THE JOURNAL OF BIOLOGICAL CHEMISTRY
18835–18840, 1998
© 1998 by The American Society for Biochemistry and Molecular Biology, Inc.
Syd, a SecY-interacting Protein, Excludes SecA from the SecYE
Complex with an Altered SecY24 Subunit*
(Received for publication, April 13, 1998, and in revised form, May 11, 1998)
Ei-ichi Matsuo‡, Hiroyuki Mori‡, Takashi Shimoike‡§, and Koreaki Ito‡¶
From the ‡Institute for Virus Research, Kyoto University, Sakyo-ku, Kyoto 606-8507, Japan
Syd is an Escherichia coli cytosolic protein that inter-
acts with SecY. Overproduction of this protein causes a
number of protein translocation-related phenotypes, in-
cluding the strong toxicity against the secY24 mutant
cells. Previously, this mutation was shown to impair the
interaction between SecY and SecE, the two fundamen-
tal subunits of the membrane-embedded part of protein
translocase. We have now studied in vitro the mecha-
nisms of the Syd-directed inhibition of protein translo-
cation. Pro-OmpA translocation into inverted mem-
brane vesicles (IMVs) prepared from the secY24 mutant
cells as well as the accompanied translocation ATPase
activity of SecA were rapidly inhibited by purified Syd
protein. In the course of protein translocation, high af-
finity binding of preprotein-bearing SecA to the trans-
locase on the IMV is followed by ATP-driven insertion of
the 30-kDa SecA segment into the membrane. Our exper-
iments using 125I-labeled SecA and the secY24 mutant
IMV showed that Syd abolished both the high affinity
SecA binding and the SecA insertion. Syd was even able
to release the inserted form of SecA that had been sta-
bilized by a nonhydrolyzable ATP analog. Syd affected
markedly the proteolytic digestion pattern of the IMV-
integrated SecY24 protein, suggesting that Syd exerts
its inhibitory effect by interacting directly with the
SecY24 protein. In accordance with this notion, a SecY24
variant with a second site mutation (secY249) resisted
the Syd action both in vivo and in vitro. Thus, Syd acts
against the SecY24 form of translocase, in which SecY-
SecE interaction has been compromised, to exclude the
SecA motor protein from the SecYE channel complex.
Protein translocation across the E. coli plasma membrane is
facilitated by multiple Sec factors (for reviews, see Refs. 1 and
2). A membrane-interacting ATPase, SecA, binds with high
affinity to the membrane containing the functional SecY/SecE
components (3). This is then followed by the ATP-dependent
insertion of a SecA segment into the membrane (4). ATP
hydrolysis-dependent release of the preprotein and deinsertion
of SecA allow the next cycle of polypeptide movement (4). SecY,
SecE, and SecG are the essential core components in the mem-
* This work was supported by grants from the Ministry of Education,
Science and Culture, Japan; the Human Frontier Science Program
Organization; and CREST, Japan Science and Technology Corp. (to
K. I.) and a Japan Society for the Promotion of Science Research Fel-
lowship for Young Scientists (to E. M.). The costs of publication of this
article were defrayed in part by the payment of page charges. This
article must therefore be hereby marked “advertisement” in accordance
with 18 U.S.C. Section 1734 solely to indicate this fact.
§ Present address: Dept. of Virology II, National Institute of Infec-
tious Diseases, 1-23-1, Toyama, Shinjuku-ku, Tokyo 162-8640, Japan.
¶ To whom correspondence should be addressed: Inst. for Virus Re-
search, Kyoto University, Sakyo-ku, Kyoto 606-8507, Japan. Tel.: 81-
75-751-4015; Fax: 81-75-771-5699 or 81-75-761-5626; E-mail: kito@
virus.kyoto-u.ac.jp.
This paper is available on line at http://www.jbc.org
This is an Open Access article under the CC BY license.
Vol. 273, No. 30, Issue of July 24, pp.
Printed in U.S.A.
brane, having 10, 3, and 2 transmembrane segments, respec-
tively. They not only provide the high affinity binding site for
SecA (3) but allow productive SecA insertion (4, 5), and they are
thought to provide a channel-like pathway for the movement
of polypeptide chain. These membrane proteins form a trim-
eric complex (6 – 8) in which SecY interacts independently
with SecE and SecG (9). Proteoliposomes reconstituted only
from SecY and SecE exhibit some basic translocation activi-
ties in conjunction with SecA (10). In addition, the inclusion
of SecG markedly stimulates the translocation activities of
the SecYE proteoliposomes (8, 11).
In vivo, SecY requires SecE as the stabilizing partner (12,
13). Thus, uncomplexed forms of SecY are degraded rapidly by
the FtsH proteolytic system (14). We proposed previously that
a dominant negative variant of SecY, SecY2d1, with a small
internal deletion, sequesters SecE in an inactive complex (15).
Intragenic suppressor mutations that abolish the dominant
effect preferentially occur within or around cytoplasmic domain
4 (C4) of SecY (16). The secY24 amino acid substitution (Gly240
3 Asp) in C4 (17), which also suppresses secY2d1 intrageni-
cally, abolishes the SecY-SecE affinity co-isolation (9, 16).
