(Received for publication, November 20, 1995; and in revised form, February 1, 1996)
From the
We examined in vitro translocation of pro-OmpA
derivatives with a His tag at various positions in their
mature proteins and with a c-Myc tag at their C termini across inverted
membrane vesicles of Escherchia coli. Those with a His
tag in the N-terminal region of the mature domain, which
corresponds to the ``translocation initiation domain''
proposed previously (Andersson, H., and von Heijne, G.(1991) Proc.
Natl. Acad. Sci. U. S. A. 88, 9751-9754), could not be
translocated in the presence of 100 µM Ni
, while OmpA derivatives with a His
tag in the middle of or at the C terminus did not show such
Ni
sensitivity. The inhibitory action of
Ni
on pro-3His-OmpA` (with a His
tag
after the third amino acid of the mature OmpA-c-Myc region)
translocation was exerted only during early events, after which it
became ineffective. The inhibition point of Ni
was
suggested to lie between membrane targeting and exposure of the signal
cleavage site to the periplasm since the unprocessed and membrane-bound
form of pro-3His-OmpA` was accumulated by the addition of
Ni
. The Ni
-``trapped''
precursor was released from its translocation block by 30 mM histidine, which should compete with the His
tag on
the precursor protein for formation of a Ni
chelating
complex. We propose that Ni
confers a reversible
positive charge effect on the His
-tagged initiation domain
of the pro-OmpA derivatives and inhibits an early event(s) of protein
translocation, such as presentation of the precursor to the membranous
part of the translocase. This system will be useful in dissecting early
events of the protein translocation pathway.
Translocation across the cytoplasmic membrane is the first step
of protein targeting to the cell surface in bacterial cells. This
complex biochemical reaction involving topological change of molecules
has been analyzed by combined approaches of genetics and biochemistry
in Escherichia coli (for reviews, see (1, 2, 3, 4) ). The biochemical
studies, notably purification and reconstitution of protein
translocation machinery, have revealed key players of the
translocation, translocation ATPase (SecA), a secretory
protein-specific chaperone (SecB), and an integral membrane component
(SecY-SecE-SecG
complex)(5, 6, 7, 8, 9) .
From in vitro analyses using inverted bacterial plasma
membrane vesicles, several subprocesses in the protein translocation
reaction can be envisaged: 1) recognition of preproteins by chaperones
(like SecB) that retain ``translocation-competent
conformation'' of the secretory protein precursors, 2) targeting
of the preprotein-SecB complex to SecA bound to the high affinity site
of the plasma membrane, 3) ATP binding-dependent partial insertion of
the precursors into a translocation channel, and 4) ATP
hydrolysis-coupled and -dependent bulk protein
translocation(9, 10, 11, 12, 13) .
Translocating secretory proteins are surrounded by SecA and SecY, but
not by lipid molecules(14) . Recently, Economou and Wickner (15) found that the movement of the secretory protein is
coupled with insertion and de-insertion of a 30-kDa segment of SecA.
Deep insertion of SecA into the membrane is also detected in
vivo(16) .
During the course of these analyses, several systems have been developed to trap translocation intermediates during post-translational protein translocation. Except for the cases of kinetic trapping of intermediates by low ATP concentration (12) and formation of a disulfide bond loop of precursor protein in the absence of proton-motive force(11) , most of the methods rely on some ``tightly folded'' structures that block further penetration of the preproteins into the translocase. For instance, translocation of epitope-tagged preprotein was blocked by epitope-specific antibody(13) . Covalent attachment of stable structures such as bovine pancreas trypsin inhibitor (12) or methotrexate-binding dihydrofolate reductase (14) moieties to the precursor protein also generates translocation intermediates. But, in all of these cases, the blockades were exerted during the events that occur in the middle of translocation of the bulk of the mature domain.
In the cotranslational translocation system in the eukaryotic endoplasmic reticulum, the ribosome-nascent chain complex offers an ideal experimental tool to define various translocation intermediate states, including those in quite early stages in translocation(17) . On the other hand, early biochemical events in the bacterial post-translational system have only insufficiently been investigated due to the lack of convenient methods to accumulate ``early intermediates.''
