(Received for publication, October 18, 1994)
From the
A mutant form of SecY, SecY1, was previously
suggested to sequester a component(s) of the protein translocator
complex. Its synthesis from a plasmid leads to interference with
protein export in Escherichia coli. SecE is a target of this
sequestration, and its overproduction cancels the export interference.
We now report that overexpression of another gene, termed syd,
also suppresses secY
1.
The nucleotide sequence of syd predicted that it encodes a
protein of 181 amino acid residues, which has been identified by
overproduction, purification, and determination of the amino-terminal
sequence. Cell fractionation experiments suggested that Syd is loosely
associated with the cytoplasmic surface of the cytoplasmic membrane.
SecY may be involved in the membrane association of Syd since the
association is saturable, the extent of which depends on the
overproduction of SecY. SecY is rapidly degraded in vivo unless its primary partner, SecE, is sufficiently available.
Overproduction of Syd was found to stabilize oversynthesized SecY.
However, Syd cannot stabilize the SecY
1 form of
SecY. Thus, in the presence of both secY
and secY
1, Syd increases the
effective SecY
/SecY
1 ratio in the
cell and cancels the dominant interference by the latter. We also found
that overproduction of Syd dramatically inhibits protein export in the secY24 mutant cell in which SecY-SecE interaction has been
weakened. These results indicate that Syd, especially when it is
overproduced, has abilities to interact with SecY. Possible
significance of such interactions is discussed in conjunction with the
apparent lack of phenotypic consequences of genetic disruption of syd.
Newly synthesized non-cytoplasmic proteins must traverse the membrane. Instead of directly moving through the lipid bilayer, they utilize proteinaceous components of the membrane (Joly and Wickner, 1993; Mothes et al., 1994). Genetic and biochemical studies of the Escherichia coli system revealed that several integral membrane proteins, SecY, SecE, SecD, SecF, and SecG as well as a peripheral membrane ATPase, SecA, participate in this reaction (Schatz and Beckwith, 1990; Pugsley, 1993; Nishiyama et al., 1994; Douville et al., 1994). Among them, SecY and SecE are the two central membrane-embedded components, which function in close mutual interaction (Ito, 1992).
We previously isolated a dominant negative
allele of secY, secY1 (Shimoike et al., 1992). Expression of secY
1 from a plasmid
interferes with protein export even in the presence of the wild-type secY allele on the chromosome. Evidence suggests that the
altered and inactive SecY protein, with an internal deletion of 3 amino
acid residues at the interface between cytoplasmic domain 5 and
transmembrane segment 9, competes with the wild-type SecY molecules for
the association with SecE, the limited partner of SecY in the
translocator complex (Shimoike et al., 1992; Taura et
al., 1993; Baba et al., 1994). Such a sequestration
mechanism was supported by the fact that overproduction of SecE
alleviates the dominant negative effect of secY
1 (Shimoike et
al., 1992). The dominant interference can also be alleviated by
intragenic suppressor mutations that cluster in cytoplasmic domain 4,
suggesting that this region is required for SecE binding (Baba et
al., 1994). Although SecY in the wild-type cell is stable, this
stability is due to its association with SecE (Taura et al.,
1993). Thus, most overexpressed SecY molecules are degraded immediately
upon their synthesis unless SecE is simultaneously overproduced
(Matsuyama et al., 1990; Taura et al., 1993).
In
the work reported here, we attempted to identify new SecY-interacting
factors by isolating additional multicopy suppressors of secY1. If disruption of
such a gene causes an export defect, its product should be a component
of the translocation machinery that is sequestered by
SecY
1. We now report on identification (as a
multicopy suppressor of secY
1) and
characterization of a new gene, syd, and its product. (
)Our results suggest that SecE is the only sequestration
target of the dominant negative SecY, and Syd protects wild-type SecY
molecules that have failed to associate with SecE from proteolytic
degradation. Such protection is not seen for the dominantly inactive
SecY molecules. Thus, high intracellular concentration of Syd improves
protein export in the presence of SecY
1 through
preferential stabilization of the wild-type SecY protein. In addition,
an increased Syd concentration has been shown to potently block protein
translocation that is mediated by a mutant (secY24) form of
SecY, in which interaction with SecE has been weakened.