Thus, C4 is an essential cytoplasmic domain that is important
for the interaction between SecY and SecE. The importance of
the second cytoplasmic segment (C2) of SecE for SecY-SecE
interaction was also shown (18).
The secY2d1 mutation allowed us to identify a new gene, syd,
as a multicopy suppressor against this mutation (19). The syd
gene product (Syd) is a hydrophilic protein of 181 amino acid
residues, which is associated weakly with the membrane, pre-
sumably via its interaction with the SecY protein. When Syd is
overproduced in SecY2d1-expressing cells, it preferentially sta-
bilizes the chromosomally encoded wild-type SecY protein,
which is now given a greater opportunity to associate with
SecE. This is the proposed suppression mechanism. Another
striking phenotype associated with overproduction of Syd is its
strong toxicity against the secY24 mutant cells. Presumably
because of the defect in SecY-SecE interaction, the SecY24
mutant protein is degraded at 42 °C. This degradation is pri-
marily responsible for the protein export defect (14). However,
the Syd-mediated impairment of protein export and cell viabil-
ity is not accompanied by degradation of the SecY protein (19).
Thus, Syd seems to directly interfere with the functioning of
the SecY24 mutant protein. These observations indicate that
Syd provides a useful means to investigate into the nature of
protein interactions involving SecY.
Through our reversion studies of the secY24 mutant with
respect to its susceptibility to Syd overproduction, we identified
three new alleles of secY (20). While two of them affected
residue 240 (the residue altered by secY24) of SecY, the third
mutation, termed secY249 (for Ala249 3 Val) alleviated the Syd
sensitivity of the secY24 alteration (when placed in cis config-
uration with secY24) without restoring the temperature resist-
ance or the SecY-SecE interaction. Thus, the secY249 mutation
18835
18836 Syd and SecY-SecA Interaction
may affect the interaction between SecY and Syd.
This work was focused on the ability of Syd to inhibit protein
translocase when its SecY subunit is altered to the SecY24
mutant form. Our in vitro experiments show that Syd inter-
feres with productive interaction between SecA and the mem-
brane-integrated SecYE complex.
EXPERIMENTAL PROCEDURES
Escherichia coli Strains and Plasmids——Plasmid pST67 contained
the syd gene placed under the lac promoter and a translation-initiating
SD sequence (59-AAGGAG-39) and was used as a Syd-overproducing
plasmid. For its construction, a polymerase chain reaction was progra-
mmed on pST30 (19) with primers 59-GGGGTACCCCAAGGAGAACA-
GCTATGGACGATTTGACCGC-39 and 59-GCCCTAGGGCAATGTCTT-
CTCCGAATT-39 to produce a 0.57-kilobase pair fragment, which was
then treated with KpnI and BamHI and cloned into pUC18. The result-
ing plasmid was confirmed for the Syd-coding region by sequencing. E.
coli strain AD202 was a derivative of MC4100 (21) carrying ompT::kan
(22). EM147 was a derivative of MC4100 carrying syd::kan (19). EM166
was a derivative of MC4100 carrying syd::cat and constructed essen-
tially as described previously (19), except that it carried the chloram-
phenicol resistance determinant (cat) (instead of kan previously used)
in the middle of the syd gene. Mutants carrying the secY24 or secY24 –
249 mutation were described previously (20). These mutations were
transduced into the genetic background of strain TW155 (a DuncB uncC
derivative of AD202; Ref. 5) and named EM153 and EM152, respec-
tively. For these constructions, zhd-33::Tn10 (17) was used as a selec-
tive marker, and transductants were examined for temperature-sensi-
tivity and Syd sensitivity (20); an isogenic secY1 strain was named
EM154. Their further derivatives that received syd::cat from EM166
were named EM168 (secY24 syd::cat), EM167 (secY24 –249 syd::cat),
and EM169 (secY1syd::cat). IMVs1 from strains of a particular secY
phenotype exhibited essentially indistinguishable activities whether
they were from strains of the syd1 or the syd::cat background.
Purification of Syd—Syd-overproducing cells (EM147/pST67 or
CU141 (23)/pST67) were grown at 37 °C to a middle to late log phase in
1.5 liters of peptone medium (20) supplemented with 0.017 M KH2PO4,
0.072 M K2HPO4, and ampicillin (50 mg/ml). Isopropyl b-D-thiogalacto-
pyranoside (1 mM) was added to induce the lac promoter-directed over-
expression of syd. Syd was purified essentially as described previously
(19), desalted by a NAP-25 column (Amersham Pharmacia Biotech)
equilibrated with 50 mM Tris-HCl (pH 7.5) and 10% glycerol and stored
at 280 °C. Protein concentration was determined by a Micro BCA
protein assay kit (purchased from Pierce) using bovine serum albumin
as a standard.