Mutants affected in the translocation processes provide useful clues about the early events in vivo. Especially the prl mutations in secY and secE loci, which broaden the specificity of signal sequence recognition, suggested a direct interaction between signal sequence and SecY/SecE, the main membranous subunits of the E. coli translocase. Silhavy and co-workers (18, 19) found a striking clustering of prlA mutations in the first periplasmic domain and in the seventh and tenth transmembrane domains, which they proposed are essential for SecY's recognition of signal sequence and SecE. Our isolation of cold-sensitive and dominant sec mutations in secY suggested that the region C-terminal to transmembrane domain 8 is important for translocation facilitation and that the fourth cytoplasmic region is required for interaction with SecE(20, 21, 22) . To obtain an integrated picture of translocation in molecular terms, it is essential to analyze the nature of the early interaction between precursors and the SecY-SecE-SecG complex on the membrane in vitro. More specifically, it is highly desired to devise a new method to ``trap'' translocation intermediates in the early stages in vitro.
In this report, we exploited the technique of
hexahistidine tagging to use His-tagged precursor proteins
for easy purification as well as for generation of a new type of
translocation intermediates. We found that the pro-OmpA derivatives
with a His
tag in their N-terminal regions of their mature
proteins could not be translocated in the presence of a low
concentration of Ni
. Ni
acted only
on pro-OmpA derivatives with a His
tag in the N-terminal
region of the mature sequence. This inhibition occurs only at an early
stage(s) of the translocation reaction and can be released by adding
histidine, which competes for chelating Ni
with the
His
tag in the preprotein. Ni
did not
inhibit, but rather enhanced, membrane association of
His
-tagged OmpA, suggesting that it acts just after the
membrane targeting step. This system will be suitable for dissecting
the early events in bacterial protein translocation.
To construct pTYE005, a His fusion vector, the ``His
oligonucleotides''
5`-GGAATTCATCGAAGGCCGTCACCATCACCATCACCACATCGATGG-3` and
5`-CCATCGATGTGGTGATGGTGATGGTGACGGCCTTCGATGAATTCC-3` were annealed,
digested with EcoRI and ClaI, and cloned into
pBluescript SK(-) digested with the same enzymes. For the
construction of a c-Myc fusion vector, pTYE006, the ``c-Myc
oligonucleotides''
5`-CCATCGATGAAGAACAGAAACTCATCTCCGAAGAGGACCTGCTGCGCAAACGTTAAGGTACCC-3`
and
5`GGGTACCTTAACGTTTGCGCAGCAGGTCCTCTTCGGAGATGAGTTTCTGTTCTTCATCGATGG-3`
were annealed and cloned between the ClaI and KpnI
sites of pBluescript SK(-). pTYE007, a His
-c-Myc
fusion vector, was then constructed by ligating a 1.12-kb ClaI-ScaI fragment of pTYE005 and a 1.84-kb ClaI-ScaI fragment of pTYE006. The His
and c-Myc oligonucleotides are designed to encode IEGRHHHHHH
(factor Xa site followed by a His
tag) and
EEQKLISEEDLLRKR-ocher (c-Myc monoclonal antibody 9E10
epitope(24) ), respectively. When these double-strand
oligonucleotides were cloned into pBluescript SK(-) as described
above, they did not disrupt the lacZ
open reading frame,
and the tags were encoded on another reading frame. Therefore, the lacZ assay can be used for cloning an exogenous DNA fragment
into these three vectors.
To construct the OmpA-His-Myc-expressing
plasmid, a 1.23-kb SspI-PstI fragment of pRD87
covering the ompA open reading frame was cloned into pTYE007
to obtain pTYE008. A 0.25-kb BglII-EcoRI fragment of
pTYE008 was replaced with a 0.15-kb fragment of the 3`-terminal region
of the ompA gene amplified by polymerase chain reaction with
the primers 5`-AAAGGTATCCCGGCAGAC-3` and
5`-GGAATTCAGCCTGCGGCTGAGTTAC-3` and digested with BglII and EcoRI. The resulting plasmid, pTYE009, encoded an in-frame
fusion between OmpA and the His-c-Myc tag. A 2.94-kb EcoRI-ScaI fragment from pTYE009 was ligated with a
1.16-kb EcoRI-ScaI fragment from pTYE006 to yield
pTYE018, encoding OmpA-c-Myc (abbreviated as OmpA`) fusion protein. To
insert a His
tag at various locations in the OmpA mature
domain, pTYE018 was mutagenized with the mutagenic primers described
below.
