©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Product of a New Gene, syd, Functionally Interacts with SecY when Overproduced in Escherichia coli(*)

(Received for publication, October 18, 1994)

Takashi Shimoike Tetsuya Taura Akio Kihara Tohru Yoshihisa Yoshinori Akiyama Kurt Cannon (§) Koreaki Ito (¶)

From the Department of Cell Biology, Institute for Virus Research, Kyoto University, Kyoto 606-01, Japan

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

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 secY1. 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 SecY1 form of SecY. Thus, in the presence of both secY and secY1, Syd increases the effective SecY/SecY1 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.


INTRODUCTION

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 secY1 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 secY1 (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 SecY1. We now report on identification (as a multicopy suppressor of secY1) and characterization of a new gene, syd, and its product. (^1)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 SecY1 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.


MATERIALS AND METHODS

Bacterial Strains

E. coli K12 strain MC4100 was described by Silhavy et al.(1984). KI438 (Shimoike et al., 1992) was a derivative of CSH26 (Miller, 1972) carrying the secA-lacZ-f181 fusion gene and a prophage PR9 (Riggs et al., 1988). FS1576 (Stahl et al., 1986) was a recD1009 strain used for linear DNA transformation. KI297 and KI298 (Ito and Akiyama, 1991) were derivatives of IQ85 (MC4100, secY24 zhd-33::Tn10) and IQ86 (secY), respectively, carrying F` lac lacI^Q. KI267 was a derivative of CSH26 carrying F` lacI^Q lacPL8 lacZYA pro. TW130 (Taura et al., 1993) was a ompT::kan zhd-33::Tn10 rpsE derivative of MC4100. AD208 (Baba et al. 1990) was a secY39 (cs) ompT::kan zhd-33::Tn10 rpsE derivative of MC4100.

Media and Reagents

L medium (Silhavy et al., 1984) and minimal medium M9 (Miller, 1972) that was supplemented with glycerol (0.4%), amino acids (20 µg/ml each, other than methionine and cysteine), and thiamine (2 µg/ml) were used. Ampicillin (50 µg/ml), chloramphenicol (20 µg/ml), and kanamycin (25 µg/ml) were added for growing plasmid-bearing cells. All the reagents except when indicated were of the highest reagent grade obtained from either Nakalai Tesque or Wako Pure Chemicals. Enzymes for DNA manipulations were obtained from either Takara Shuzo or Toyobo. [S]Methionine (29 Tbq/mmol) was purchased from American Radiolabeled Chemicals. Anti-Syd serum was raised in rabbit against a synthetic peptide, DDLTAQALKDFTARY (corresponding to the amino-terminal residues 2-16 of Syd), that had been conjugated with Keyhole Limpet Hemocyanin (this preparation was carried out by Takara Shuzo Co.). Anti-SecY antisera were similarly prepared in this laboratory against MAKQPGLDFQSAKGGLGELKRRC, corresponding to the amino terminus of SecY, as well as against CYESALKKANLKGYGR, corresponding to its carboxyl terminus. Anti-OmpA serum was provided by Y. Anraku, and anti-maltose-binding protein serum was prepared in rabbit using purified protein. Anti-beta-galactosidase was purchased from 5 Prime 3 Prime, Inc.

Plasmids

pKY241, carrying secY1 placed under the lac promoter control, was previously described (Shimoike et al., 1992). pKY247 (carrying secY1 cloned into a pACYC184-derived vector) and pKY248 (carrying secY) were described by Taura et al. (1993). pST6, which carries the syd gene cloned onto pACYC184 vector, was obtained in this work from library screening. pST30, pST32, and pST34 were constructed by cloning a 0.6-kb (^2)NspI-BglII fragment from pST6 (after blunt end formation by T4 DNA polymerase) into the SmaI site of pSTV29 (see below), pUC19 (Vieira and Messing, 1982), or pNO1575H (a pBR322-derived vector) (Ueguchi and Ito, 1992), respectively. pSTV29 was constructed by cloning 0.4-kb multicloning site region of pUC119 (Vieira and Messing, 1987) (digested with HaeII and blunt ended) into pACYC184, which had been digested with HindIII and HincII and blunt ended. pKY4 carrying secY and pKY262 carrying secY39 were described by Akiyama and Ito(1987) and Taura et al.(1994), respectively. pKY259 (carrying secY1-lacZalpha gene fusion) and pKY271 (carrying secE) were described by Baba et al.(1994). pST42 (carrying syd::kan) was constructed as follows. Tn5 was first allowed to transpose onto pST6 from Tn5 (b221 rex::Tn5 cI857 Pam Oam) (Kleckner et al., 1978) by selecting colonies resistant to 250 µg/ml kanamycin. A plasmid from one of the clones was shown to have a Tn5 insertion after nucleotide 77 of syd (see Fig. 1). This plasmid was digested with HapI, and about a 5-kb fragment containing most of the Tn5 sequence was replaced by the kanamycin resistance determinant prepared from pUC4K (Vieira and Messing, 1982) by PstI digestion.


