(Received for publication, March 24, 1995; and in revised form, June 27, 1995)
From the
The Escherichia coli SecB protein binds newly
synthesized precursor maltose-binding protein (preMBP) and promotes its
rapid export from the cytoplasm. Site-directed mutagenesis of two
regions of SecB was carried out to better understand factors governing
the SecBpreMBP interaction. 30 aminoacyl substitution mutants
were analyzed, revealing two distinct classes of secB mutants.
Substitutions at the alternating positions Phe-74, Cys-76, Val-78, or
Gln-80 reduced the ability of SecB to form stable complexes with
preMBP, but caused only mild defects in the rate of MBP export from
living cells. The pattern revealed by this class of mutants suggests
that a primary binding site for preMBP is hydrophobic and contains
-sheet secondary structure. In contrast, substitutions at Asp-20,
Glu-24, Leu-75, or Glu-77 caused a severe slowing in the rate of MBP
export but did not disrupt SecB
preMBP complex formation. These
largely acidic residues may function to regulate the opening of a
preprotein binding site, allowing both high affinity preprotein binding
and rapid dissociation of SecB
preprotein complexes at the
membrane translocation site.
The interaction of the export chaperone SecB with nascent or
newly synthesized preproteins is an early event in Escherichia coli protein export(1) . SecB binding prevents the premature
folding or aggregation of preproteins and maintains them in an
export-competent
conformation(2, 3, 4, 5) . SecB also
greatly stimulates the rate of protein export, possibly through a
``targeting'' activity that involves conveying preprotein to
the export apparatus in a specific conformation(6) . The best
studied natural SecB ligand is maltose-binding protein (MBP), ()the periplasmic maltose receptor required by E. coli for maltose utilization(7) . SecB binds to nonoverlapping
regions within a central portion of unfolded MBP(8) . Exported
proteins that do not require the chaperone activity of SecB for export
may interact with the GroEL-GroES or DnaK-DnaJ-GrpE heat shock
chaperones prior to export(9, 10) .
There are
differences between the SecBpreprotein interaction and the
interaction of the heat shock chaperones with their substrates. The
heat shock chaperones have broad substrate-binding specificities, and
they bind and hydrolyze ATP in order to release bound
proteins(11) . In vitro, SecB binds with high affinity
to a variety of peptides and unfolded proteins, many of which are not
natural SecB substrates(12, 13) . SecB is not known to
bind or hydrolyze ATP. Dissociation of SecB
preprotein complexes
may involve preprotein binding to SecA, a peripheral membrane protein
required for general protein export. SecA binds and hydrolyzes ATP (14) and, when membrane-bound, has a high affinity for
SecB
proOmpA complexes formed in vitro(15) .
Mutational studies could be used to identify SecB residues that function in preprotein binding. Previously, chemically induced amino acid substitution mutations were shown to cluster in two regions of SecB(16, 17) . Mutations in region 1 were tightly clustered at positions Leu-75, Cys-76, and Glu-77 (Fig. 1). Mutations in the amino-terminal region 2 occurred at the acidic residues Asp-20 and Glu-24. Two region 1 mutants studied in detail, secBL75Q and secBE77K, are both strongly defective in promoting the rapid export of MBP from the cytoplasm. However, purified mutant SecB protein containing either alteration retains the ability to interact with unfolded MBP and prevent folding in vitro(16) . These two mutations cause SecB to exhibit apparently enhanced binding to unfolded MBP, suggesting that region 1 may play a role in preprotein binding. To better understand the roles of regions 1 and 2 in SecB function, a new set of mutations was generated in both regions. We describe here the effect these mutations exert on both the kinetics of MBP export and SecB:preMBP complex formation.
Figure 1: Single residue substitutions in SecB. The topsequence shows the wild-type SecB amino acid sequence. Columnsbelow represent single residue substitutions at each position. Mutations were generated using corresponding oligonucleotides containing low level random base substitutions (see ``Materials and Methods''). Mutants were divided into 3 classes using a colony color assay as a semi-quantitative measure of secB activity. Class I mutants were the least defective in secB activity, while Class III mutants were extremely defective in secB activity.