Figure 1:
Chromosomal segment around syd. pST6 is one of the plasmids obtained as multicopy
suppressors against the secY1 mutation. The 543-base pair open reading frame (syd) is
indicated. Also shown are the sequenced segment (1402 base pairs) as
well as segment carried on plasmids pST30 and pST42. Plac and kan represent the lac promoter and the kanamycin
resistance determinant that had been inserted within the syd gene on pST42, respectively.
Subcloning experiments suggested
that the multicopy suppression activity resided within the 1.3-kb EcoRI-SalI interval (Fig. 1). We sequenced
this region for 1402 residues and identified one open reading frame of
181 codons with a GUG initiation ( Fig. 1and Fig. 2). We
constructed a plasmid (pST30) that carried, under the lac promoter, a 642-base pair fragment containing the open reading
frame. It suppressed secY1. We designated this open reading frame syd (suppressor of secY dominance).
The nucleotide
and the deduced amino acid sequences did not show any extensive
homology of considerable length with the current data base entries (see
``Discussion'' for the possible existence of a SecE motif).
Figure 2:
Nucleotide and deduced amino acid
sequences of the syd region. The carboxyl-terminal region of
Syd protein is predicted to assume an -helical secondary
structure. Among this region, the residues underlined can form
a consecutive hydrophobic patch on one side of the helical wheel
projection, whereas the other side is mostly hydrophilic or charged.
The dottedunderline indicates the region of possible
local homology with SecE (see Fig. 11).
Figure 11:
Possible local homology between Syd and
SecE. The alignment includes a gap/insertion. Identical
amino acids are indicated by reversetype, and those
of similar characters are outlined.
Although the above screening specifically yielded the syd clones, we repeated similar screenings but this time used secY1 at a reduced
expression level from a pACYC184-based plasmid (pKY247) and a library
constructed under the lac promoter on a pBR322-based plasmid.
It was estimated that pKY241 and pKY247 express secY at levels
about 20- and 5-fold the chromosomally encoded secY expression, respectively (Taura et al., 1993). We
obtained several clones that apparently suppressed secY
1. However, all of
them were weak in that they (after recloning into a pACYC184-based
vector) could not suppress secY
1, which was
expressed from the pBR322-derived plasmid. In addition, many of them
caused retardation of the host cell growth and apparently contained
only incomplete open reading frames. The suppression in these cases
seemed to be nonspecific and somehow caused by abnormal proteins or by
slow-growing conditions. The suppression by yajC overexpression (Taura et al., 1994) is also weak in that
it cannot suppress secY
1 on the pBR322-based plasmid. (
)Thus, we have been able
to identify two genes, secE (Shimoike et al., 1992)
and syd, which can exert significantly strong multicopy
suppression of the export interference caused by secY
1.
Figure 3: Immunological detection of the syd gene product. Cells of MC4100/pST6 (syd) (lanes1 and 2), SI13-11 (syd::kan) (lane3), or MC4100 (lane4) were grown, and total cell extracts were separated by SDS-PAGE and electrophoretically blotted onto an Immobilon polyvinylidene difluoride membrane filter, which was then stained with anti-Syd serum. In lane2, immunodetection was carried out in the presence of the antigen peptides (2 µg/ml). Note that lanes1 and 2 received 20 µg of proteins and lanes3 and 4 received 46 µg of proteins. Arrowheads indicate Syd. The band just above the Syd band is a nonspecific background.
The
overall amino acid composition of Syd indicates that it is a
hydrophilic protein. Its carboxyl-terminal 18 residues were predicted
to form an amphiphilic helix (see Fig. 2). Syd contains 4
cysteine residues. Although the mobility of Syd in SDS-PAGE varies
under reducing (apparent molecular mass, 26 kDa) and non-reducing
(apparent molecular mass, 21 kDa) conditions, iodoacetoamide treatment
before addition of SDS rendered Syd migrate always as the reduced form.
Thus, cysteines in Syd are normally reduced, consistent with its
localization to the cytoplasmic surface of the membrane (see below).
