Altered translation initiation factor 2 in the cold-sensitive ssyG mutant affects protein export in Escherichia coli.

The Escherichia coli gene secY (pr1A) codes for an integral membrane protein that plays an essential role in protein export. We previously isolated cold-sensitive mutations (ssy) as extragenic suppressors of temperature-sensitive secY24 mutation. Now we show that the ssyG class of mutations are within infB coding for the translation initiation factor IF2. The mutants produce altered forms of IF2 with a cold-sensitive in vitro activity to form a translation initiation complex. The mutation suppresses not only secY24 but also other secretion-defective mutations such as secA51 and rp10215. The beta-galactosidase enzyme activity of the MalE-LacZ 72-47 hybrid protein is strikingly reduced in the ssyG mutant at the permissive high temperature, while the hybrid protein itself is normally synthesized. This effect, which was observed only for the hybrid protein with a functional signal sequence, may result from some alteration in the cellular localization of the protein. These results suggest that IF2 or the translation initiation step can modulate protein export reactions. The isolation of cold-sensitive ssyG mutations in infB provides genetic evidence that IF2 is indeed essential for normal growth of E. coli cells.     

Mutation that suppresses the protein export defect of the secY mutation and causes cold-sensitive growth of Escherichia coli.

A cold-sensitive mutant was isolated among temperature-resistant revertants of the secY24 mutant defective in secretion of envelope proteins across the cytoplasmic membrane at 42 degrees C. A single mutation, designated ssyA3, is responsible both for the extragenic suppression of secY and for the cold-sensitive growth. In contrast to the parental secY24 mutant, the suppressed cells do not accumulate precursors of envelope proteins at any temperatures. The cells containing the ssyA3 mutation, whether in combination with secY24 or not, show an optimal growth at 42 degrees C and a very poor growth at 30 degrees C. At the low temperature, protein synthesis is generally slowed down, probably at the step of chain elongation. The gene ssyA was mapped at a new locus between hisS and glyA on the chromosome. It is possible that the product of this gene interacts both with the protein secretion system and the protein synthesizing system.     

The last RNA-binding repeat of the Escherichia coli ribosomal protein S1 is specifically involved in autogenous control

The ssyF29 mutation, originally selected as an extragenic suppressor of a protein export defect, has been mapped within the rpsA gene encoding ribosomal protein S1. Here, we examine the nature of this mutation and its effect on translation. Sequencing of the rpsA gene from the ssyF mutant has revealed that, due to an IS10R insertion, its product lacks the last 92 residues of the wild-type S1 protein corresponding to one of the four homologous repeats of the RNA-binding domain. To investigate how this truncation affects translation, we have created two series of Escherichia coli strains (rpsA(+) and ssyF) bearing various translation initiation regions (TIRs) fused to the chromosomal lacZ gene. Using a beta-galactosidase assay, we show that none of these TIRs differ in activity between ssyF and rpsA(+) cells, except for the rpsA TIR: the latter is stimulated threefold in ssyF cells, provided it retains at least ca. 90 nucleotides upstream of the start codon. Similarly, the activity of this TIR can be severely repressed in trans by excess S1, again provided it retains the same minimal upstream sequence. Thus, the ssyF stimulation requires the presence of the rpsA translational autogenous operator. As an interpretation, we propose that the ssyF mutation relieves the residual repression caused by normal supply of S1 (i.e., that it impairs autogenous control). Thus, the C-terminal repeat of the S1 RNA-binding domain appears to be required for autoregulation, but not for overall mRNA recognition.     

Inositol monophosphatase activity from the Escherichia coli suhB gene product.

