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.     

SecA specificity for different signal peptides

SecA performs a critical function in the recognition, targeting, and transport of secretory proteins across the cytoplasmic membrane of Escherichia coli. In this study we investigate the substrate specificity of SecA, including the influence of the early mature region of the preprotein on SecA interactions, and the extent to which SecA recognizes targeting signals from different transport pathways. A series of fusion proteins were generated which involved the tandem expression of GST, signal peptide, and the first 30 residues from alkaline phosphatase. These were purified and evaluated for their ability to promote SecA ATPase activity. No significant difference in the stimulation of SecA-lipid ATPase activity between the synthetic wild-type alkaline phosphatase signal peptide and a fusion that also contains the first 30 residues of alkaline phosphatase was observed. The incorporation of sequence motifs in the mature region, which confer SecB dependence in vivo, had no impact on SecA activation in vitro. These results suggest that the early mature region of alkaline phosphatase does not affect the interactions between SecA and the signal peptide. Sec, Tat, and YidC signal peptide fusions were also assayed for their ability to stimulate SecA ATPase activity in vitro and further analyzed in vivo for the Sec dependence of the transport of the corresponding signal peptide mutants of alkaline phosphatase. Our results demonstrate that E. coli Sec signals give the highest level of SecA activation; however, SecA-signal peptide interactions in vitro are not the only arbiter of whether the preprotein utilizes the Sec pathway in vivo.     

A signal sequence is not required for protein export in prlA mutants of Escherichia coli.

The prlA/secY gene, which codes for an integral membrane protein component of the Escherichia coli protein export machinery, is the locus of the strongest suppressors of signal sequence mutations. We demonstrate that two exported proteins of E.coli, maltose-binding protein and alkaline phosphatase, each lacking its entire signal sequence, are exported to the periplasm in several prlA mutants. The export efficiency can be substantial; in a strain carrying the prlA4 allele, 30% of signal-sequenceless alkaline phosphatase is exported to the periplasm. Other components of the E.coli export machinery, including SecA, are required for this export. SecB is required for the export of signal-sequenceless alkaline phosphatase even though the normal export of alkaline phosphatase does not require this chaperonin. Our findings indicate that signal sequences confer speed and efficiency upon the export process, but that they are not always essential for export. Entry into the export pathway may involve components that so overlap in function that the absence of a signal sequence can be compensated for, or there may exist one or more means of entry that do not require signal sequences at all.     

Interaction of SecB and SecA with the N-terminal region of mature alkaline phosphatase on its secretion in Escherichia coli

Export-specific chaperone SecB and translocational ATPase SecA catalyze the cytoplasmic steps of Sec-dependent secretion in Escherichia coli. Their effects on secretion of periplasmic alkaline phosphatase (PhoA) were shown to depend on the N-terminal region of the mature PhoA sequence contained in the PhoA precursor. Amino acid substitutions in the vicinity of the signal peptide (positions +2, +3) not only dramatically inhibited secretion, but also reduced its dependence on SecB and SecA. Immunoprecipitation reported their impaired binding with mutant prePhoA. The results testified that SecB and SecA interact with the mature PhoA region located close to the signal peptide in prePhoA.     

Interaction of SecB and SecA with the N-Terminal Region of Mature Alkaline Phosphatase on Its Secretion in Escherichia coli

Export-specific chaperone SecB and translocational ATPase SecA catalyze the cytoplasmic steps of Sec-dependent secretion in Escherichia coli. Their effects on secretion of periplasmic alkaline phosphatase (PhoA) were shown to depend on the N-terminal region of the mature PhoA sequence contained in the PhoA precursor. Amino acid substitutions in the vicinity of the signal peptide (positions +2, +3) not only dramatically inhibited secretion, but they also reduced its dependence on SecB. Immunoprecipitation reported their impaired binding with mutant prePhoA. The results testified that SecB and SecA interact with the mature PhoA region located close to the signal peptide in prePhoA.     