In Vitro Translocation Assay—IMVs were prepared as described
previously (24) from cells of the specified strains that had been grown at
30 °C. They were treated with 6 M urea for 1 h at 0 °C (25) when
specified. Pro-OmpA was synthesized in vitro in the presence of
[35S]methionine as described previously (26). The transcription-trans-
lation mixture was then mixed with chloramphenicol (100 mg/ml), so-
dium succinate (5 mM), IMV (100 mg of protein/ml), and SecA (40 mg/ml).
Various concentrations of Syd were added before SecA when specified.
After incubation at 37 °C, samples were placed on ice and treated with
proteinase K (100 mg/ml) at 0 °C for 30 min. Reactions were stopped by
the addition of 1 mM phenylmethylsulfonyl fluoride followed by trichlo-
roacetic acid precipitation. Samples were separated by SDS-PAGE and
visualized by autoradiography as described previously (26), and the
bands were quantified by a Fuji BAS2000 phosphor imager.
SecA Translocation ATPase Assay—The reaction mixture (50 ml)
contained urea-washed IMV (100 mg/ml), SecA (40 mg/ml) and ATP (4
mM) in buffer composed of 50 mM Tris-HCl (pH 7.2), 30 mM KCl, 30 mM
NH4Cl, 2.5 mM magnesium acetate, and 0.5 mg/ml bovine serum albu-
min (fatty acid-free, from Nacalai Tesque). It was further supplemented
with pro-3His-OmpA9 (66 mg/ml; Ref. 24) and/or Syd (100 mg/ml) as
specified. After incubation at 37 °C, inorganic phosphate liberated was
quantified (5, 27). To obtain translocation ATPase activities, values in
the absence of pro-3His-OmpA9 were subtracted from those in its
presence.
SecA Insertion and Binding Assays—SecA was iodinated with 125I
and subjected to the insertion assay essentially as described previously
(4, 5), except that Syd was added when indicated. SecA binding assays
1
The abbreviations used are: IMV, inverted inner membrane vesicle;
AMP-PNP, adenosine 59-(b,g-imino)triphosphate; PAGE, polyacryl-
amide gel electrophoresis.
were similarly performed according to previously published procedures
(3, 5).
Trypsin Digestion of IMV——Urea-treated IMVs were incubated in
the presence or absence of Syd (100 mg/ml) with various concentrations
of trypsin at 0 °C for 20 min. A purified preparation of pro-OmpA (26
mg/ml) (5, 28) was added in some experiments, but it did not affect the
digestion patterns. Reactions were stopped by the addition of 1 mM
phenylmethylsulfonyl fluoride followed by trichloroacetic acid precipi-
tation. The precipitates were washed with acetone, solubilized in SDS
sample buffer at 37 °C for 5 min, and separated by SDS-PAGE (19).
SecY and SecE were then visualized by immunoblotting (19), using
antiserum against the N-terminal 22 residues of SecY (19) and anti-
serum against residues 64 – 81 of SecE, respectively. The latter anti-
serum, a custom product of Sawady Technology, was raised in a rabbit
using the synthetic peptide conjugated with keyhole limpet hemocya-
nin. Kaleidoscope prestained standards (purchased from Bio-Rad) were
used as molecular weight markers in electrophoresis.
RESULTS
Syd Inhibits Protein Translocation into IMVs with the Mu-
tated SecY24 Subunit of Protein Translocase—Overproduction
of Syd in the secY24 mutant cells leads to the severest extent of
inhibition of protein export known for E. coli sec mutants (19)
and appeared to merit investigations in vitro. We prepared
IMVs from wild-type and secY24 mutant cells grown at 30 °C,
the permissive temperature for the mutant. An in vitro trans-
lation product of pro-OmpA, labeled with [35S]methionine, was
subjected to posttranslational translocation into IMV. When
IMVs from secY24 mutant cells were preincubated at 0 °C with
increasing concentrations of Syd, translocation of pro-OmpA
was progressively inhibited (Fig. 1). In contrast, pro-OmpA
translocation into the wild-type IMV was not appreciably af-
fected by Syd of up to 100 mg/ml (Fig. 1). Dose-dependent
inhibition of translocation was also observed when a crude
cytosol fraction from the Syd-overproducing cells was added to
the reaction with the SecY24 IMV. Immunoblotting experi-
ments showed that the concentrations of Syd protein required
for a particular level of inhibition were similar between the
purified sample and the crude cytosolic fraction, indicating that
Syd had not been inactivated during the purification proce-
dures (data not shown). Neither bovine serum albumin, up to 1
mg/ml, nor heat-treated Syd exerted any appreciable inhibitory
effect (data not shown). Thus, this inhibition was due to the
functional Syd protein and not to some nonspecific factors such
as increased protein concentration.
When the SecY24 IMV was first incubated with excess Syd,
sedimented, and subjected to the translocation assay, substan-
tial levels of translocation of pro-OmpA were observed. Syd
concentration in the second incubation mixture was about 15%
of that before centrifugation. We suggest that Syd inhibits
translocation while in an equilibrium of association and disso-
ciation with IMV. In fact, preincubation was not necessary,
since Syd added after the initiation of translocation reaction
also prevented the subsequent increase of the translocated
molecules (data not shown). None of the translocase subunits
was proteolyzed during the pretreatment with Syd or after the
incubation at 37 °C with Syd, as confirmed by direct immuno-
blotting experiments (data not shown).