To further clarify the roles of region 1 and region 2, we generated a new collection of single-residue substitution mutants in both regions and characterized their effects on SecB function. To circumvent the limitations associated with chemical mutagenesis, mutations were targeted to region 1 and region 2 using synthetic oligonucleotides containing low level random base substitutions.
To identify secB mutants, we employed a red/white colony color assay that monitors the localization of MBP to the periplasm and thereby reflects cellular secB activity(26) . This assay relies on the expression of preMBP(18-1), a form of MBP containing a partially defective signal sequence. Using this assay, a mutant screen was set up to identify new secB mutants based on their failure to efficiently complement a chromosomal secBL75Q mutation. Employing this screen, 20 unique single residue substitutions were recovered in region 1 (Fig. 1). Small insertion or deletion mutations, as well as stop-codon mutations, were recovered at other positions within region 1, indicating that mutagenesis occurred throughout the targeted region. 10 unique substitutions were identified in region 2, all occurring at positions Asp-20, Ser-22, and Glu-24 (Fig. 1). A number of double, triple, and quadruple substitutions were also recovered in region 2. However, each of these contained at least one change at Asp-20, Ser-22, or Glu-24 (data not shown).
This collection of mutants was divided into three classes by estimating the levels of secB activity using the red/white colony color assay (Fig. 1). In these experiments, plasmids expressing mutant SecB supplied the only source of cellular SecB. Class I mutants retained substantial secB activity and included all of the substitutions at Phe-74, Cys-76, Val-78, plus E77D and S22A in region 2. Class II mutants showed a more severe secB deficiency and included most substitutions at Leu-75 and Glu-77 in region 1 and all of the substitutions at Asp-20 and Glu-24 in region 2. Three class III mutants, each the result of proline substitution, occurred in region 1. These mutants lacked secB activity completely because they failed to support growth of E. coli on rich media, a characteristic of E. coli strains devoid of SecB(27) . Western blot analysis of numerous region 1 and region 2 mutants revealed that only the proline substitutions in region 1 prevented high-level accumulation of SecB in growing cells (data not shown). Proline substitution in this region probably prevents normal folding, leading to rapid proteolytic degradation.
Figure 2:
Kinetics of maltose-binding protein export
in secB mutant strains. secB mutant plasmids were
introduced into strain CK1961 (malEsecB::Tn5 recA1). MBP export kinetics were determined
by monitoring the rate of cleavage of the preMBP leader peptide.
Cultures were pulse-labeled for 15 s with Tran
S-label as
described. A pulse sample was taken, and the chase was begun by adding
chloramphenicol (3.4 µg/ml) and unlabeled methionine (0.1 mg/ml)
simultaneously. Chase samples were collected 30 and 60 s after the
addition of chase. Samples were immunoprecipitated with anti-MBP
antiserum and separated by SDS-polyacrylamide gel electrophoresis.
Relative amounts of preMBP and mature MBP were determined using a
Molecular Dynamics PhosphorImager; percent of total MBP in the
precursor form is shown. Experiments were carried out in duplicate, and
each bar represents the average of two
measurements.
PreMBP(18-1) was coimmunoprecipitated
with SecB using anti-SecB antisera (Fig. 3A, lane2). Other SecB ligands, such as preLamB and proOmpA, were
not detected because they contain functional signal sequences and are
rapidly exported from the cytoplasm. PreMBP(18-1) did not
fortuitously cross-react with SecB antibodies because it was not
precipitated from extracts lacking SecB due to mutation (Fig. 3A, lane1). Co-precipitation
of SecB and preMBP(18-1) was reduced if 5 µg of unlabeled
purified competitor SecB was added to the extracts prior to the
addition of antisera, indicating that SecB and preMBP(18-1) were
present in these extracts as soluble complexes (Fig. 3B, lanes1 and 3).