5`-GTAGCGCAGGCCGCTCCGAAACACCATCACCATCACCATATCGATAACACCTGGTACACTGG-3`
was used for the construction of pTYE050, encoding 3His-OmpA`, which
has a His
-Ile insert after the third amino acid of the OmpA
mature sequence;
5`-GATAACACCTGGTACCACCATCACCATCACCATAGTACTGGTGCTAAACTG-3` for pTYE086,
encoding 8His-OmpA`, which has His
-Ser after the eighth
amino acid; 5`-TCCCAGTACCATGATCACCATCACCATCACCATAGTACTGGTTTCATCAAC-3`
for pTYE098, encoding 20His-OmpA`, which has His
-Ser after
the 20th amino acid; and
5`-CGTTTATGGTAAAAACCACCATCACCATCACCATGTCGACACCGGCGTTTCTCC-3` for
pTYE112, encoding 114His-OmpA`, which has His
-Val after
His-114. Mutations were confirmed by the presence of new restriction
sites, underlined in the oligonucleotides (ClaI, ScaI, ScaI, and SalI, respectively). To
construct SecB-overproducing plasmid pTYE025, a 1.24-kb BamHI-PvuII secB fragment derived from
pAK330 (43) was subcloned into pBluescript KS(-) digested
with BamHI and EcoRV.
SecA was prepared from CU148/pKY173, ()a SecA overproducer, by a combination of Matrex gel red A
dye binding column and DEAE-Sepharose Fast Flow chromatography. SecB
was overproduced in TYE126 (JM109(DE3)/pTYE025) from the T7 promoter
and purified as described (26) up to the Q-Sepharose column
step, and the sample was further purified by butyl-Sepharose column
chromatography.
S-Labeled pro-OmpA derivatives were
prepared by in vitro transcription and translation using
appropriate plasmids, E. coli S130, and
[
S]Met(27) . Proteins were then
precipitated with 5% trichloroacetic acid (final concentration) and
dissolved in HU buffer (50 mM HEPES/KOH, pH 8.0, 1 M NaCl, 8 M urea, 10 mM
-mercaptoethanol).
SecA ATPase
was assayed as described in (7) . Protein concentration was
determined by the Bio-Rad protein assay solution with bovine
-globulin as a standard.
Figure 1:
His- and c-Myc-tagged OmpA
derivatives. The OmpA derivatives used in this study are summarized. Hatched and open boxes represent signal and mature
domains of OmpA, respectively. Shaded and filled boxes indicate c-Myc and His
tags, respectively. Numbers above the filled boxes indicate insertion positions of
the His
tag. a. a. r., amino acid
residues.
Figure 2:
Accumulation, purification, and
translocation of His-tagged pro-OmpA derivatives.
Pro-3His-OmpA` and pro-OmpA-His` were expressed from pTYE050 and
pTYE009, respectively, in TYE055, a secY24 strain that
overproduces Syd, a strong secretion inhibitor in this strain. A, the cells expressing 3His-OmpA` were disrupted by
sonication in the presence of 8 M urea and fractionated by
centrifugation at 100,000
g for 1 h. Materials
equivalent to 0.66 Klett unit of culture were subjected to Western
blotting with anti-c-Myc epitope antibody. Lane 1, total
lysate; lane 2, supernatant; lane 3, pellet. Similar
results were obtained for OmpA-His`. p, precursor; m,
mature protein. B, pro-OmpA-His` and pro-3His-OmpA` were
purified as described under ``Experimental Procedures,'' and
0.2 µg each was analyzed on 12.5% SDS-polyacrylamide gel and
stained with Coomassie Brilliant Blue. Lane M, molecular mass
standards; lane 1, pro-OmpA-His`; lane 2,
pro-3His-OmpA`. There is a slight contamination of mature OmpA-His` in lane 1. C, translocation of purified pro-OmpA-His`
was tested using INV prepared from TYE024. Precursor protein (1.32
µg in 8 M urea) was diluted into translocation reaction
mixture (40 µg/ml SecA, 20 µg/ml SecB, 1.6 mg/ml INV in
standard assay buffer) and incubated at 37 °C for 30 min. Lanes indicated +ATP received an ATP/ATP regeneration
system and 5 mM NADH (final concentration). After the
incubation, three 7-µl aliquots were withdrawn and subjected to
mock treatment, trypsin treatment (0.25 mg/ml TPCK-treated trypsin), or
trypsin/Triton X-100 treatment (0.25 mg/ml TPCK-treated trypsin, 0.2%
Triton X-100), as indicated, at 0 °C for 10 min. The
trypsin-resistant portions of OmpA derivatives were visualized by
Western blotting with anti-c-Myc antibody.