Syd Rapidly Uncouples SecA Functions from Protein Trans-
location into the SecY24 IMV—The ATP hydrolysis activity of
SecA that is stimulated by a precursor protein and membrane
vesicles with intact SecYEG complex is termed translocation
ATPase (29). We found that Syd was a potent inhibitor of
translocation ATPase when SecA was combined with the
SecY24 IMV. Whereas Syd (100 mg/ml) inhibited this activity
almost completely (Fig. 2, solid triangles), the same concentra-
tion of Syd only negligibly affected the activity in combination
with the wild-type IMV (Fig. 2, solid circles). The inhibitory
effect of Syd was specific for ATP hydrolysis that was coupled
 Syd and SecY-SecA Interaction
FIG. 1. In vitro effects of Syd on translocation of pro-OmpA. A,
in vitro synthesized and 35S-labeled pro-OmpA was subjected to post-
translational translocation reaction at 37 °C for 40 min into IMVs
prepared from strains EM169 (secY1), EM168 (secY24), and EM167
(secY24 –249) as indicated. IMVs had been premixed with the indicated
concentration of purified Syd (Syd conc.) at 0 °C before the initiation of
the assays. After proteinase K digestion and SDS-PAGE, 35S-labeled
OmpA was visualized by autoradiography. p and m represent the pre-
cursor and mature forms, respectively. B, the result in A is graphically
depicted after quantification by a Fuji BAS 2000 phosphor imager.
Open circles, wild type; solid triangles, secY24; open squares, secY24 –
249. Translocation efficiency into the wild type IMV in the absence of
Syd was about 80%.
FIG. 2. Effects of Syd on SecA translocation ATPase activity.
SecA translocation ATPase activities were assayed using urea-washed
IMVs from the wild type (EM169; circles) or the secY24 (EM168; trian-
gles) cells, in the presence (solid symbols) of Syd (100 mg/ml) or in its
absence (open symbols).
with translocation, such that neither the membrane ATPase
activity (in the absence of preprotein) nor the intrinsic ATPase
activity (without any other macromolecules) was affected sig-
nificantly (data not shown). The addition of Syd into the reac-
tion mixture in which SecA translocation ATPase was already
activated by pro-OmpA and SecY24 IMV resulted in almost
instantaneous inhibition of the ongoing reaction (Fig. 3).
18837
FIG. 3. Effects of Syd on ongoing reactions of SecA transloca-
tion ATPase. Two reactions of SecA translocation ATPase using IMVs
from the secY24 mutant (EM168) were initiated at 37 °C. After 5 min,
Syd (100 mg/ml) was added to one reaction (solid triangles), whereas the
other reaction received the same volume of the buffer (open triangles).
We examined the SecA insertion reaction (4) using 125I-
labeled SecA and urea-washed wild-type and SecY24 IMVs. In
the absence of added Syd, both wild-type IMV and SecY24 IMV
supported the pro-OmpA- and ATP-dependent formation of a
membrane-protected 30-kDa fragment (Fig. 4, lane 4), al-
though the efficiency was slightly lower for the SecY24 IMV. A
nonhydrolyzable ATP analog, AMP-PNP, supported apparently
pro-OmpA-independent insertion of SecA (Fig. 4, lane 6) as
reported previously (5, 30). When Syd was included in these
reactions, the SecY24 IMV did not support any detectable in-
sertion of SecA, irrespective of whether ATP or AMP-PNP was
used (Fig. 4, middle panel, lanes 1–3). In contrast, Syd inhib-
ited SecA insertion into the wild-type IMV only slightly (Fig. 4,
upper panel, lanes 1–3). These results indicate that Syd uncou-
ples the SecA functions from the SecY24-mediated protein
translocation reaction.
Syd Interferes with High Affinity Binding of SecA to the
SecY24 IMV—To define the target reaction of the Syd inhibi-
tion, we carried out SecA binding assays. Various concentra-
tions of SecA and IMV were incubated at 0 °C, and the mem-
brane-bound SecA was isolated for the Scatchard analysis of
the binding. SecA binding to the SecY24 IMV was found to be
abolished almost completely by Syd. Thus, the high affinity
phase of the binding was hardly observed in the presence of Syd
(Fig. 5B, solid triangles). High affinity binding of SecA to the
wild-type IMV was lowered by Syd to only a small extent (Fig.
5A, compare open and solid circles). These results suggest that
the Syd action to the SecY24 IMV can essentially be ascribed to
the elimination of the high affinity SecA binding.