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.



Isolation of Multicopy Suppressors of secY1

Genomic libraries carrying 5-15-kb fragments of the wild-type E. coli chromosomal DNA were constructed as previously described (Ueguchi and Ito, 1992). The mixtures of plasmids were introduced into cells of KI438 (secA-lacZ)/pKY241(secY1). Transformants were selected on peptone agar plates supplemented with 5-bromo-4-chloro-3-indolyl-beta-D-galactopyranoside (40 µg/ml), phenylethyl-thiogalactopyranoside (0.5 mM), ampicillin (50 µg/ml), and chloramphenicol (20 µg/ml) (Riggs et al., 1988; Shimoike et al., 1992). White colonies, which appeared at frequencies of about 10, were saved for further analysis of the plasmids they carried.

Determination of the syd Nucleotide Sequence

Appropriate restriction fragments of pST6 were subcloned into pKY225 (Taura et al., 1993). Single-stranded DNAs were prepared (Vieira and Messing, 1987) and sequenced by the chain termination method (Sanger et al., 1977), using Sequenase 7-deaza-dGTP sequencing kit (U. S. Biochemicals). Synthetic primers were used for further elongation.

Construction of syd Disruptant

The 14-kb ScaI-XbaI fragment from pST42 (containing the syd::kan mutation described above) was used to transform FS1576 (recD)/pST34 to kanamycin (10 µg/ml) resistance. P1vir lysate was prepared on one of the transformants and used to introduce the kan marker into KI458, a derivative of KI437 (MC4100, secA-lacZ) (Baba et al., 1994) carrying fuc::Tn10 (Singer et al., 1989). The fuc marker was cotransduced at a frequency of about 63%. One of transductants with the preserved fuc::Tn10 mutation was saved as strain SI13-11. Southern hybridization experiments, using the chromosomal DNA and a probe of the EcoRI fragments of pST6 confirmed that the 1.1-kb EcoRI fragment was shifted to 2.7 kb in the syd::kan disruptants.

Cell Fractionation

Cells of CSH26 were harvested, washed with 10 mM Tris-HCl (pH 8.1), and resuspended in 20% sucrose, 30 mM Tris-HCl (pH 8.1), followed by addition of volume of 1 mg/ml lysozyme dissolved in 0.1 M EDTA (pH 8.0) and incubation for 45 min on ice (Yamato et al., 1975). Centrifugation at 26,000 times g for 10 min yielded supernatants and pellets. Cells of this genetic background lyse by this mild treatment such that the supernatant quantitatively contains both the periplasmic and cytoplasmic proteins (Akiyama and Ito, 1985). In the case of strains other than CSH26, the above procedure yielded separate periplasmic (supernatant) and spheroplast (pellet) preparations. The latter was suspended in 10% sucrose, 3 mM EDTA, 1 mM dithiothreitol, disrupted either by French press (7500 p.s.i.) or sonication (5 times 20-s bursts using a Heat Systems sonicator), diluted 2-fold with the same buffer, and centrifuged first at 7,000 times g for 10 min and then at 95,000 times g for 60 min. The final supernatant and pellets were used as the cytoplasmic and the total membrane fractions. Further separation of the membranes into the inner membrane and the outer membrane was by sucrose isopycnic centrifugation (Osborn and Munson, 1974).