We found that the purified Syd protein in Tris-HCl buffer tends to
stick to the plastic tubes during storage. Our estimation by
immunoblotting experiments showed that the cellular abundance of the
Syd protein is less than 1 10
molecules per cell.
Figure 4:
Cellular localization of Syd. A,
cells of MC4100 were grown in L broth to a mid-log phase and treated
with lysozyme-sucrose to yield the periplasmic fraction (Peri, lane1) and spheroplasts, which were subsequently
disrupted by sonication to yield the membrane (Memb, lane2) and the cytoplasmic (Cyto, lane3) fractions. Syd in each fraction was detected by
immunoblotting following SDS-PAGE. B, spheroplasts of MC4100
prepared as in A were disrupted by French press, and crude
membranes were separated into inner membrane (IM, lane1) and outer membrane (OM, lane2) fractions. The amount of Syd found in the inner
membrane fraction (lane1) was estimated to be about
10% of the total. C, cells of KI267
(CSH26/F`lac lacI
) (lanes1 and 2), KI267/pST30 (syd) (lanes3 and 4), or KI267/pST30/pKY4 (secY
) (lanes 5 and 6) were
grown in L-broth supplemented with 1 mM IPTG and appropriate
antibiotics and treated with lysozyme (0.1 mg/ml) in the presence of
20% (w/v) sucrose. CSH26 cells are known to release the cytoplasmic
contents by the above treatment (Akiyama and Ito, 1985). Samples were
centrifuged, and supernatant (S, lanes1, 3, and 5) and pellets (P, lanes2, 4, and 6) were subjected to SDS-PAGE
and immunoblotting. The upper part of the filter was stained with
antiserum against
-galactosidase (
-gal), and the
lower part was stained with anti-Syd serum.
Syd molecules that were overproduced from
a plasmid were largely (about 90%) soluble even in the lysozyme-treated
KI267 cells (Fig. 4C, lanes3 and 4). This suggests that the weak binding of Syd to the membrane
is saturable. When the secY gene was simultaneously
overexpressed, about 70% of overproduced Syd molecules became
sedimentable, whereas the localization of the cytoplasmic control,
-galactosidase, was not affected (Fig. 4C, lanes5 and 6). This result is
consistent with a notion that Syd interacts with the membrane-bound
SecY protein in the cell.
Figure 5:
Effects of Syd overproduction on export
of maltose-binding protein (MBP) and OmpA. Cells were grown at
37 °C to a mid-log phase in M9 medium supplemented with glycerol
(0.4%), maltose (0.4%), and amino acids except methionine and cysteine,
pulse labeled for 1 min with [S]methionine, and
subjected to immunoprecipitation with anti-maltose-binding protein and
anti-OmpA sera, followed by SDS-PAGE. Cells used were as follows: lane1, MC4100/pKY241 (secY
1)/pSTV29 (vector); lane 2, MC4100/pKY241/pST6 (syd); lane 3,
MC4100/pKY241/pST30 (syd); lane 4, TW130/pKY262 (secY39)/pSTV29; lane 5, TW130/pKY262/pST30; lane
6, AD208 (secY39)/pSTV29; lane 7, AD208/pST30. p and m indicate precursor and mature forms,
respectively.
We also introduced the syd plasmid into other secY mutants (Taura et al., 1994). As described later, Syd overproduction was found to be incompatible with the secY24 (Ts) mutation. Protein export in none of the other secY mutants was affected by Syd. Overproduced Syd improves protein export in the presence of wild-type SecY protein whose function has been compromised by certain dominant negative mutations.