We then examined whether Syd interferes with the SecA-
SecY interaction passively, for instance by steric hindrance, or
if it acts more actively, for instance to change the subunit
arrangement of the SecY-SecE complex. For this purpose, we
studied the effect of Syd on a state of SecA that had already
been inserted and stabilized as such by AMP-PNP. 125I-Labeled
SecA was first allowed to insert into wild-type or SecY24 IMV
in the presence of AMP-PNP and then subjected to the second
incubation with either Syd or buffer alone. The second incuba-
tion in the presence of Syd resulted in the disappearance of the
30-kDa fragment inserted into the SecY24 IMV (Fig. 6, lower
panel, lane 1), whereas SecA inserted into the wild-type IMV
persisted (Fig. 6, upper panel, lane 1). Incubation without Syd
did not cause any change in the intensity of the inserted SecA
18838 Syd and SecY-SecA Interaction

FIG. 4. Effects of Syd on SecA insertion into IMVs. 125I-Labeled

SecA (4 mg) and urea-treated IMVs (60 mg of protein) from secY1
(EM154; upper panel), secY24 (EM153; middle panel), and secY24 –249
(EM167; lower panel) cells were preincubated at 0 °C in a total volume
of 230 ml, and IMV-SecA complex was isolated by centrifugation,
resuspended, and divided into six portions, each of which was then
subjected to a SecA insertion reaction at 37 °C for 15 min in a total
volume of 50 ml containing the 125I-SecA-bound IMV (100 mg/ml),
pro-3His-OmpA9 (66 mg/ml; lanes 1 and 4), either ATP (2 mM; lanes 1,
2, 4, and 5) or AMP-PNP (2 mM; lanes 3 and 6), and Syd (100 mg/ml;
lanes 1–3). The samples were treated with trypsin (1000 mg/ml at 0 °C
for 15 min), and the 30-kDa fragment was visualized by SDS-PAGE
and autoradiography.
FIG. 5. Effects of Syd on SecA high affinity binding to IMVs.
SecA (1–200 nM, in which 125I-SecA occupied 1 nM) was mixed with
(compare Fig. 4, lane 6, and Fig. 6, lane 2). Thus, Syd is able to urea-washed IMVs (100 mg/ml) from secY1 (EM154; A) or secY24
dissociate SecA that is otherwise locked in the inserted state by (EM153; B) cells, in a 50-ml reaction. Syd (100 mg/ml) was also included
the lack of ATP hydrolysis. We also noted that the AMP-PNP- for the reactions shown by solid symbols. Samples were incubated at
0 °C for 30 min and centrifuged to pellet down the IMV-SecA complex.
stabilized SecA was partially released from the SecY24 IMV,
Radioactivities of the pellet and the supernatant were determined by an
but not from the wild-type IMV, when unlabeled excess SecA LKB g-ray counter, and the results are shown as Scatchard plots. The
was added, suggesting that SecA insertion occurs somewhat apparent Kd values for high affinity binding of SecA were 14.9 and 25.6
weakly against the mutant IMV. nM for the SecY1 IMV in the absence and presence of Syd, respectively.
SecY24 Alteration of Subunit Arrangements of Translocase The value for the SecY24 IMV was 15.4 nM in the absence of Syd, while
the high affinity binding was not clearly observed in the presence of
and Further Evidence for the Syd Interaction with SecY—The Syd.
secY24 alteration in SecY weakens the interaction between
SecY and SecE such that the SecY24-SecE complex comes
apart after solubilization with a nonionic detergent (9, 26). We
examined trypsin digestion patterns of SecY and SecE in the
membrane-integrated state. Wild-type and SecY24 IMV were
treated with increasing concentrations of trypsin, and SecY
and SecE were detected by immunoblotting using antisera
against the N-terminal sequence of SecY and the central cyto-
plasmic region of SecE, respectively. Treatment of the wild-
type IMV with low concentrations of trypsin produced an N-
terminal fragment of about 21 kDa (Fig. 7, lanes 17 and 18). Its
apparent molecular size suggests that the site of this cleavage
was within cytoplasmic region 4 (C4). The SecY24 IMV gave
different trypsin digestion patterns in two respects. First, the
overall trypsin sensitivity as assessed by the disappearance of
the intact SecY molecules was significantly higher for the FIG. 6. Effects of Syd on the inserted form of SecA that was
stabilized by AMP-PNP. Two SecA insertion reactions were carried
SecY24 IMV than the wild-type IMV (Fig. 7, compare lanes
out in a volume of 40 ml, using either the SecY1 IMV (upper panel) or
6 –10 with lanes 16 –20). Second, the 21-kDa N-terminal frag- the SecY24 IMV (lower panel) and in the presence of AMP-PNP (2 mM),
ment produced from the SecY24 IMV (Fig. 6, lane 7) was as described in the legend to Fig. 4. They were then mixed with 10 ml of
further shortened to a slightly faster migrating product when solution containing Syd (5 mg; lane 1) or buffer alone (lane 2) and
trypsin concentrations were increased (Fig. 7, lanes 8 and 9). incubated at 37 °C for an additional 10 min. The 30-kDa SecA fragment
was visualized as described in the legend to Fig. 4.