Interdependent Effects of the Charge of the N-Terminal Region of the Signal Peptide,SecA, and SecB on Secretion of Alkaline Phosphatase in Escherichia coli

The cytoplasmic step of posttranslational secretion in Escherichia coli is catalyzed by export-specific chaperone SecB and translocational ATPase SecA. In addition, the efficiency of secretion depends on the charge of the signal peptide (SP). Replacement of positively charged Lys(–20) with uncharged Ala or negatively charged Glu in the N-terminal region of SP of the alkaline phosphatase precursor (prePhoA) was shown to decrease the PhoA secretion in the periplasm. The effect on secretion increased in the absence of SecB and was especially high on SecA inactivation. A change in SP charge strengthened the SecA and SecB dependences of secretion. On evidence of immunoprecipitation, the charge of the N-terminal region of SP had no effect on prePhoA interaction with the cytoplasmic secretion factors, suggesting no direct binding between this region and SecA or SecB. Yet the charge of the N-terminal region proved to affect the functions of SP as an intramolecular chaperone and a factor of prePhoA targeting to the membrane in cooperation with SecA and SecB.     

Interaction of effect on secretion of alkaline phosphatase from E. coli by charge of the N-terminal part of the signal peptide and proteins SecB and SecA

The cytoplasmic step of posttranslational secretion in Escherichia coli is catalyzed by export-specific chaperone SecB and translocational ATPase SecA. In addition, the efficiency of secretion depends on the charge of the signal peptide (SP). Substitution of positively charged Lys(-20) with noncharged Ala or negatively charged Glu in the N-terminal region of SP of the alkaline phosphatase (PhoA) precursor (prePhoA) was shown to decrease the PhoA secretion in the periplasm. The effect on secretion increased in the absence of SecB and was especially high on SecA inactivation. A change in SP charge strengthened the SecA and SecB dependences of secretion. On evidence of immunoprecipitation, the charge of the N-terminal region of SP had no effect on prePhoA interaction with the cytoplasmic secretion factors, suggesting no direct binding between this region and SecA or SecB. Yet the charge of the N-terminal region proved to affect the functions of SP as an intramolecular chaperone and a factor of prePhoA targeting to the membrane in cooperation with SecA and SecB.     

Effect of export-specific cytoplasmic chaperone, protein SecB, on secretion of Escherichia coli alkaline phosphatase

The efficiency of secretion of Escherichia coli alkaline phosphatase depends on the presence in cells of a cytoplasmic chaperone—protein SecB. Secretion increases in the presence of this chaperone at 30°C, which is the most favorable for the interaction of SecB with the export-initiation domain found previously in the N-terminal region of the mature enzyme. This interaction most likely occurs in the region of the export domain, which is located close to the signal peptide and in complex with a translocational ATPase—protein SecA.     

Functional implementation of the posttranslational SecB-SecA protein-targeting pathway in Bacillus subtilis

Bacillus subtilis and its close relatives are widely used in industry for the Sec-dependent secretory production of proteins. Like other Gram-positive bacteria, B. subtilis does not possess SecB, a dedicated targeting chaperone that posttranslationally delivers exported proteins to the SecA component of the translocase. In the present study, we have implemented a functional SecB-dependent protein-targeting pathway into B. subtilis by coexpressing SecB from Escherichia coli together with a SecA hybrid protein in which the carboxyl-terminal 32 amino acids of the B. subtilis SecA were replaced by the corresponding part of SecA from E. coli. In vitro pulldown experiments showed that, in contrast to B. subtilis SecA, the hybrid SecA protein gained the ability to efficiently bind to E. coli SecB, suggesting that the structural details of the extreme C-terminal region of SecA constitute a crucial SecB binding specificity determinant. Using a poorly exported mutant maltose binding protein (MalE11) and alkaline phosphatase (PhoA) as model proteins, we could demonstrate that the secretion of both proteins by B. subtilis was significantly enhanced in the presence of the artificial protein targeting pathway. Mutations in SecB that do not influence its chaperone activity but prevent its interaction with SecA abolished the secretion stimulation of both proteins, demonstrating that the implemented pathway in fact critically depends on the SecB targeting function. From a biotechnological view, our results open up a new strategy for the improvement of Gram-positive bacterial host systems for the secretory production of heterologous proteins.     