Figure 3:
In vitro translocation of
pro-3His-OmpA`, but not of pro-OmpA-His`, is sensitive to
Ni. A,
S-labeled pro-OmpA`,
pro-OmpA-His`, and pro-3His-OmpA` were synthesized in vitro and post-translationally translocated into INV in the presence of
various concentrations of NiCl
and/or NTA/Na
.
In lanes 1-3, 20, 10, and 0% of the in vitro translated precursors used in the reactions were run,
respectively. Each reaction mixture contained the reagents indicated
above the panel. Portions of translocation reactions were treated
either with trypsin (lanes 4-11) or with buffer (lanes 12-19). Lanes 4 and 12 show
negative controls of the reaction without ATP, and lanes 5 and 13 represent positive controls of the standard reaction
mixture without NiCl
or NTA/Na
. p,
precursor; m, mature protein. B, purified
pro-3His-OmpA` (lanes 1-8) and pro-OmpA-His` (lanes
9-16) were translocated in the absence (lanes 1, 5, 9, and 13) or presence (lanes 2, 6, 10, and 14) of 100 µM NiCl
. Lanes 3, 7, 11, and 15 are the minus-ATP controls, and lanes 4, 8, 12, and 16 are reactions without INV.
Samples were subjected to SDS-PAGE with (lanes 1-4 and 9-12) or without (lanes 5-8 and 13-16; one-fourth of the trypsinized samples were used)
trypsinization, and OmpA species were visualized by Western blotting. Asterisks represent unrelated
bands.
The above results
suggested that the inhibition of translocation by Ni was dependent on the position of the His
tag in the
OmpA protein. We constructed a series of OmpA derivatives with a
His
tag at various positions in the mature domain of the
OmpA protein (Fig. 1). The Ni
sensitivities of
their translocation were compared using in vitro translated
preproteins (Fig. 4). Translocation of pro-3His-OmpA` (Fig. 4A,
) and pro-8His-OmpA` (
) with
His
tags after the third and eighth amino acids of the
mature domain, respectively, was completely inhibited by 120 µM Ni
(Fig. 4A). The same
concentration of Ni
did not inhibit translocation of
pro-OmpA`, pro-OmpA-His`, or pro-114His-OmpA`. Pro-20His-OmpA` showed
an intermediate sensitivity to Ni
(Fig. 4A,
). Although 400 µM Ni
somewhat inhibited translocation of even
pro-OmpA-His` and pro-114His-OmpA`, but not of pro-OmpA`, we did not
pursue this Ni
effect further. We conclude that
20-100 µM Ni
affects translocation
of only precursor proteins with a His
tag in the N-terminal
region of the mature domain.
Figure 4:
Ni sensitivity of
various His
-tagged OmpA derivatives. The pro-OmpA
derivatives listed in Fig. 1were translated in vitro and post-translationally translocated into INV in the presence of
0, 12, 40, 120, or 400 µM NiCl
. After a 15-min
reaction, samples were trypsin-treated and run on 12.5%
SDS-polyacrylamide gel, and the amounts of the trypsin-protected
species were quantified. A, translocation is expressed as
percent of trypsin-protected OmpA species against input.
,
pro-OmpA`;
, pro-OmpA-His`;
, pro-3His-OmpA`;
,
pro-8His-OmpA`;
, pro-20His-OmpA`;
, pro-114His-OmpA`. B, inhibition efficiency of NiCl
at 120 µM on translocation of various pro-OmpA derivatives is plotted
against position of the His
tag.
The spectrum of the positional effect
in the His tag and the Ni
-dependent
translocation block (Ni
inhibition at 120
µM; summarized in Fig. 4B) reminds us of
the results of Andersson and von Heijne(31) , who found that
the first 30 amino acid residues of the mature domain of the secretory
precursor are particularly sensitive to introduction of positive
charges (6 consecutive lysines). They proposed to call this region the
``translocation initiation domain.'' The inhibitory effect of
Ni
on translocation of N-terminally
His
-tagged pro-OmpA` may also result from introduction of
positive charges in the translocation initiation domain by chelating
Ni
. Consistent with this notion, protein
translocation of pro-3His-OmpA` in the presence of 80 µM NiCl
was restored by adding NTA (Fig. 3A, lower panel, compare lanes 6 and 10), which should interact with the His
tag with high affinity through chelating
Ni
(30) . Because an NTA molecule has two
minus charges at pH 8.0, at which we performed the translocation assay,
the antagonistic activity of NTA on Ni
inhibition may
simply be explained by its charge neutralization effect. Still other
explanations remain. For instance, a steric effect caused by
introduction of Ni
in the His
tag portion
may prevent the conformational change required for the molecular
movement through the translocation channel.