Purification of Syd Protein and Determination of its Amino-terminal Sequence

Syd-overproducing cells (MC4100/pST32) were grown to a late log phase in 1 liter of peptone medium supplemented with ampicillin (50 µg/ml). Cells were harvested, washed with 10 mM Tris-HCl (pH 8.1), suspended in 10 ml of the same buffer, and sonicated. After centrifugations for 3,000 times g for 10 min and then at 540,000 times g for 30 min, the supernatant was loaded on a DEAE-Sepharose Fast Flow (Pharmacia Biotech Inc.) column and eluted by 0-0.3 M linear gradient of NaCl. Syd was detected by Coomassie Brilliant Blue R250 staining and immunoblotting after SDS-PAGE, and the peak fractions (about 0.23 M NaCl) were rechromatographed with a 0.1-0.3 M NaCl elution. After concentration, the sample was subjected to gel filtration using a Superose 12 column (Pharmacia). Syd was eluted at the estimated molecular mass of about 20 kDa and was further chromatographed through a Mono-Q column (Pharmacia) with elution with a 0.15-0.5 M NaCl gradient. About 40 µg of Syd preparation was obtained, and its purity as judged by SDS-PAGE was about 90%. For confirmation of the amino-terminal sequence, a sample at the DEAE-Sepharose step was separated by SDS-PAGE, transferred onto an Immobilon polyvinylidene difluoride membrane filter (Millipore), and stained. The Syd region of the filter was used for automated Edman degradation using a Shimadzu protein sequencer.

Pulse-Chase and Immunoprecipitation

Cells were grown in the amino acid-supplemented M9-glycerol medium. When the lac promoter was to be induced, IPTG was added to 1 mM. Cells at a mid-log phase were pulse labeled for the period indicated in each experiment with about 0.37 Bq/ml [S]methionine, and, when appropriate, chase was initiated by adding non-radioactive L-methionine (final 20 µg/ml). Samples were mixed with trichloroacetic acid (final 5%) and processed for immunoprecipitation as previously described (Baba et al., 1990; Taura et al., 1993).

SDS-PAGE

Proteins were separated by SDS, 15% acrylamide, 0.12% N,N`-methylene-bis-acrylamide gel electrophoresis (Ito, 1984) for analysis of Syd and SecY or by SDS-10% PAGE (Laemmli, 1970) for analysis of maltose-binding protein and OmpA. Radioactive proteins were visualized/quantitated by autoradiography and/or a Bioimaging Analyzer BAS2000 (Fuji Film).

Immunoblotting

A portion of a mid-log phase culture was treated with trichloroacetic acid to precipitate proteins that were collected by centrifugation, washed with acetone, and dissolved in 1% SDS, 1 mM EDTA, 50 mM Tris-HCl (pH 8.1). After determination of protein concentration, at least two known amounts were separated by SDS-PAGE and electrophoretically blotted onto Immobilon polyvinylidene difluoride membrane filter (Millipore) for Syd detection or onto Zeta-Probe membrane filter (Bio-Rad) for SecY detection (Taura et al., 1993). The filter was blocked with 5% skim milk dissolved in phosphate-buffered saline containing 0.1% Tween 20 as well as sodium azide (0.02%) at 42 °C for 2 h, incubated with appropriately diluted antisera in the same buffer at room temperature for 1 h, washed twice with phosphate-buffered saline/Tween 20, and treated with goat anti-rabbit immunoglobulin G that had been conjugated with horseradish peroxidase (Bio-Rad) in the same buffer for 1 h at room temperature. After washing for three times, peroxidase activity was detected by ECL Western detection reagents (Amersham Corp.) and quantitated by Discovery Series Densitometer (PDI).


