TEXT

Top Abstract Text References

SecB is a molecular chaperone which is required for the efficient transport of a subset of proteins in Escherichia coli. A tetramer of identical 16-kDa subunits, it functions by binding some nascent secretory proteins as they emerge from the ribosome in the cytosol. SecB is thought to interact with the mature region of the preprotein (16, 24, 27) to prevent its premature folding and to target the preprotein to the membrane in a transport-competent state (4, 14). The SecB-preprotein complex binds SecA at the gateway of translocation sites, and this interaction is thought to facilitate the transfer of the preprotein to SecA with the subsequent release of SecB (10, 14). SecA, in turn, initiates the passage of the preprotein through the translocon via its associated ATPase activity (3, 4).

The rules which govern a requirement for SecB assistance in protein transport are unknown. A variety of outer membrane-bound and periplasmic proteins require SecB for efficient transport (e.g., OmpA, OmpF, PhoE, MBP) while others do not (e.g., PhoA, RBP, -lactamase). Moreover, SecB exhibits a very broad substrate specificity involving either charged or hydrophobic regions of preproteins (19, 22), suggesting that SecB utilization is not dictated by a requirement for specific high-affinity binding sites. Nor does the mere presence of SecB-binding sites determine whether a preprotein is inherently SecB dependent; modifications in SecB-independent proteins can lead to SecB utilization, indicating that at least some proteins are endowed with the ability to employ SecB whether they need to or not (17, 18). Furthermore, no consensus sequence or region consistent among proteins has been found responsible for conferring SecB dependence (1, 12, 30). One model suggests that the requirement for SecB depends on the partitioning of the preprotein between transport-productive and aggregation-prone, nonproductive pathways (6, 13); while a functional signal peptide does not directly bind SecB (24), it functions to impede the folding of the preprotein so that SecB can bind (6, 23), while a defective signal peptide does not. This model has been questioned, however, because SecB can also bind fully folded proteins (26) and the rate of SecB-ligand association can be much faster than the rate of folding of SecB-independent proteins (9).

Alkaline phosphatase is a normally SecB-independent protein with which we have made a series of small well-defined adjustments to show its incremental conversion to a SecB-dependent protein. The results indicate that SecB plays a fundamental role in enhancing the efficiency with which the amino-terminal domain of the preprotein engages the transport pathway.

Discrete changes in the early mature region of alkaline phosphatase gradually shift the equilibrium toward SecB utilization. We have previously shown that a 10-residue motif, including a cluster of basic amino acids in the mature region of alkaline phosphatase, confers SecB dependence and that the impact of the basic motif parallels the accessibility of its location within the protein to the secretion apparatus (18). In order to determine the extent to which the basic residues are specifically responsible for the SecB dependence, a series of mutant alkaline phosphatases were generated in which the lysines were successively replaced with asparagines within the motif inserted at residue 13 of the mature region of the preprotein (Fig. 1A). secB⁺ and secB null cells were grown in Luria-Bertani medium and then transferred to MOPS (morpholinepropanesulfonic acid) medium lacking phosphate to induce expression of the mutant alkaline phosphatases as described previously (18). The cells were then pulse labeled with [³⁵S]methionine (30 s) followed by incubation with excess cold methionine for an additional 30 s to ensure the progression of protein synthesis. Alkaline phosphatase was immunoprecipitated and the relative ratio of the precursor to mature forms was assessed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and quantified with a phosphorimager (18). The ratio of precursor to mature forms of alkaline phosphatase provides a quantifiable earmark of the relative extent to which each mutant accomplishes protein transport in the presence and absence of SecB. In every case, when the mature form of the protein was generated it was localized in the periplasm (data not shown). As shown in Fig. 1B and C, the presence of the basic motif results in little or no accumulation of precursor when expressed in the presence of SecB. However, in the absence of SecB there is a direct correlation between the number of basic residues and the accumulation of the precursor form of the protein. Although these mutants are processed in the presence of SecB with transport kinetics comparable to those of the wild-type SecB-dependent protein, MBP (18), transport of mutants with more lysine residues is more sensitive to the loss of SecB.

View larger version (27K): [in this window] [in a new window]   FIG. 1.   SecB dependence of mutant alkaline phosphatases carrying different numbers of basic residues in the early mature region and expressed in secB+ and secB null cells. (A) The amino acid sequences of the inserted motifs and the flanking regions. Alkaline phosphatase is shown diagrammatically with the signal sequence region darkly shaded, the mature region is shown as an open bar, and the signal peptide cleavage site is marked by an arrow. The sequences inserted at the thirteenth residue of the mature protein (checkered) and the flanking sequences are shown. The inserted sequences are shown in boldface, and the amino acids generated during the cloning procedure are shown in italics. (B) Precursor processing of the mutant alkaline phosphatases expressed in secB+ (AW1043) and secB null (CK1953) cells. The positions of the precursor (p) and mature (m) forms of alkaline phosphatase are indicated. Strain CK1953 has a chromosomal copy of the alkaline phosphatase gene, the product of which is indicated with the open arrowhead. (C) Quantification of the extent of processing of the mutant alkaline phosphatases from the experiment depicted in panel B. Darkly shaded bars represent the extent of processing in the secB+ strain, and hatched bars represent the processing in the secB null strain.