The suhB gene is located at 55 min on the Escherichia coli chromosome and encodes a protein of 268 amino acids. Mutant alleles of suhB have been isolated as extragenic suppressors for the protein secretion mutation (secY24), the heat shock response mutation (rpoH15), and the DNA synthesis mutation (dnaB121) (K. Shiba, K. Ito, and T. Yura, J. Bacteriol. 160:696-701, 1984; R. Yano, H. Nagai, K. Shiba, and T. Yura, J. Bacteriol. 172:2124-2130, 1990; S. Chang, D. Ng, L. Baird, and C. Georgopoulos, J. Biol. Chem. 266:3654-3660, 1991). These mutant alleles of suhB cause cold-sensitive cell growth, indicating that the suhB gene is essential at low temperatures. Little work has been done, however, to elucidate the role of the product of suhB in a normal cell and the suppression mechanisms of the suhB mutations in the aforementioned mutants. The sequence similarity shared between the suhB gene product and mammalian inositol monophosphatase has prompted us to test the inositol monophosphatase activity of the suhB gene product. We report here that the purified SuhB protein showed inositol monophosphatase activity. The kinetic parameters of SuhB inositol monophosphatase (Km = 0.071 mM; Vmax = 12.3 mumol/min per mg) are similar to those of mammalian inositol monophosphatase. The ssyA3 and suhB2 mutations, which were isolated as extragenic suppressors for secY24 and rpoH15, respectively, had a DNA insertion at the 5' proximal region of the suhB gene, and the amount of SuhB protein within mutant cells decreased. The possible role of suhB in E. coli is discussed.     

A Mutation Affecting the Regulation of a Seca-Lacz Fusion Defines a New Sec Gene

It was shown previously that the secA gene of Escherichia coli is derepressed in cells that have a defect in protein export. Here it is demonstrated that the beta-galactosidase produced by a secA-lacZ gene fusion strain is regulated in the same way. Studies on the fusion strain reveal that the promoter or a site involved in regulation of the secA gene is located considerably upstream from the structural gene. The properties of the fusion strain provide a new selection for mutants that are defective in protein export. Selection for increased lac expression of a secA-lacZ fusion strain yields mutations in three of the known sec genes, secA, secD and prlA/secY. In addition, mutations in several genes not previously known to affect secA expression were obtained. A mutation in one of these genes causes a pleiotropic defect in protein export and a cold-sensitive growth defect; this gene, which maps at approximately 90 min on the bacterial chromosome, has been named secE.     

Insertional disruption of the nusB (ssyB) gene leads to cold-sensitive growth of Escherichia coli and suppression of the secY24 mutation

Summary The Escherichia coli gene ssyB was cloned and sequenced. The ssyB63 (Cs) mutation is an insertion mutation in nusB, while the nusB5 (Cs) mutation suppresses secY24, indicating that inactivation of nusB causes cold-sensitive cell growth as well as phenotypic suppression of secY24. The correct map position of nusB is 9.5 min rather than I I min as previously assigned. It is located at the distal end of an operon that contains a gene showing significant homology with a Bacillus subtilis gene involved in riboflavin biosynthesis.     

Genetic analysis of an essential cytoplasmic domain of Escherichia coli SecY based on resistance to Syd, a SecY-interacting protein

We previously described a dominant negative secY ^-d 1 allele in Escherichia coli, whose product interferes with protein export, presumably by sequestering SecE, the stabilizing partner of SecY. Syd is the product of a multicopy suppressor of the secY ^-d 1 phenotype, and its overproduction preferentially stabilizes the wild-type SecY protein. In contrast, overproduction of Syd is toxic to the secY24 mutant, which shows a partial defect in SecY-SecE interaction. We isolated Syd-resistant revertants from the secY24 mutant. Pseudo-reversions mapped to sites at or near the secY24 mutation site (Gly240→Asp). The secY249 mutation (Ala249→Val) intragenically suppressed Syd sensitivity, but not the temperature-sensitive Sec phenotype of the secY24 mutation. The SecY249 mutant protein shows a reduced capacity to be stabilized by Syd, suggesting that the mutation weakens the SecY-Syd interaction. The other two mutations changed residue 240 (the site of the secY24 alteration) to Asn (secY245) or Ala (secY241) and restored the ability of the cell to export protein. Although the secY245 mutant retained some sensitivity␣to Syd overproduction, the secY241 mutant was completely Syd-resistant. Furthermore, the secY241 mutation seemed to represent a “hyper reversion” with respect to the SecY-SecE interaction. Protein export in this mutant was no longer sensitive to SecY^-d1. When the secY ^-d 1 mutation was combined intragenically with secY241, the resulting double mutant gene (secY ^-d 1–241) showed an increased ability to interfere with protein export. On the basis of our model for SecY^-d1, these results suggest that the secY241 alteration enhances SecY-SecE interaction. These results indicate that residue 240 of SecY is crucial for the interaction between the cytosolic domains of SecY and SecE required for the establishment of the translocase complex. Received: 20 October 1997 / Accepted: 1 December 1997     

