal recognition factor for protein export in E. coli

A purified 64 kd protein, consisting of four identical subunits of the 16 kd SecB, binds to the signal sequence of preproteins prior to their translocation across inverted vesicles (INV) derived from the E. coli plasma membrane. The purified SecB tetramer competes with canine signal recognition particle (SRP) in signal sequence binding and thus behaves as a prokaryotic equivalent of SRP. As shown by cell fractionation and immunoblot analysis with anti-SecB antibodies, SecB is a cytosolic protein. An E. coli supernatant depleted of SecB after passage through an anti-SecB Sepharose column retains full translation activity but is unable to support translocation into added INV. Translocation into INV is fully restored by readdition of purified SecB.     

Kinetics and energetics of the translocation of maltose binding protein folding mutants

Protein translocation in Escherichia coli is mediated by the translocase that, in its minimal form, comprises a protein-conducting pore (SecYEG) and a motor protein (SecA). The SecYEG complex forms a narrow channel in the membrane that allows passage of secretory proteins (preproteins) in an unfolded state only. It has been suggested that the SecA requirement for translocation depends on the folding stability of the mature preprotein domain. Here we studied the effects of the signal sequence and SecB on the folding and translocation of folding stabilizing and destabilizing mutants of the mature maltose binding protein (MBP). Although the mutations affect the folding of the precursor form of MBP, these are drastically overruled by the combined unfolding stabilization of the signal sequence and SecB. Consequently, the translocation kinetics, the energetics and the SecA and SecB dependence of the folding mutants are indistinguishable from those of wild-type preMBP. These data indicate that unfolding of the mature domain of preMBP is likely not a rate-determining step in translocation when the protein is targeted to the translocase via SecB.     

Positive charges in the cytoplasmic domain of Escherichia coli leader peptidase prevent an apolar domain from functioning as a signal.

Leader peptidase, an integral transmembrane protein of Escherichia coli, requires two apolar topogenic elements for its membrane assembly: a 'hydrophobic helper' and an internal signal. The highly basic cytoplasmic region between these domains is a translocation poison sequence, which we have shown blocks the function of a preceding signal sequence. We have used oligonucleotide-directed mutagenesis to remove positively charged residues within this polar domain to determine if it is the basic character in this region that has the negative effect on translocation. Our results show that mutations that remove two or more of the positively charged residues within the polar region no longer block membrane assembly of leader peptidase. In addition, when the translocation poison domain (residues 30-52) is replaced with six lysine residues, the preceding apolar domain cannot function as an export signal, whereas it can with six glutamic acids. Thus, positively charged residues within membrane proteins may have a major role in determining the function of hydrophobic domains in membrane assembly.