Figure 6:
SecY-stabilizing ability of Syd. Cells
were pulse labeled at 37 °C for 30 s with
[S]methionine and chased with unlabeled
methionine for indicated periods. Samples were processed for
immunoprecipitation of SecY (opensymbols) and
SecY
1 (filledsymbols) that were
overproduced from pKY248 or pKY247, respectively. After SDS-PAGE,
relative intensities of the corresponding bands were quantitated. The
value at the chase time (0.5 min) was taken as unity for each strain;
without chase, lower labelings were obtained due to insufficient
polypeptide completion. Strains used were as follows:
,
TW130/pKY248 (secY
)/pNO575H (vector);
, TW130/pKY248/pST34 (syd);
,
TW130/pKY248/pKY271 (secE);
, TW130/pKY247 (secY
1)/pNO1575H;
,
TW130/pKY247/pST34;
, TW130/pKY247/pKY271. The upper and lowerparts of this figure should have been
superimposed but are shown separately for
clarity.
The above
experiments did not discriminate between the chromosomally encoded
SecY and the plasmid-encoded SecY species.
Nevertheless, it is inferred that Syd may suppress the dominant effects
of SecY
1 by preferentially stabilizing the
chromosomally encoded SecY
molecules. To demonstrate
this mechanism of suppression, we used a derivative of
SecY
1 in which a sequence derived from the
polylinker-LacZ
region of pUC19 had been attached to the carboxyl
terminus (this construction is referred to as
SecY
1
) (Taura et al., 1993; Baba et al., 1994). This attachment does not abolish the dominant
interfering ability of SecY
1 (Baba et al.,
1994) but enables its electrophoretic separation from
SecY
. Cells carrying plasmid pKY259 encoding
SecY
1
were pulse-chased, and SecY species were
immunoprecipitated. As shown in Fig. 7A, the presence
of pKY259 (secY
1
)
destabilized SecY
(open circles),
due presumably to a competition for binding with the SecE molecules
that are limited in number (Matsuyama et al., 1992; Taura et al., 1993). Simultaneous overproduction of Syd prevented
this degradation of SecY
(Fig. 7B, opensquares), while degradation of
SecY
1
continued or even accelerated (Fig. 7B, filledsquares). Steady
state accumulation of SecY
and
SecY
1
was examined by immunoblotting (Fig. 7, A and B, insets). Clearly,
accumulation of SecY
was preferred over that of
SecY
1
in the presence of the Syd plasmid (Fig. 7B).
Figure 7:
Demonstration of preferential Syd
stabilization of SecY over
SecY
1
. Pulse-chase of cells,
immunoprecipitation, and quantitation of SecY species were done as
described for the experiment shown in Fig. 6, except that SecY
species overproduced was the SecY
1-LacZ
fusion
protein (SecY
1
). Cells used were TW130/pKY259 (secY
1
)/pSTV29
(vector) in A and TW130/pKY259/pST30 (syd) in B. Opensymbols (
and
)
represent chromosomally encoded SecY
, whereas filledsymbols (
and
) represent
SecY
1
protein synthesized from the plasmid. Insets indicate steady state accumulation of
SecY
1
(upperband) and
SecY
(lowerband), as detected by
immunoblotting.
Plasmid pST30, in which syd expression is controlled
by the lac promoter, could be introduced into a secY24 derivative strain (KI297) carrying the lac repressor
overproducing mutation (lacI). The resulting
transformants were sensitive to IPTG, an inducer of the lac system. Cell growth was slowed down soon after addition of IPTG
and stopped at about 150 min (Fig. 8, opencircles). We observed a striking loss of viability that
started a few minutes after the induction, well before the growth
cessation (Fig. 8, closedcircles).
Eventually, the cells underwent partial lysis.
Figure 8:
Syd overproduction is toxic to the secY24 mutant cell. Cells were grown at 30 °C in amino
acid-supplemented M9-glycerol medium. IPTG (1 mM) was added as
indicated to overexpress syd. Cell growth was monitored by
measuring turbidity with a Klett colorimeter (filter 54). , KI297 (secY24 lacI
)/pST30;
, KI298 (secY
lacI
)/pST30. Viable
(colony-forming) cells were counted by plating appropriate dilutions
onto glucose-chloramphenicol agar, which was then incubated at 30
°C.
, KI297/pST30;
,
KI298/pST30.