SecBpreMBP(18-1) complexes were also detected when anti-MBP
antisera was used in place of anti-SecB antisera (Fig. 3A, lane5).
Figure 3:
Detection of SecB:preMBP(18-1)
complexes in radiolabeled cell extracts. Extracts were prepared and
incubated with anti-SecB antiserum or anti-MBP antiserum as described.
Immune complexes were isolated and fractionated by SDS-polyacrylamide
gel electrophoresis; fluorograms are shown. M, anti-MBP
antiserum; B, anti-SecB antiserum. A, extracts were
prepared from [S]methionine-labeled cells
expressing SecB, preMBP(18-1), or both, as indicated by ±
symbols. The relevant genotypes are as follows: lanes1 and 4, secB::Tn5 malE18-1; lanes2 and 5, secB::Tn5
malE18-1 pHK205(secB
); lanes2 and 6, secB::Tn5
malB101 pHK205(secB
). B, the secB::Tn5 malE18-1
pHK205(secB
) strain was labeled with
Tran
S-label. Extracts were prepared and immunoprecipitated
as in panelA. For lane3, 5 µg
of purified His-tagged SecB protein was added to the extract prior to
incubation with anti-SecB antiserum. C,
SecB:preMBP(18-1) complex formation in secB mutant
strains. secB mutant plasmids were transformed into the secB::Tn5 malE18-1 strain HK57. The resulting
strains were labeled with Tran
S-label. Extraction and
immunoprecipitation were as in panelA.
The results of co-precipitation experiments with five region 1 mutants are shown in Fig. 3C. Complexes between SecBF74I and preMBP(18-1) were not detected using either anti-SecB or anti-MBP antisera (Fig. 3C, lanes1 and 2). Similarly, complexes involving SecBC76Y or SecBQ80R were observed at greatly reduced levels using either anti-SecB or anti-MBP antisera (Fig. 3C, lanes5 and 6 and lanes9 and 10). In contrast, complexes involving SecBL75R or SecBE77V were observed at levels comparable with that seen with normal SecB (Fig. 3C, lanes3 and 4 and lanes7 and 8).
While approximately 12-15% of the total
precipitable preMBP(18-1) was observed in SecBpreMBP
complexes with normal SecB, most or all of the normal SecB exists in
SecB
preMBP(18-1) complexes (Fig. 3B, lanes1 and 2). Quantitation of these
co-precipitation results yielded estimated SecB
preMBP(18-1)
stoichiometries of 4-5:1 for normal SecB, SecBL75R, and SecBE77V,
indicating that SecBL75R and SecBE77V share the same binding
stoichiometry as normal SecB. Since SecB is probably a
tetramer(28) , the complexes we observe appear to be composed
of one SecB tetramer and one preMBP(18-1) chain. Since each SecB
monomer contains one peptide binding site(12) ,
SecB
preMBP(18-1) complexes may consist of one SecB tetramer
bound at multiple points along one preMBP chain.
Overall, region 1 mutants showed a distinct pattern of preMBP(18-1) binding defects (Fig. 4). Substitutions at alternating residues Phe-74, Cys-76, Val-78, and Gln-80 abolished or greatly reduced preMBP binding. In contrast, diverse substitutions at Leu-75 and Glu-77 all failed to disrupt preMBP complex formation. There are two exceptions to this pattern in region 1: SecBF74Y stably bound preMBP(18-1), whereas three Phe-74 substitution mutants involving the branched chain amino acids did not, indicating that an aromatic side chain at position 74 is essential for preMBP binding. Also, SecBV78G formed complexes with preMBP(18-1) but SecBV78F did not, indicating that only changes that introduce bulky side chains at Val-78 are sufficient to disrupt preMBP binding.
Figure 4: Summary of SecB:preMBP(18-1) co-precipitation results.