RESULTS

Multicopy Suppressors of secY1, a Dominant Negative Mutation

For the initial purpose of identifying new components of the protein translocation machinery, we searched for plasmid clones that suppressed the export interference caused by a dominant negative secY mutation. Plasmid pKY241 carries the secY1 allele of secY (Shimoike et al., 1992) and is driven by the replication system of pBR322. Cells of KI438 (secA-lacZ) harboring this plasmid are defective in export of secretory proteins, but the export interference by SecY1 is moderate and does not lead to cessation of growth or loss of viability. Thus, they form blue colonies on agar plates containing 5-bromo-4-chloro-3-indolyl-beta-D-galactopyranoside. This is because the expression of secA increases in response to a defect in protein translocation, and, accordingly, the beta-galactosidase activity increases, which in turn hydrolyzes the chromogenic substrate. This method of discriminating between the colonies of export-deficient and export-proficient cells had been developed by Riggs et al.(1988). To identify E. coli gene(s) that can overcome the dominant export defect in multicopy states, we introduced a chromosomal gene library into cells of KI438/pKY241. This library had been constructed using pACYC184 vector. From a total of about 10^4 transformants, we obtained 15 white colonies. Among them, 9 were shown to be independent clones with respect to the restriction maps of plasmids they carried. All of them were shown to share 1.1- and 1.8-kb EcoRI fragments (see Fig. 1). Cross-hybridization experiments showed that they indeed carried the same two fragments (data not shown). These EcoRI fragments were mapped to minute 60 on the E. coli chromosome, as they hybridized with the Kohara clones 10B5 and 8C5 (Kohara et al., 1987; data not shown).

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).^1 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 alpha-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.^5 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 secY1. However, all of them were weak in that they (after recloning into a pACYC184-based vector) could not suppress secY1, 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 secY1 on the pBR322-based plasmid. (^3)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 secY1.

Identification of the syd Gene Product

To detect the product of the syd gene, we first prepared antiserum against a synthetic peptide of sequence corresponding to the 2nd to the 16th codons. Whole cell extracts were subjected to SDS-PAGE and immunoblotting. A band with an electrophoretic mobility corresponding to about 26 kDa was detected (Fig. 3). We conclude that this protein band represents the syd gene product for the following reasons. (i) Its reaction with the antibodies was competed by the antigen peptide (Fig. 3, compare lanes1 and 2); (ii) its intensity increased markedly in cells carrying a syd plasmid (lane1); (iii) the 26-kDa protein was overproduced and purified as described under ``Materials and Methods,'' and its amino-terminal sequence was determined to be MDDLTAQALKDFTARY, in complete agreement with the nucleotide sequence; and (iv) the band was undetectable in the strain with its syd gene disrupted (lane3).


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 alpha 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 times 10^3 molecules per cell.

Cellular Localization of Syd

When wild-type cells were disrupted by sonication and fractionated, Syd was almost exclusively recovered from the soluble cytoplasmic fraction (Fig. 4A, lane3). When a French pressure cell was used to break the cell, about 10% of Syd was recovered from the cytoplasmic membrane fraction but none from the outer membrane fraction (Fig. 4B). Cells of strain CSH26 are peculiar in that they release the cytoplasmic contents just by treatment with EDTA-lysozyme in the presence of 20% sucrose (Akiyama and Ito, 1985). We subjected cells of KI267 (CSH26/F`lac) to this mild lysis. Syd was largely recovered from the low speed centrifugation pellet while beta-galactosidase in the same cells was quantitatively released into the supernatant (Fig. 4C, lanes1 and 2). Also, a majority of cellular proteins (stained with Coomassie Brilliant Blue) were recovered from the supernatant (data not shown). These results indicate that Syd is associated with rapidly sedimenting cellular materials, and this association is preserved only under the mild lysis conditions. Since a fraction of Syd molecules is found in the cytoplasmic membrane after the French press disruption (Fig. 4B), we assume that Syd is weakly associated with the cytoplasmic membrane.


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^Q) (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 beta-galactosidase (beta-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, beta-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.

Effects of Syd Overproduction on Protein Export

Plasmids carrying syd effectively suppressed the protein export-interfering ability of SecY1 as assessed from the in vivo processing (translocation) kinetics of OmpA and maltose-binding protein (Fig. 5, compare lanes2 and 3 with lane1). It was previously reported that some of the cold-sensitive secY mutations in cytoplasmic domain 5 exhibited dominant export interference (Taura et al., 1994). The secY39 allele was one of them. When cells carrying secY on the chromosome and secY39 on a plasmid (pKY262) were further transformed with pST30 (syd), the transformants exhibited significantly improved export of OmpA and maltose-binding protein (Fig. 5, lane5) in comparison to the control (Fig. 5, lane4). Interestingly, the same syd plasmid did not improve protein export of a strain with the haploid configuration of the secY39 mutation (Fig. 5, lanes6 and 7).


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 (secY1)/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.