At 16 min after the induction, when Syd accumulated considerably (Fig. 9B, lane5), export of OmpA and maltose-binding protein was already retarded (Fig. 9A, lane5). The export inhibition reached the maximum at about 30 min post-induction (Fig. 9A, lane6). Fig. 10shows kinetics of conversion of precursors to mature forms at 60 min after induction. In the induced cells (Fig. 10A, lanes5-8), only small fractions of OmpA and maltose-binding protein were converted to the mature form even after the 30-min chase point. This is in contrast to the cases of most sec mutations, which generally allow slow translocation/processing even under non-permissive conditions. It should be noted that even the uninduced cells (Fig. 8A, lanes1-4) contained a small amount of precursor maltose-binding protein that persisted during the chase. It appears that when Syd molecules exceed a certain level, they inactivate the mutationally altered translocation machinery that contains SecY24. Precursor molecules thus aborted appear to be in a dead-end state.
Figure 9:
Protein export in the secY24 mutant is severely blocked by Syd overproduction. A,
cells of KI297/pST30 were grown at 30 °C as described in Fig. 8. At 0 (lane1), 2 (lane2), 4 (lane3), 8 (lane4), 16 (lane5), and 30 (lane6) min after addition of IPTG, cells were pulse labeled
for 1 min with [S]methionine. Maltose-binding
protein (MBP) and OmpA were immunoprecipitated, separated by
SDS-PAGE, and visualized by a Bioimaging Analyzer BAS2000 (Fuji Film). p and m indicate precursor and mature forms,
respectively. B, non-radioactive whole cell proteins at each
time point were separated by SDS-PAGE, and Syd was visualized by
immunoblotting. About 5 µg of total proteins were used in each lane.
Figure 10:
Effects of syd overproduction on
export kinetics of maltose-binding protein (MBP) and OmpA in secY24 cells. Cells grown at 30 °C in the absence of IPTG (lanes1-4) or those induced for 60 min with
IPTG (lanes5-8) were pulse labeled for 30 s
with [S]methionine followed by chase with
unlabeled methionine. Samples were withdrawn at 0 (lanes1 and 5), 2 (lanes2 and 6), 8 (lanes3 and 7), and 30 (lanes4 and 8) min after initiation of chase and
processed for immunoprecipitation of maltose-binding protein and OmpA,
which were analyzed by SDS-PAGE. A, KI297 (secY24)/pST30 (syd); B, KI297/pSTV29
(vector); C, KI298 (secY
)/pST30. p and m indicate precursor and mature forms,
respectively.
Dominant negative mutation secY1 can be suppressed
in several different ways. First, it can be suppressed intragenically
by alterations in cytoplasmic domain 4 of SecY (Baba et al.,
1994). Second, overproduction of SecE can effectively cancel the
dominant export interference. Finally, as described in this paper,
overproduction of Syd can suppress the mutation.
We previously
hypothesized that the secY1 mutation inactivates some essential translocation-facilitating
activity of SecY but preserves the ability of SecY to associate with
other component(s) of the system, hence making the altered gene product
sequester such factors (Shimoike et al., 1992; Ito, 1992).
Thus, we originally anticipated that we might be able to identify a new
translocation factor by identifying multicopy suppressors. However, it
is now clear that loss of functions of either Syd or YajC (Taura et
al., 1994) does not lead to an export defect, excluding these
factors as a sequestration target. We now conclude that the main and
possibly sole target of SecY
1 sequestration is SecE,
whose loss of function confers an export-defective phenotype (Schatz et al., 1991). This is in concert with the finding that the secY24 alteration within the C4 domain intragenically
suppresses secY
1 and
weakens SecY-SecE interaction (Baba et al., 1994).
Our
estimation indicates that cellular abundance of Syd is comparable with
those of the Sec factors (Matsuyama et al., 1992). When
overproduced, it exhibits several phenotypes that are all consistent
with the notion that Syd can interact with SecY. First, its
overproduction improves protein export that has been dominantly
compromised by the secY1 mutation. Second, overproduction of Syd stabilizes SecY molecules
that are otherwise unstable due to the unbalanced synthesis. Thirdly,
localization of excess Syd in the cell can be affected when SecY is
also overproduced. Finally, overproduction of Syd shows specific
incompatibility with the secY24 mutation, giving a severest
degree of export defect known in E. coli.