Asparagine-linked carbohydrate does not determine the cellular location of yeast vacuolar nonspecific alkaline phosphatase

The nonspecific alkaline phosphatase of Saccharomyces sp. strain 1710 has been shown by phosphatase cytochemistry to be exclusively located in the vacuole, para-Nitrophenyl phosphate-specific alkaline phosphatase is not detected by this procedure because the activity of this enzyme is sensitive to the fixative agent, glutaraldehyde. To determine whether the oligosaccharide of nonspecific alkaline phosphatase is necessary to transport the enzyme into the vacuole, protoplasts were derepressed in the absence or in the presence of tunicamycin, an antibiotic which interferes with the glycosylation of asparagine residues in proteins. The location of the enzyme in the tunicamycin-treated protoplasts, as determined by electron microscopy and subcellular fractionation, was identical to its location in control protoplasts. In addition, carbohydrate-free alkaline phosphatase was found in vacuoles from tunicamycin-treated protoplasts. Our findings indicate that the asparagine-linked carbohydrate moiety does not determine the cellular location of the enzyme.     

Alkaline phosphatase activity does not mediate phosphate transport in the renal-cortical brush-border membrane.

We studied (1) the effect of primary modulators of phosphate transport, namely the hypophosphataemic mouse mutant (Hyp) and low-phosphorus diet, on alkaline phosphatase activity in mouse renal-cortex brush-border membrane vesicles and (2) the effect of several primary inhibitors of alkaline phosphatase on phosphate transport. Brush-border membrane vesicles from Hyp-mouse kidney had 50% loss of Na+-dependent phosphate transport, but only 18% decrease in alkaline phosphatase activity. The low-phosphorus diet effectively stimulated Na+/phosphate co-transport in brush-border membrane vesicles (+ 118%), but increased alkaline phosphatase activity only slightly (+13%). Levamisole (0.1 mM) and EDTA (1.0 mM) inhibited brush-border membrane-vesicle alkaline phosphatase activity of 82% and 93% respectively, but had no significant effect on Na+/phosphate co-transport. We conclude that alkaline phosphatase does not play a direct role in phosphate transport across the brush-border membrane of mouse kidney.     

2-Mercaptoacetate administration depresses the beta-oxidation pathway through an inhibition of long-chain acyl-CoA dehydrogenase activity.

In an investigation of the link between Pi transport and alkaline phosphatase in mammalian small intestine, the characteristics of Pi uptake by brush-border membrane vesicles prepared from rat intestine were compared with the properties of the tissue alkaline phosphatase. The NaCl-dependent Pi uptake had a Km of 0.1 mM at pH 7.5 and was inhibited totally by 1 mM-arsenate and by 1 mM-vanadate. These compounds are also potent competitive inhibitors of the alkaline phosphatase activity of the vesicles, with Ki values less than 5 microM at pH 7.5. When the effect on Pi uptake of several other potent inhibitors of alkaline phosphatase, including phosphonates and phosphate analogues, was tested, however, it was found that there was little, if any, inhibition of transport under conditions in which the inhibition of phosphatase activity was total. Incubation of the vesicles for 20 min with oxidized adenosine 5'-[beta gamma-imido]triphosphate followed by rapid gel filtration to remove the inhibitor resulted in an irreversible loss of phosphatase activity, but left Pi transport unimpaired. Conversely, a similar prolonged incubation with adenosine 5'-[beta-thio]diphosphate or adenosine 5'-[gamma-thio]triphosphate had no effect on alkaline phosphatase activity but resulted in a permanent partial loss of transport capability. The failure to demonstrate an inhibition of Pi transport resulting from inhibition of alkaline phosphatase and the different responses of enzymic activity and Pi transport to irreversible inhibition make it very unlikely that the enzyme is directly involved in the transport system.     

Evidence that SecB enhances the activity of SecA

In Escherichia coli, SecA is a critical component of the protein transport machinery which powers the translocation process by hydrolyzing ATP and recognizing signal peptides which are the earmark of secretory proteins. In contrast, SecB is utilized by only a subset of preproteins to prevent their premature folding and chaperone them to membrane-bound SecA. Using purified components and synthetic signal peptides, we have studied the interaction of SecB with SecA and with SecA-signal peptide complexes in vitro. Using a chemical cross-linking approach, we find that the formation of SecA-SecB complexes is accompanied by a decrease in the level of cross-linking of SecA dimers, suggesting that SecB induces a conformational change in SecA. Furthermore, functional signal peptides, but not dysfunctional ones, promote the formation of SecA-SecB complexes. SecB is also shown to directly enhance the ATPase activity of SecA in a concentration-dependent and saturable manner. To determine the biological consequence of this finding, the influence of SecB on the signal peptide-stimulated SecA/lipid ATPase was studied using synthetic peptides of varying hydrophobicity. Interestingly, the presence of SecB can sufficiently boost the response of signal peptides with moderate hydrophobicity such that it is comparable to the activity generated by a more hydrophobic peptide in the absence of SecB. The results suggest that SecB directly enhances the activity of SecA and provide a biochemical basis for the enhanced transport efficiency of preproteins in the presence of SecB in vivo.     