Figure 5:
Ni affects only early
step(s) of translocation reaction. Two types of translocation reactions
were performed as summarized above the graph. In reaction scheme
I, a 150-µl reaction was started at 37 °C by adding 0.6
µg (14.6 pmol) of
I-labeled pro-3His-OmpA`. At the
indicated times, a 10-µl aliquot was withdrawn and transferred to a
new tube containing 1 µl of 1.1 mM NiCl
and
further incubated for a total of 25 min. Another 150-µl reaction
was carried out as described above, and a 10-µl aliquot was cooled
in ice water to measure the amounts of translocated 3His-OmpA` at each
time point (scheme II). After the reaction, samples were
trypsin-treated and subjected to SDS-PAGE. A, translocation
efficiencies of the two reaction schemes at each time point are
plotted.
, scheme I;
, scheme II. Translocation efficiency
is expressed by the amount of trypsin-resistant pro-OmpA and mature
OmpA derivatives in a 25-µl standard reaction for comparison with
other experiments. B, efficiency of Ni
inhibition after its addition was calculated from the data at
each time point in A by the following formula: % inhibition
efficiency = (((scheme I value) - (scheme II
value))/((scheme I value at 25 min) - (scheme II value)))
100.
Although it
has not been established to what extent the in vitro translocation reaction using the E. coli INV system is
synchronized and how long it takes for a single precursor molecule to
complete translocation, the following considerations will be useful for
interpretation of the data obtained. If one assumes that a single cycle
reaction occurs synchronously and that an inhibitory action is exerted
at a specific substep of the reaction, addition of the inhibitor prior
to the inhibition step in the scheme I reaction completely blocks the
final yield of translocation, whereas after the inhibition step, the
inhibitor is totally ineffective in lowering the final yield. As shown
in Fig. 5A (), the total amount of translocation
(scheme II) increased steadily up to the 25-min incubation period
examined. The translocation yields in the scheme I reaction (
)
were significantly higher that those in scheme II, except for the 0-
and 1-min time points. The effectiveness of the Ni
inhibition after its addition is shown in Fig. 5B. It was found that the inhibitory action was
gradually lost during the course of this in vitro reaction.
This indicates that Ni
does not inhibit the reaction
uniformally at every step or, at least, the final step of
translocation. Rather, the inhibition point(s) should be located early
in the translocation pathway. This interpretation is also supported by
the fact that Ni
blocked the signal cleavage of
pro-3His-OmpA`, which occurs early in translocation(12) . The
lack of a clear ``cutoff'' point in Fig. 5B may be due to asynchrony in the initial process as well as to
possible random slowing down during late steps of translocation.
Therefore, we conclude that Ni
acts early in the
translocation event(s).
Figure 6:
Ni does not inhibit, but
rather enhances, membrane targeting of pro-His-OmpA`. 1 µg of
pro-3His-OmpA` or pro-OmpA-His` was incubated in 100 µl of
translocation reaction mixture at 0 °C for 15 min in the presence (+Ni
lanes) or
absence (control lanes) of 100 µM NiCl
. Preproteins associated with urea-treated INV
were recovered by sedimentation through a 20% sucrose cushion (see
``Experimental Procedures''). Localization of preproteins,
SecA, and SecY (INV marker) in soluble (upper phase of the gradient)
and membrane (pellet) fractions was analyzed by Western blotting. T, total; S, soluble fraction; P, pellet
(membrane fraction).
We then subjected the membrane-targeted pro-OmpA preparation to the
translocation reaction. Pro-3His-OmpA` that had been targeted to the
membrane in the presence of Ni was competent for the
subsequent translocation. This translocation was
Ni
-sensitive as shown in Fig. 7. Slight
translocation of pro-3His-OmpA` was noted in the presence of
Ni
; possibly a small fraction of pro-3His-OmpA` had
escaped from the Ni
-sensitive step during sample
manipulations. These results imply that NiCl
inhibits a
step that occurs subsequent to the recruitment of the precursor to the
membrane. It might result in the apparent accumulation of
pro-3His-OmpA` in INV through stabilizing or ``locking'' the
membrane-bound state of the precursor.