Although the SecY24 amino acid alteration (Gly240 to Asp) is
also in C4 domain of SecY, it is unrelated to any amino acid
residues directly relevant to the trypsin-mediated hydrolysis of SecE between the wild-type IMV and the SecY24 IMV. SecE in
peptide bonds. Thus, this sequence alteration may have caused the former IMV resisted trypsin of the highest concentration
a change in local conformation of SecY or in subunit arrange- examined (Fig. 7, lower panel, lanes 16 –20), although some
ments of the SecYEG complex. decrease in the intensity of SecE, in parallel with that of the
We found a striking difference in the trypsin susceptibility of intact SecY, was observed at higher trypsin concentrations
 Syd and SecY-SecA Interaction 18839

FIG. 7. Effects of the secY24 mutation and Syd on trypsin digestion patterns of IMV-integrated SecY and SecE. Urea-treated IMVs
prepared from the secY1 (EM154; lanes 11–20) and secY24 (EM153; lanes 1–10) cells were treated with increasing concentrations of trypsin in the
presence of Syd (100 mg/ml; lanes 1–5 and 11–15) or in its absence (lanes 6 –10 and 16 –20), at 0 °C for 20 min. The concentrations of trypsin used
were as follows: 0 (lanes 1, 6, 11, and 16), 1 (lanes 2, 7, 12, and 17), 10 (lanes 3, 8, 13, and 18), 100 (lanes 4, 9, 14, and 19), and 1000 (lanes 5, 10,
15, and 20) mg/ml. Note, however, that trypsin in the stock solution used in this particular experiment had been partially inactivated due to
repeated freezings and thawings, and essentially identical results were obtained in confirmatory experiments using a freshly prepared trypsin
solution of concentrations about 1⁄5 those used in this experiment. Samples were separated by SDS-PAGE and immunologically stained with
antiserum against the N-terminal part of SecY (upper panel) and antiserum against the central part of SecE (lower panel). The solid arrowheads
indicate the positions of the full-length SecY (upper panel) and SecE (lower panel), respectively, and the open arrowheads indicate the positions
of N-terminal fragments of SecY.

(Fig. 7, lanes 11–20, compare upper and lower panels). In con-
trast, SecE in the SecY24 IMV was digested almost completely
by trypsin (Fig. 7, lower panel, lanes 9 and 10). SecE was more
trypsin-sensitive than SecY24 itself (compare upper and lower
panels for lane 5 of Fig. 7). These results lend further support
to the notion that subunit arrangements of the SecYEG com-
plex have been altered by the SecY24 amino acid alteration,
resulting in significantly impaired SecY-SecE interaction.
We then repeated the trypsin digestion experiments in the
presence of an excess of Syd protein. While the addition of Syd
did not appreciably affect the trypsin digestion profiles of SecY
or SecE in the wild-type IMV, it markedly changed the pattern
of SecY cleavage in the SecY24 IMV. The 21-kDa N-terminal
fragment was now shifted to an apparent molecular mass of 27
kDa (Fig. 7, upper panel, lanes 3 and 4). Thus, the original two
cleavage sites (presumably in C4) were somehow protected by
Syd. Thus, Syd interacts specifically with SecY24. However, it
did not exert any protecting effect on SecE (Fig. 7, lower panel,
lanes 4 and 5).
A Second Site Mutation, secY249, Cancels the Inhibitory Ef-
fects of Syd—Previously, we isolated Syd-resistant variants
from the secY24 mutant (20). The mutations converged into
three alleles in addition to the true reversion. Two of the alleles
change residue 240 affected by the secY24 mutation, restoring
the SecY-SecE interaction (20). The other allele, secY249 (for
an Ala249 to Val change), is an intragenic suppressor mutation
that suppresses only the sensitivity to overproduction of Syd,
without any effect on the temperature-sensitive protein export
defect of the secY24 mutation (20). IMVs prepared from the
secY24 –249 double mutant supported pro-OmpA translocation
with an efficiency similar to that of the SecY24 IMV. However,
it was only slightly inhibited by Syd (Fig. 1). The double mu-
tant IMV also supported the translocation ATPase activity of
SecA even in the presence of Syd (data not shown). Insertion of
the 30-kDa fragment into the double mutant IMV was not
inhibited by Syd (Fig. 4, lower panel, lane 1). The SecY24 –249
IMV gave tryptic digestion patterns of SecY and SecE that were
essentially identical to those of the SecY24 IMV. However, they
were unaffected by the presence of excess Syd (data not shown).
Thus, the effects of the secY249 mutation on the phenotypes of
the secY24 mutation have been reproduced in vitro. These
results suggest that the Syd effect is brought about through
specific interaction between SecY and Syd, in which the residue
249 of SecY plays an essential role.
DISCUSSION
Syd, originally identified as a multicopy suppressor of the
secY2d1 mutation (19), directly interacts with SecY. Overpro-
duction of Syd stabilizes the wild type SecY protein when it
alone is overproduced or when it fails to associate with SecE
because of the presence of the excess amounts of SecY2d1, the
dominant negative SecY variant. The association of Syd with
the plasma membrane is saturable such that SecY overproduc-
tion increases the membrane-bound state of Syd. We demon-
strated in this study that Syd markedly altered the trypsin
digestion pattern of SecY24 in IMV, lending further support to
the notion that Syd interacts with SecY.