Effects of Syd Overproduction on Stability of SecY

SecY is very unstable when singly overexpressed from a plasmid (see Fig. 6, opencircles), but it is stable if SecE is overproduced as well (Matsuyama et al., 1990; Taura et al., 1993; see also Fig. 6, opentriangles). We examined by pulse-chase and immunoprecipitation experiments whether Syd can affect the stability of SecY. As shown in Fig. 6, overproduction of Syd was found to stabilize oversynthesized SecY molecules (opensquares), rendering further support to the notion that Syd can interact with SecY. When the SecY1 form of SecY was expressed from a plasmid, it was degraded with kinetics slightly slower than SecY (Fig. 6, filledcircles). Our basic assumption is that SecY1 competes with SecY for the formation of stable complex with SecE; this competition should occur between newly synthesized molecules (Taura et al., 1993; Joly et al., 1994). We found that, in contrast to wild-type SecY, overexpressed SecY1 was not stabilized at all by the Syd overproduction (Fig. 6, filledsquares). Both SecY and SecY1 were stabilized by SecE (Fig. 6, triangles). In contrast, another mutant form of SecY, SecY24, is stabilized by Syd but not by SecE (Baba et al., 1994). These results indicate that stabilization of SecY by Syd is selective, excluding trivial possibilities such as nonspecific saturation of the proteolytic system.


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 SecY1 (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: circle, TW130/pKY248 (secY)/pNO575H (vector); box, TW130/pKY248/pST34 (syd); up triangle, TW130/pKY248/pKY271 (secE); bullet, TW130/pKY247 (secY1)/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 SecY1 by preferentially stabilizing the chromosomally encoded SecY molecules. To demonstrate this mechanism of suppression, we used a derivative of SecY1 in which a sequence derived from the polylinker-LacZalpha region of pUC19 had been attached to the carboxyl terminus (this construction is referred to as SecY1alpha) (Taura et al., 1993; Baba et al., 1994). This attachment does not abolish the dominant interfering ability of SecY1 (Baba et al., 1994) but enables its electrophoretic separation from SecY. Cells carrying plasmid pKY259 encoding SecY1alpha were pulse-chased, and SecY species were immunoprecipitated. As shown in Fig. 7A, the presence of pKY259 (secY1alpha) 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 SecY1alpha continued or even accelerated (Fig. 7B, filledsquares). Steady state accumulation of SecY and SecY1alpha was examined by immunoblotting (Fig. 7, A and B, insets). Clearly, accumulation of SecY was preferred over that of SecY1alpha in the presence of the Syd plasmid (Fig. 7B).


Figure 7: Demonstration of preferential Syd stabilization of SecY over SecY1alpha. 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 SecY1-LacZalpha fusion protein (SecY1alpha). Cells used were TW130/pKY259 (secY1alpha)/pSTV29 (vector) in A and TW130/pKY259/pST30 (syd) in B. Opensymbols (circle and box) represent chromosomally encoded SecY, whereas filledsymbols (bullet and ) represent SecY1alpha protein synthesized from the plasmid. Insets indicate steady state accumulation of SecY1alpha (upperband) and SecY (lowerband), as detected by immunoblotting.



Incompatibility of Syd Overproduction with the secY24 Mutation

The mutation secY24, a Gly to Asp substitution in cytoplasmic domain 4 of SecY, results in temperature-sensitive protein export and growth (Shiba et al., 1984). It weakens the interaction of SecY with SecE, leading to gradual proteolysis of the altered SecY at high temperature (Baba et al., 1994). We initially failed to introduce any Syd-overproducing plasmid into the secY24 mutant cells even at the permissive temperature (30 °C), unless the mutation had been complemented by a secY plasmid. Rare transformants obtained contained deletions in the syd region on the plasmid. Thus, the secY24 mutant cells are sensitive to overproduction of Syd.

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^Q). 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). circle, KI297 (secY24 lacI^Q)/pST30; , KI298 (secY lacI^Q)/pST30. Viable (colony-forming) cells were counted by plating appropriate dilutions onto glucose-chloramphenicol agar, which was then incubated at 30 °C. bullet, 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.