Figure 7:
Membrane-targeted pro-3His-OmpA` in
presence of Ni is competent for subsequent
translocation in absence of NiCl
.
I-Labeled
pro-3His-OmpA` (0.35 µg, 8.5 pmol) was fist incubated with INV in a
175-µl reaction mixture at 0 °C for 15 min in the presence (1st incubation, +Ni
)
or absence (1st incubation, control) of 100
µM NiCl
. The vesicles were recovered as
described in the legend of Fig. 6; resuspended in standard
reaction mixture; and further incubated with ATP (control),
with ATP and 100 µM NiCl
(+Ni
), or without ATP (-ATP) at 37 °C for 15 min (2nd
incubation). The amounts of the translocated OmpA species were
assessed by trypsin treatment, SDS-PAGE, autoradiography, and its
quantification. Translocation is expressed as the increase in
trypsin-resistant OmpA species (femtomoles)/minute/standard 25-µl
reaction volume.
Figure 8:
Translocation inhibition by
Ni is released by histidine. A 150-µl reaction
was started by adding 0.6 µg (14.6 pmol) of
I-labeled
pro-3His-OmpA` in the absence (
) and presence (
and
)
of 100 µM NiCl
(final concentration). As
marked by the arrows, a final concentration of 30 mM histidine HCl, pH 8.0, was added to the reactions with
Ni
at 4 min (
) or at 10 min (
). Also
shown is a 25-min reaction in the presence of 100 µM NiCl
without receiving histidine (
). 10-µl
aliquots were withdrawn at each time point, and the amounts of
translocated 3His-OmpA` were determined after trypsin treatment,
SDS-polyacrylamide gel electrophoresis, and quantitative
autoradiography.
We do not believe, however, that the
apparent translocation rates shown in Fig. 5and Fig. 8represent the kinetics of translocation of individual
precursor molecules. They must represent a sum of the heterogeneous
population at different stages of the reactions. The fact that the
Ni trap only shortened the initial lag period but did
not enhance the apparent translocation rate upon release may suggest
that there are multiple ``bottleneck'' processes in vitro and that some of them occur after the
Ni
-sensitive step. Although we need a further
investigation of the molecular nature of the
Ni
-trapped intermediate, it is a new type of
``reversible'' inhibition of an early event of translocation
in the bacterial system.
In this study, we made use of the His tag method
developed by Bush et al.(29) not only to purify
chemical amounts of E. coli pro-OmpA derivatives, but also to
dissect their translocation across INV of E. coli in vitro.
Our system using a combination of secY24 mutation and
overexpression of syd(28) will be useful to
accumulate bacterial precursor proteins in E. coli cells. We
found that a His
tag introduced into the N-terminal region
of the OmpA mature domain confers Ni
sensitivity to
its translocation. Ni
inhibits only the early step(s)
of the translocation reaction, which is after the association of
precursor with the membrane, but before the signal cleavage. Inhibition
can be released by addition of histidine, which breaks the
His
Ni
chelating complex.
It is
likely that the effect of Ni is due to introduction
of positive charges to the N-terminal mature region of the precursor
protein. The position-specific Ni
effects are
difficult to explain in terms of nonspecific jamming of the
translocation machinery, damage to the
µ
generating system by heavy
metal ion, or the molecular size of the
His
Ni
chelating complex. The fact
that Ni
inhibition was observed only when the
His
tag was positioned within the first 20 residues or so
of the mature domain of pro-OmpA suggests that the chelating complex
affects some specific event(s) where the translocase interacts with
this particular N-terminal region of the precursor protein. Toxicity of
positively charged amino acids in the N-terminal mature domain has been
reported in several secretory proteins in vivo and in
vitro(32, 33, 34, 35) .