Evidence indicates that the secY24 mutation impairs SecY-
SecE interaction. The temperature sensitivity of this mutant is
partially suppressible by overproduction of SecE,2 and the mu-
tation can act as an intragenic suppressor against the SecE-
sequestering effect of the secY2d1 mutation (16, 20). After
solubilization of the membrane with a nonionic detergent, a
complex between SecY24 and SecE cannot be isolated (9, 16).
The temperature-sensitive protein export defect of the secY24
mutant is due to the FtsH-mediated degradation of the SecY24
protein at 42 °C (14, 16). These observations suggest that the
impaired SecY-SecE interaction is the primary cause of the
secY24 defect. In further support of this notion, we found that
SecE in the SecY24 IMV was more accessible to trypsin diges-
tion than that in the wild-type IMV. The temperature sensitiv-
ity of the secY24 mutant could be due to temperature-depend-
ent exacerbation of the interaction defect, temperature-
dependent activation of the proteolytic system, or both.
When Syd is overproduced in the secY24 mutant, protein
export is rapidly blocked followed by the loss of viability even at
30 °C (19). The severity of this block makes it the most useful
2
E. Matsuo, T. Taura, Y. Akiyama, and K. Ito, unpublished
observations.
18840 Syd and SecY-SecA Interaction
system to accumulate chemical amounts of a precursor protein
with uncleaved signal sequence within the cell (24). The rapid-
ity and the lack of accompanied SecY proteolysis suggest that
this inhibition is brought about by direct interference by Syd
with the translocase functions.
We reproduced the inhibitory effect of Syd in the in vitro
translocation reaction of pro-OmpA. The concentration of Syd
required for 50% inhibition of translocation was 5–10 mg/ml (or
250 –500 nM) in a reaction containing IMV of 100 mg/ml pro-
teins. Assuming that a cell contains 500 molecules of SecY (31)
and that 10% of total proteins are located in the inner mem-
brane, the above concentration of IMV corresponds to 250
ng/ml (or 5 nM) of SecY. Thus, numbers of the Syd molecules
required for efficient inhibition far exceed those of SecY mole-
cules. We have shown that Syd inhibits the high affinity bind-
ing of SecA to the IMV containing the SecY24 form of the
translocase subunit. This could be due to a steric hindrance by
the SecY-bound Syd with respect to SecA binding. There may
be a competition between Syd and SecA for the interaction with
SecYEG translocase. Indeed, increased concentrations of SecA
acted to partially overcome the inhibition caused by a low
concentration (10 mg/ml) of Syd. Although quantitative discus-
sion requires knowledge about the Kd value of Syd-IMV bind-
ing, our IMV re-isolation experiments suggested that the dis-
sociation rate of Syd is considerably high. Thus, real time
binding/dissociation measurements will be required to deter-
mine the Kd value.
While the simple steric hindrance discussed above is possi-
ble, we believe it is more likely that Syd causes more drastic
structural changes to the SecYE complex. Syd was found to
inhibit the SecA insertion reactions. We previously noted that
the SecY205 mutant form of SecY (32) did not allow the pre-
protein- and ATP-dependent mode of SecA insertion, while it
still allowed SecA insertion in the presence of AMP-PNP (5).
Our present results show, in contrast, that Syd abolishes both
of these modes of SecA insertion reactions. Thus, it is conceiv-
able that the SecY205 defect lies in a specific process of the
SecA insertion reaction cascade, whereas Syd impairs the sys-
tem more generally or inhibits a very early event. Indeed, Syd
inhibited the high affinity binding between SecA and the
SecY24 IMV. Furthermore, it acted to dissociate the inserted
SecA segment that had been stabilized by AMP-PNP. In these
cases, the subunit arrangement of the SecYE complex may
have been altered, leading to the elimination of both the high
affinity SecA binding site and the structural element (or chan-
nel) required for maintaining the “inserted” state of SecA.
Previous studies suggested that the high affinity binding of
SecA requires both SecY and SecE (3, 33, 34). Thus, it seems to
be a logical explanation that the presence of a high concentra-
tion of Syd acts to dissociate the SecY-SecE complex that is
already compromised by the SecY24 alteration. An interesting
possibility here is that Syd may bind preferentially to an un-
complexed state of SecY. This is consistent with the known
ability of Syd to stabilize overproduced SecY (19). Although the
physiological role of Syd is elusive (19), some speculations may
be useful. For instance, Syd may act as a reservoir for unas-
sembled forms of SecY, thus preventing its deleterious effects
on the cell (14). Alternatively, it may serve as a regulatory
factor that negatively controls the translocase function by in-
terfering with either SecY-SecE or SecY-SecA binding. At any
rate, interaction between Syd and SecY seems specific in that it
is disrupted by the secY249 alteration of residue 249 of SecY.