Phenotypes of syd Disruption

To assess the role of Syd in normal cells, we examined phenotypes associated with disruption of the chromosomal syd gene. We first inserted a kanamycin resistance determinant into syd that was carried on a plasmid (pST42). The linearized syd::kan fragment was then used to transform a recD strain (FS1576). One of transformants was confirmed by Southern hybridization experiments to have its chromosomal syd disrupted. The syd::kan region could be transduced into strains without a complementing plasmid. The Syd protein was undetectable in such a strain (Fig. 3, lane3). We examined growth rates as well as protein export under various conditions, but we have been unable to detect any significant difference between the wild-type and the syd::kan cells. Stability and cellular abundance of SecY do not decrease in the syd-disrupted cells (data not shown).


DISCUSSION

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 SecY1 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 secY1 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 SecY1 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. (^4)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^4.

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). (^5)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 SecY1 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. (^6)Many of them are still able to stabilize SecY and to suppress SecY1. 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 secY1 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 alpha 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.^4 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.


FOOTNOTES

*
This work was supported by grants from the Ministry of Education, Science, and Culture, Japan, from Naito Foundation, from Yamada Science Foundation, and from Mitsubishi Kasei Corp. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBank(TM)/EMBL Data Bank with accession number(s) D38520[GenBank].

§
Present address: Yale University School of Medicine, New Haven, CT 06510.

To whom correspondence should be addressed. Tel.: 81-75-751-4015; Fax: 81-75-771-5699 or 81-75-761-5626; kito{at}virus.kyoto-u.ac.jp.

(^1)
In the previous publications, we (Ito, 1992; Taura et al., 1993, 1994; Baba et al., 1994) and others (Pugsley, 1993) discussed a portion of this work, and the gene symbol of ``ydr'' was used in these publications. However, since an E. coli gene symbol starting with a letter ``y'' may be confusing with the nomenclature proposed for unidentified open reading frames (Rudd, 1993), we give an alternative nomenclature, syd, to this gene. We thank Kenn Rudd for pointing out the issue.

(^2)
The abbreviations used are: kb, kilobase pair(s); IPTG, isopropyl-beta-D-thiogalactopyranoside; PAGE, polyacrylamide gel electrophoresis.

(^3)
T. Taura, unpublished results.

(^4)
T. Shimoike, unpublished results.

(^5)
We thank Arnold Driessen for drawing attention to this sequence motif.

(^6)
K. Cannon, T. Shimoike, and K. Ito, unpublished results.


ACKNOWLEDGEMENTS

We thank Arnold Driessen for pointing out, by visual inspection of the Syd sequence, the possible existence of the SecE motif in it, Kazuko Kagawa for advice in use of the protein sequencer, Hirotada Mori and Keiko Takemoto for oligonucleotides as well as help in homology search, Satoshi Kishigami for preparing a DNA library, Tadashi Baba for discussion, and Kiyoko Mochizuki, Junko Kataoka, and Kuniko Ueda for technical and secretarial assistance.