Systematic insertion of Lys
after the first or second
transmembrane domain of leader peptidase indicates that the 30-40
amino acid residues following the signal sequence or the signal anchor
sequence form a special domain that cannot tolerate the positive
charges(31) . Andersson and von Heijne (31) termed this
region the translocation initiation domain. The location of the
His
tag that confers Ni
-sensitive
translocation on OmpA coincided well with the translocation initiation
domain. The antagonistic effect of NTA can be explained by its minus
charges and ability to form a ternary complex with the His
motif and Ni
, although we do not have direct
evidence of the translocation of this ternary complex. Another
possibility might be that the N-terminal region of the mature protein
is quite sensitive to a conformational constraint. For instance, the
initial formation of the hairpin loop structure in the N terminus of
preproteins could be important for translocation initiation, and the
local conformation of the His
Ni
complex may prevent the formation of such a structure.
Previously, the effect of positive charges in the translocation
initiation domain had been discussed from the point of view of the
electrochemical nature of the membrane, such as interaction of the
N-terminal region of preprotein with acidic phospholipids and
determination of its orientation according to the ``positive
inside
rule''(31, 32, 33, 34, 35) .
However, the existence of positive charges themselves in this domain,
but not charge balance flanking the signal sequence core, is essential
for inhibition(34) . Both in the bacterial plasma membrane and
in the eukaryotic endoplasmic reticulum, which have homologous
translocation machinery, compelling evidence suggests that
proteinaceous pores composed of SecY-SecE-SecG or
Sec61-Sec61
-Sec61
complexes lead preproteins across the
membrane(14, 36, 37, 38, 39) .
A tempting and serious possibility is that the translocation initiation
domain directly interacts with some part of the translocase and that
this interaction itself is sensitive to positively charged amino acids
in this domain.
The existence of prlA and prlG mutants is one piece of strong evidence for direct interaction
between membranous translocase subunits and precursor
protein(2, 18, 19) . Certain prlA mutants can also translocate mutant preprotein with positive
charges in the translocation initiation domain in
vivo(33) . We found that pro-3His-OmpA` translocation in vitro was less sensitive to Ni when INV
prepared from the prlA3 mutant was used. (
)We
suppose that Ni
affects translocation of N-terminally
His
-tagged pro-OmpA through the step of precursor protein
recognition by the SecY-SecE-SecG complex. A similar signal sequence
recognition event by translocase is also proposed in the mammalian
Sec61 system, as revealed in a reaction in the absence of signal
recognition particle(40) . Actually, translocation across the
endoplasmic reticulum is also sensitive to positives charges in the
N-terminal portion of the mature protein albeit its lower sensitivity
compared with the prokaryotic system(41) .
While signal
sequence recognition by SecY could be regarded as essentially a
proofreading activity that rejects nonfunctional precursor
proteins(18) , the fact that histidine addition can restore
translocation without a short lag may indicate that a
translocon-associated precursor can be reactivated on site, i.e. no rejection occurs on the membrane. We detected efficient
cross-linking between pro-3His-OmpA` on the membrane with SecA
irrespective of the existence of Ni,
supporting this notion. Interaction of SecY(-SecE-SecG) with the
signal sequence and the N-terminal portion of the mature protein may be
required for some intrinsic mechanism of translocation, such as gate
opening of the translocation channel(18, 40) . It is
interesting to point out that the prlA3 and other prlA alleles that suppress the translocation defects caused by the
basic amino acids (33) or the
His
Ni
conjugate (this study) reside
in the first periplasmic domain of the SecY
protein(18, 42) . The idea that this region of SecY
acts to accept or reject the early mature part (3) is
reasonable in terms of topological consideration of SecY and
preprotein; this SecY domain may recognize preprotein during or after
the insertion of its hairpin loop structure composed of the signal
sequence and the translocation initiation domain. But our results imply
that the Ni
-inhibited precursor can still remain
associated with the membrane. On the other hand, the His
tag portion of the Ni
-trapped precursor on the
membrane should not be completely buried in the translocation
machinery. It is accessible to exogenous histidine added from the
cytoplasmic surface. Either the intermediate-bearing translocation
channel may be open to the cytoplasmic side, or this intermediate is in
a fast equilibrium between a membrane-embedded state and a
water-accessible state.
Since most translocation intermediate traps
developed so far confer a translocation block at its middle or late
stages(11, 12, 13, 14) , the
HisNi
-tagged precursor system is a
novel tool for analyzing initial translocation events. It will be
useful for investigating the nature of signal recognition by the
SecY-SecE-SecG complex through in vitro analysis of translocon
mutants, especially prlA mutants. Also in vitro characterization of the secY mutants with lowered
secretory efficiency (22) with the
His
Ni
-tagged precursor system will
be promising in identifying secY mutants with a deficiency in
the early events.