Syd will be useful to probe the architecture of protein translo-
case from the functional point of view.
Acknowledgments—We thank Tohru Yoshihisa and Yoshinori
Akiyama for useful comments; Gen Matsumoto for suggestions in the
SecA insertion and binding experiments; and Kiyoko Mochizuki,
Yusuke Shimizu, and Toshiki Yabe for technical support.
REFERENCES
1. Wickner, W., and Leonard, M. R. (1996) J. Biol. Chem. 271, 29514 –29516
2. Ito, K. (1996) Genes Cells 1, 337–346
3. Hartl, F.-U., Lecker, S., Schiebel, E., Hendrick, J. P., and Wickner, W. (1990)
Cell 63, 269 –279
4. Economou, A., and Wickner, W. (1994) Cell 78, 835– 843
5. Matsumoto, G., Yoshihisa, T., and Ito, K. (1997) EMBO J. 16, 6384 – 6393
6. Brundage, L., Hendrick, J. P., Schiebel, E., Driessen, A. J. M., and Wickner, W.
(1990) Cell 62, 649 – 657
7. Joly, J. C., Leonard, M. R., and Wickner, W. T. (1994) Proc. Natl. Acad. Sci.
U. S. A. 91, 4703– 4707
8. Brundage, L., Fimmel, C. J., Mizushima, S., and Wickner, W. (1992) J. Biol.
Chem. 267, 4166 – 4170
9. Homma, T., Yoshihisa, T., and Ito, K. (1997) FEBS Lett. 408, 11–15
10. Akimaru, J., Matsuyama, S., Tokuda, H., and Mizushima, S. (1991) Proc. Natl.
Acad. Sci. U. S. A. 88, 6545– 6549
11. Nishiyama, K., Mizushima, S., and Tokuda, H. (1993) EMBO J. 12, 3409 –3415
12. Matsuyama, S., Akimaru, J., and Mizushima, S. (1990) FEBS Lett. 269,
96 –100
13. Taura, T., Baba, T., Akiyama, Y., and Ito, K. (1993) J. Bacteriol. 175,
7771–7775
14. Kihara, A., Akiyama, Y., and Ito, K. (1995) Proc. Natl. Acad. Sci. U. S. A. 92,
4532– 4536
15. Shimoike, T., Akiyama, Y., Baba, T., Taura, T., and Ito, K. (1992) Mol. Micro-
biol. 6, 1205–1210
16. Baba, T., Taura, T., Shimoike, T., Akiyama, Y., Yoshihisa, T., and Ito, K. (1994)
Proc. Natl. Acad. Sci. U. S. A. 91, 4539 – 4543
17. Shiba, K., Ito, K., Yura, T., and Cerretti, D. P. (1984) EMBO J. 3, 631– 635
18. Pohlschröder, M., Murphy, C., and Beckwith, J. (1996) J. Biol. Chem. 271,
19908 –19914
19. Shimoike, T., Taura, T., Kihara, A., Yoshihisa, T., Akiyama, Y., Cannon, K.,
and Ito, K. (1995) J. Biol. Chem. 270, 5519 –5526
20. Matsuo, E., and Ito, K. (1998) Mol. Gen. Genet. 258, 240 –249
21. Casadaban, M. J. (1976) J. Mol. Biol. 104, 541–555
22. Akiyama, Y., and Ito, K. (1990) Biochem. Biophys. Res. Commun. 167, 711–715
23. Akiyama, Y., Ogura, T., and Ito, K. (1994) J. Biol. Chem. 269, 5218 –5224
24. Yoshihisa, T., and Ito, K. (1996) J. Biol. Chem. 271, 9429 –9436
25. Taura, T., Yoshihisa, T., and Ito, K. (1997) Biochimie 79, 517–521
26. Baba, T., Jacq, A., Brickman, E., Beckwith, J., Taura, T., Ueguchi, C.,
Akiyama, Y., and Ito, K. (1990) J. Bacteriol. 172, 7005–7010
27. Lill, R., Dowhan, W., and Wickner, W. (1990) Cell 60, 271–280
28. Sato, K., Mori, H., Yoshida, M., Tagaya, M., and Mizushima, S. (1997) J. Biol.
Chem. 272, 20082–20087
29. Lill, R., Cunningham, K., Brundage, L. A., Ito, K., Oliver, D., and Wickner, W.
(1989) EMBO J. 8, 961–966
30. Economou, A., Pogliano, J. A., Beckwith, J., Oliver, D. B., and Wickner, W.
(1995) Cell 83, 1171–1181
31. Matsuyama, S., Fujita, Y., Sagara, K., and Mizushima, S. (1992) Biochim.
Biophys. Acta 1122, 77– 84
32. Taura, T., Akiyama, Y., and Ito, K. (1994) Mol. Gen. Genet. 243, 261–269
33. Hanada, M., Nishiyama, K., Mizushima, S., and Tokuda, H. (1994) J. Biol.
Chem. 269, 23625–23631
34. Duong, F., and Wickner, W. (1997) EMBO J. 16, 2756 –2768