Titration of signal peptide hydrophobicity directly parallels a requirement for SecB in the transport process. We considered the possibility that SecB rescues transport of alkaline phosphatase with the basic motif by overcoming an unfavorable or inefficient interaction with the transport machinery. This model suggests that alkaline phosphatase with signal sequence defects which are known to reduce the interaction of the preprotein with components of the transport pathway should be SecB dependent. To examine this possibility, we employed a series of alkaline phosphatase signal peptide mutants in which the core region is replaced with various combinations of leucine and alanine residues (Fig. 2A). Earlier studies have demonstrated a clear correlation between the hydrophobicity of these signal peptides, the efficiency of in vivo transport (7, 15), and cross-linking with P48 and with SecA (28) in vitro and with binding to SecA in reconstituted systems (21, 29).

View larger version (23K): [in this window] [in a new window]   FIG. 2.   SecB dependence of mutant alkaline phosphatases carrying signal peptides of different hydrophobicity and expressed in secB+ and secB null cells. (A) The amino acid sequences of the signal peptide region of the preproteins. Alkaline phosphatase is shown diagrammatically with the signal peptide core region represented by the hatched area within the darkly shaded signal peptide, the mature region is represented by an open bar, and the signal peptide cleavage site is marked by an arrow. The sequences of the signal peptides are given below with the hydrophobic core region shown in boldface. (B) Precursor processing of the mutant alkaline phosphatases expressed in secB+ (AW1043) and secB null (CK1953) cells. The positions of the precursor (p) and mature (m) forms of alkaline phosphatase are indicated. Expression of a chromosomal copy of the phoA gene was minimized by 40 mM phosphate. (C) Quantification of the extent of processing of the mutant alkaline phosphatases from the experiment depicted in panel B. Darkly shaded bars represent the extent of processing in the secB+ strain, and hatched bars represent the processing in the secB null strain. (D) A chloramphenicol-resistant plasmid carrying the wild type (WT) or 4L6A mutant was introduced into strain HS101(pAB5) or HS101(pE63). SecB or GpsA expression was induced by 0.2% arabinose. Alkaline phosphatase was expressed as shown in panel B.

Signal peptides optimized for transport override the requirement for SecB conferred by the basic motif. If SecB rescues transport of alkaline phosphatase with the basic motif by overcoming an unfavorable or inefficient interaction with the transport machinery, then this unfavorable interaction should be stabilized by enhancing the degree of the preprotein interaction by other means. This possibility was tested using the series of alkaline phosphatase mutants which correspond to the signal peptide mutants described above but which also carry the basic motif in the early mature region of the protein (Fig. 3A). Again, secB⁺ and secB null cells expressing these mutant alkaline phosphatases were pulse labeled with [³⁵S]methionine and analyzed for the extent of precursor processing as described for Fig. 1. As shown in Fig. 3B and C for the preprotein carrying the basic motif, as the hydrophobicity of the signal peptide increases the dependence on SecB decreases. For preproteins with the 3L7A and 4L6A signal peptides, no transport is observed. Interestingly, transport of alkaline phosphatase carrying the basic motif with the 5L5A signal peptide is severely restricted relative to that with the same signal peptide in the absence of the motif, pointing to the interplay between these two regions in achieving transport efficiency. In contrast, alkaline phosphatases with the 6L4A, 7L3A, and 9L1A signal peptides are transported efficiently irrespective of the presence of the basic motif, with only the 6L4A mutant requiring SecB assistance. The most hydrophobic signal peptides apparently ameliorate the problem caused by the basic motif, and SecB is no longer required for efficient entry into the transport pathway. At the other extreme, however, SecB cannot rescue those preproteins with very weak signal peptides. It seems reasonable that a preprotein must have an affinity for the transport pathway that at least meets some threshold level in order for SecB to be effective. This provides a mechanism for excluding cytoplasmic proteins from SecB-mediated entrance into the transport pathway.

View larger version (30K): [in this window] [in a new window]   FIG. 3.   SecB dependence of mutant alkaline phosphatases with signal peptides of different hydrophobicity and with the basic motif (K5L5) in the early mature region. (A) Alkaline phosphatase is shown diagrammatically as in Fig. 1A and 2A. The hydrophobic core region (hatched) of the signal peptide was replaced with various hydrophobic sequences while maintaining the K5L5 motif (checkered) in the early mature region. (B) Precursor processing of the mutant alkaline phosphatases expressed in secB+ (AW1043) and secB mutant (CK1953) cells. The positions of the precursor (p) and mature (m) forms of alkaline phosphatase are indicated. (C) Quantification of the extent of processing of the mutant alkaline phosphatases from the experiment depicted in panel B. Darkly shaded bars represent the extent of processing in the secB+ strain and hatched bars represent processing in the secB mutant strain.