REFERENCES

  1. Akiyama, Y., and Ito, K. (1985) EMBO J. 4, 3351-3356 [Abstract]
  2. Akiyama, Y., and Ito, K. (1987) EMBO J. 6, 3465-3470 [Abstract]
  3. Baba, T., Jacq, A., Brickman, E., Beckwith, J., Taura, T., Ueguchi, C., Akiyama, Y., and Ito, K. (1990) J. Bacteriol. 172, 7005-7010 [Medline] [Order article via Infotrieve]
  4. Baba, T., Taura, T., Shimoike, T., Akiyama, Y., Yoshihisa, T., and Ito, K. (1994) Proc. Natl. Acad. Sci. U. S. A. 91, 4539-4543 [Abstract]
  5. Douville, K., Leonard, M., Brundage, L., Nishiyama, K., Tokuda, H., Mizushima, S., and Wickner, W. (1994) J. Biol. Chem. 269, 18705-18707 [Abstract/Free Full Text]
  6. Ito, K. (1984) Mol. & Gen. Genet. 197, 204-208
  7. Ito, K. (1992) Mol. Microbiol. 6, 2423-2428 [Medline] [Order article via Infotrieve]
  8. Ito, K., and Akiyama, Y. (1991) Mol. Microbiol. 5, 2243-2253 [Medline] [Order article via Infotrieve]
  9. Joly, J. C., and Wickner, W. (1993) EMBO J. 12, 255-263 [Abstract]
  10. Joly, J. C., Leonard, M., and Wickner, W. T. (1994) Proc. Natl. Acad. Sci. U. S. A. 91, 4703-4707 [Abstract]
  11. Kleckner, N. D., Barker, D. F., Ross, D. G., and Botstein, D. (1978) Genetics 90, 427-450 [Abstract/Free Full Text]
  12. Kohara, Y., Akiyama. K., and Isono, K. (1987) Cell 50, 495-508 [Medline] [Order article via Infotrieve]
  13. Laemmli, U. K. (1970) Nature 227, 680-685 [Medline] [Order article via Infotrieve]
  14. Matsuyama, S., Akimaru, J., and Mizushima, S. (1990) FEBS Lett. 269, 96-100 [CrossRef][Medline] [Order article via Infotrieve]
  15. Matsuyama, S., Fujita, Y., Sagara, K., and Mizushima, S. (1992) Biochim. Biophys. Acta 1122, 77-84 [Medline] [Order article via Infotrieve]
  16. Miller, J. H. (1972) Experiments in Molecular Genetics , Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY
  17. Mothes, W., Prehn, S., and Rapoport, T. A. (1994) EMBO J. 13, 3973-3982 [Abstract]
  18. Murphy, C. K., and Beckwith, J. (1994) Proc. Natl. Acad. Sci. U. S. A. 91, 2557-2561 [Abstract]
  19. Nishiyama, K., Hanada, M., and Tokuda, H. (1994) EMBO J. 13, 3272-3277 [Abstract]
  20. Osborn, M. J., and Munson, R. (1974) Methods Enzymol. 31, 642-653 [Medline] [Order article via Infotrieve]
  21. Pugsley, A. (1993) Microbiol. Rev. 57, 50-108 [Abstract]
  22. Riggs, P. D., Derman, A. I., and Beckwith, J. (1988) Genetics 118, 571-579 [Abstract/Free Full Text]
  23. Rudd, K. (1993) ASM News 59, 335-341
  24. Sanger, F., Nicklen, S., and Coulson, A. R. (1977) Proc. Natl. Acad. Sci. U. S. A. 74, 5463-5467 [Abstract]
  25. Schatz, P. J., and Beckwith, J. (1990) Annu. Rev. Genet. 24, 215-248 [CrossRef][Medline] [Order article via Infotrieve]
  26. Schatz, P. J., Bieker, K. L., Ottemann, K. M., Silhavy, T. J., and Beckwith, J. (1991) EMBO J. 10, 1749-1757 [Abstract]
  27. Shiba, K., Ito, K., Yura, T., and Cerretti, D. P. (1984) EMBO J. 3, 631-635 [Abstract]
  28. Shimoike, T., Akiyama, Y., Baba, T., Taura, T., and Ito, K. (1992) Mol. Microbiol. 6, 1205-1210 [Medline] [Order article via Infotrieve]
  29. Silhavy, T. J., Berman, M. L., and Enquist, L. W. (1984) Experiments with Gene Fusions , Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY
  30. Singer, M., Baker, T., Schnitzler, C., Deischel, S. M., Goel, M., Dove, W., Jaacks, K. J., Grossman, A. D., Erickson, J. W., and Gross, C. (1989) Microbiol. Rev. 53, 1-24
  31. Stahl, F. W., Kobayashi, I., Thaler, D., and Stahl, M. M. (1986) Genetics 113, 215-227 [Abstract/Free Full Text]
  32. Taura, T., Baba, T., Akiyama, Y., and Ito, K. (1993) J. Bacteriol. 175, 7771-7775 [Abstract]
  33. Taura, T., Akiyama, Y., and Ito, K. (1994) Mol. & Gen. Genet. 243, 261-269
  34. Ueguchi, C., and Ito, K. (1992) J. Bacteriol. 174, 1454-1461 [Abstract]
  35. Vieira, J., and Messing, J. (1982) Gene (Amst.) 19, 259-268 [CrossRef][Medline] [Order article via Infotrieve]
  36. Vieira, J., and Messing, J. (1987) Methods Enzymol. 153, 3-11 [Medline] [Order article via Infotrieve]
  37. Yamato, I., Anraku, Y., and Hirosawa, K. (1975) J. Biochem. (Tokyo) 77, 705-718 [Abstract]

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