It is striking that mutations at Phe-74/Cys-76/Val-78/Gln-80 appear to strongly destabilize preMBP(18-1) binding but result in only mild defects in the rate of MBP export in living cells. During the course of our binding assay, preMBP(18-1) may continue to fold into a mature-like form that is not a substrate for SecB binding(13) . When a competing folding reaction removes preMBP substrate, binding defects due to lowered affinity could be greatly magnified, leading to an apparent all-or-none binding pattern. The high level synthesis of SecB, which occurs in these strains from multi-copy plasmids, may overcome or suppress export defects caused by a decreased affinity for preprotein.
These results
suggest that the side chains at Phe-74/Cys-76/Val-78/Gln-80 are part of
a hydrophobic preMBP binding pocket, or are required for the formation
of such a binding pocket. The alternating pattern of binding-deficient
mutations further suggests that region 1 is one strand of -sheet
secondary structure. Region 1 could be one strand of a stable
-sheet, analogous to the large
-sheet that forms the floor of
the peptide binding site in class I MHC protein, a protein that binds a
wide variety of peptides(29) . This site may be identical to
the hydrophobic site described by Randall as capable of binding the
fluorescent compound 1-anilinonaphthalene-8-sulfonate(12) .
Intermixed with the binding site residues are Leu-75 and Glu-77. If
region 1 is one strand of -sheet secondary structure, then the
Leu-75 and Glu-77 side chains would project out onto the side opposite
that displaying the Phe-74/Cys-76/Val-78/Gln-80 side chains. In such an
arrangement, Leu-75 and Glu-77 are unlikely to participate in the same
hydrophobic preprotein binding site. Consistent with this proposal, we
found that diverse substitutions at both sites did not impair complex
formation (Fig. 4). Nevertheless, changes at Leu-75 and Glu-77
resulted in more severe kinetic defects in MBP export than did changes
at the binding site residues Phe-74/Cys-76/Val-78/Gln-80 (Fig. 2). Excluding the proline substitution at Leu-75,
substitutions at Leu-75 and Glu-77 did not prevent high level
accumulation of SecB in cells, indicating that changes at these
positions do not grossly disturb SecB structure. Taken together, these
observations indicate that Leu-75 and Glu-77 are essential for normal
SecB function, but do not directly participate in preprotein binding.
Region 2 has several features in common with region 1. First, the
alternating pattern of mutations suggests that region 2 also is
composed of -sheet secondary structure. Second, changes at the
acidic residues Asp-20 and Glu-24 did not disrupt preMBP(18-1)
binding but resulted in slowed rates of MBP export similar to those
associated with changes at Leu-75 and Glu-77 in region 1 (Fig. 2). Also, the two glutamyl residues Glu-24 and Glu-77
share the property that neither tolerates the conservative substitution
by aspartic acid. An essential difference between the two regions is
the apparent absence of binding site residues in region 2.
The overall similarities in mutant phenotypes caused by changes at Asp-20-Glu-24 and Leu-75-Glu-77 suggests that these residues function together in a SecB-mediated activity distinct from preprotein binding. Clues to the roles of Asp-20, Glu-24, Leu-75, and Glu-77 in SecB function come from previous biochemical studies of SecB protein altered at two of these positions. Purified SecB protein carrying the L75Q or E77K substitution retained the ability to interact with chemically unfolded MBP and prevent its rapid refolding(16) . In those studies, both mutant SecB proteins were more effective than normal SecB at blocking the refolding of MBP, indicating that certain alterations at Leu-75 and Glu-77 result in SecB with a higher affinity for unfolded MBP.
Residues Leu-75 and Glu-77, together with Asp-20 and Glu-24, may function to facilitate a conformational change leading to the opening of a closely associated hydrophobic preprotein binding site. Randall has reported evidence suggesting that positively charged regions of an unfolded preprotein are first bound at a site on SecB, and then a conformational change occurs and a second hydrophobic site on SecB becomes acccessible(12) . According to this two-step model of preprotein binding, Leu-75 and Glu-77 mutants could be locked in a normally short-lived high affinity conformation.