The first three phenotypes of Syd overproduction may be interrelated and relevant to the mechanism of suppression. Syd should possess an ability to bind to the wild-type SecY molecules, and this binding somehow antagonizes the rapid proteolysis of SecE-free SecY molecules. This SecY-Syd interaction may have been reflected in the greater degree of membrane association of excess Syd observed in the presence of SecY overproduction.
In contrast to the wild-type SecY, the
SecY1 form of SecY does not respond to Syd
overproduction with respect to its stability in vivo. Thus,
the 3-residue internal deletion caused by this mutation may abolish the
Syd-binding ability of SecY. SecY-SecE binding in normal cells will
occur only among newly synthesized molecules (Joly et al.,
1994; Taura et al., 1993), and chromosomally encoded
SecY
and plasmid encoded SecY
1 will
compete with each other for binding to SecE. Under such dominant
interference conditions, the preferential stabilization of
SecY
by Syd will give greater opportunity to the
chromosomally encoded wild-type SecY molecules of association with
SecE. It should be noted that Syd and SecE might be competitive with
each other to some extent (see below), but wild-type SecY molecules
will soon bind to SecE due to their high affinity to SecE.
The secY24 mutant cells are extremely sensitive to overproduction
of Syd. The mutant form of SecY has been impaired in its interaction
with SecE such that the SecY24-SecE complex cannot be
co-immunoprecipitated (Baba et al., 1994). The secY24 mutant protein should be able to interact with Syd, since
overproduced SecY24 protein is stabilized by Syd (but not by SecE; Baba et al., 1994). Since the toxic effect of Syd against the secY24 cells appears early after induction, it probably
interferes directly with the functioning of the mutated SecY protein.
This has been supported by our in vitro translocation assays. ()The SecY24 protein remains stable even when its function
has been blocked by Syd overproduction (data not shown). Taken
together, it may be conceivable that increased concentration of Syd
leads to dissociation of the SecY24-SecE complex, which is held
together only weakly, and to the possible formation of the SecY24-Syd
complex. In principle, such competition between SecE and Syd could
occur in the secY
cells as well. In some
experiments, we observed slight retardation of protein export in the
Syd-overproducing secY
cells
.
It was shown that cytoplasmic domain 2 of SecE is important for its
functioning, and a sequence motif in this region has been conserved
during evolution (Murphy and Beckwith, 1994). It is interesting to note
that a segment of Syd contains a sequence that is somewhat similar to
the SecE signature sequence (Fig. 11). ()It remains
to be established whether this limited sequence similarity is of any
functional significance. The opposite specificity of SecE and Syd with
respect to their stabilization of SecY24 versus SecY
1 is not readily explainable in terms of
simple similarity between Syd and SecE.
We isolated syd mutants that had lost the toxic effect against the secY24 mutant cells. ()Many of them are still able to
stabilize SecY
and to suppress
SecY
1. These observations suggest that the two
functions of Syd, SecY stabilization and SecY24 inhibition, are
genetically separable, if not independent. Syd-SecY interaction that
results in SecY stabilization may involve the site of the secY
1 mutation
(cytoplasmic domain 5), whereas the interaction that results in the
toxicity against SecY24 may overlap the SecE-SecY interaction. To fully
understand such multiple interactions between Syd and SecY, the
putative amphiphilic
helix at the carboxyl terminus of Syd might
also have to be taken into consideration. When this segment was
deleted, the Syd protein was destabilized in vivo.
The observed stickiness of Syd to plastic tubes appears to be
consistent with its membrane (SecY)-interacting properties.
In contrast to overproduction of Syd, which is accompanied by multiple phenotypes, depletion of Syd due to gene disruption is silent as far as we have examined. What then is the role of Syd in normal cells? It is conceivable that Syd controls activity of the translocation machinery by intervening against the SecY-SecE complex or stabilizing a transient state of SecY and that such a control mechanism operates under some extreme conditions. It is also possible that cells contain a functional substitute for Syd. These possibilities should be examined by physiological and genetic approaches.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBank(TM)/EMBL Data Bank with accession number(s) D38520[GenBank].