The mature portion of Escherichia coli maltose-binding protein (MBP) determines the dependence of MBP on SecB for export.

The product of the secB gene is required for export of a subset of secreted proteins to the outer membrane and periplasm of Escherichia coli. Precursor maltose-binding protein (MBP) accumulates in the cytoplasm of secB-carrying mutants, but export of alkaline phosphatase is only minimally affected by secB mutations. When export of MBP-alkaline phosphatase hybrid proteins was analyzed in wild-type and secB-carrying mutant strains, the first third of mature MBP was sufficient to render export of the hybrid proteins dependent on SecB. Substitution of a signal sequence from a SecB-independent protein had no effect on SecB-dependent export. These findings show that the first third of mature MBP is capable of conferring export incompetence on an otherwise competent protein.     

分子伴侣过量表达对蛋白质分泌及可溶性的影响

 通过过量表达大肠杆菌分子伴侣 Sec B和 Gro EL,研究了它们对靶蛋白的分泌及可溶性的影响 .在过量表达 Sec B的宿主菌中 ,周质空间分泌蛋白总量较对照组提高了约 71 % ,GL- 7- ACA酰化酶在周质空间酶的活力较对照组提高了约 1 .5倍 ,碱性磷酸酯酶在周质空间酶的活力较对照组提高了约 54% ;在过量表达 Gro EL的宿主菌中 ,周质分泌蛋白总量较对照组提高了约 52 % ,青霉素 G酰化酶在周质空间酶的活力较对照组提高了约 76% ,鲑鱼降钙素六聚体的可溶性组分的比例由原来的 45%增加到约 90 % ,而 MS2 -人白介素 - 3融合蛋白的包涵体有约 1 5%转变为可溶性组份 .上述结果表明 ,分子伴侣 Sec B和 Gro EL的过量表达促进了靶蛋白的分泌 ,Gro EL增加了靶蛋白的可溶性     

SecB-binding does not maintain the translocation-competent state of prePhoE

The role of SecB protein in the export of the precursor of outer membrane protein PhoE and mutant forms of this precursor was studied in vitro. When synthesized in the absence of SecB, translocation-competent prePhoE was observed post-translationally, but addition of SecB was required for efficient translocation into inner membrane vesicles. The translocation competency of in vitro synthesized prePhoE diminished with a similar half-life during incubations in the presence or absence of SecB. The loss of translocation competency of prePhoE, synthesized in the presence of SecB, was not due to dissociation of prePhoE-SecB complexes as could be demonstrated in co-immunoprecipitation experiments with anti-SecB serum. Apparently, SecB does not maintain the translocation-competent conformation of prePhoE, but is mainly required for efficient targeting of this precursor to the export apparatus.     

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.     

Investigation of alkaline phosphatase activity in the serum of pregnant rats

An investigation was undertaken to determine if the placental alkaline phosphatase of the rat enters the maternal circulation and to study some of its characteristics. Unlike human, rat placental alkaline phosphatase was found to be heat labile and the alkaline phosphatase activity in the serum of both pregnant and non-pregnant rats was also found to be heat labile. Also unlike the human, the alkaline phosphatase activity in rat serum does not increase as pregnancy progresses to term. In an endeavour to establish if the rat placental enzyme is present in the serum of the pregnant rat, the characteristics of the enzyme in both placental extracts and serum of non-pregnant and 1-, 2- and 3-week pregnant rats were studied using the techniques of heat stability at 56°, gel filtration through Sephadex columns, disc gel electrophoresis, and L-phenylalanine inhibition. The presence of rat placental alkaline phosphatase in maternal serum could not be positively demonstrated by any of these procedures, suggesting that rat placental alkaline phosphatase does not enter the maternal serum.     

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.