Genetic analysis of an essential cytoplasmic domain of Escherichia coli SecY based on resistance to Syd, a SecY-interacting protein

We previously described a dominant negative secY ^-d 1 allele in Escherichia coli, whose product interferes with protein export, presumably by sequestering SecE, the stabilizing partner of SecY. Syd is the product of a multicopy suppressor of the secY ^-d 1 phenotype, and its overproduction preferentially stabilizes the wild-type SecY protein. In contrast, overproduction of Syd is toxic to the secY24 mutant, which shows a partial defect in SecY-SecE interaction. We isolated Syd-resistant revertants from the secY24 mutant. Pseudo-reversions mapped to sites at or near the secY24 mutation site (Gly240→Asp). The secY249 mutation (Ala249→Val) intragenically suppressed Syd sensitivity, but not the temperature-sensitive Sec phenotype of the secY24 mutation. The SecY249 mutant protein shows a reduced capacity to be stabilized by Syd, suggesting that the mutation weakens the SecY-Syd interaction. The other two mutations changed residue 240 (the site of the secY24 alteration) to Asn (secY245) or Ala (secY241) and restored the ability of the cell to export protein. Although the secY245 mutant retained some sensitivity?to Syd overproduction, the secY241 mutant was completely Syd-resistant. Furthermore, the secY241 mutation seemed to represent a “hyper reversion” with respect to the SecY-SecE interaction. Protein export in this mutant was no longer sensitive to SecY^-d1. When the secY ^-d 1 mutation was combined intragenically with secY241, the resulting double mutant gene (secY ^-d 1–241) showed an increased ability to interfere with protein export. On the basis of our model for SecY^-d1, these results suggest that the secY241 alteration enhances SecY-SecE interaction. These results indicate that residue 240 of SecY is crucial for the interaction between the cytosolic domains of SecY and SecE required for the establishment of the translocase complex.     

Characterization of cold-sensitive secY mutants of Escherichia coli.

Mutations which cause poor growth at a low temperature, which affect aspects of protein secretion, and which map in or around secY (prlA) were characterized. The prlA1012 mutant, previously shown to suppress a secA mutation, proved to have a wild-type secY gene, indicating that this mutation cannot be taken as genetic evidence for the secA-secY interaction. Two cold-sensitive mutants, the secY39 and secY40 mutants, which had been selected by their ability to enhance secA expression, contained single-amino-acid alterations in the same cytoplasmic domain of the SecY protein. Protein export in vivo was partially slowed down by the secY39 mutation at 37 to 39 degrees C, and the retardation was immediately and strikingly enhanced upon exposure to nonpermissive temperatures (15 to 23 degrees C). The rate of posttranslational translocation of the precursor to the OmpA protein (pro-OmpA protein) into wild-type membrane vesicles in vitro was only slightly affected by reaction temperatures ranging from 37 to 15 degrees C, and about 65% of OmpA was eventually sequestered at both temperatures. Membrane vesicles from the secY39 mutant were much less active in supporting pro-OmpA translocation even at 37 degrees C, at which about 20% sequestration was attained. At 15 degrees C, the activity of the mutant membrane decreased further. The rapid temperature response in vivo and the impaired in vitro translocation activity at low temperatures with the secY39 mutant support the notion that SecY, a membrane-embedded secretion factor, participates in protein translocation across the bacterial cytoplasmic membrane.     

A mutation in secY that causes enhanced SecA insertion and impaired late functions in protein translocation

A cold-sensitive secY mutant (secY125) with an amino acid substitution in the first periplasmic domain causes in vivo retardation of protein export. Inverted membrane vesicles prepared from this mutant were as active as the wild-type membrane vesicles in translocation of a minute amount of radioactive preprotein. The mutant membrane also allowed enhanced insertion of SecA, and this SecA insertion was dependent on the SecD and SecF functions. These and other observations suggested that the early events in translocation, such as SecA-dependent insertion of the signal sequence region, is actually enhanced by the SecY125 alteration. In contrast, since the mutant membrane vesicles had decreased capacity to translocate chemical quantity of pro-OmpA and since they were readily inactivated by pretreatment of the vesicles under the conditions in which a pro-OmpA translocation intermediate once accumulated, the late translocation functions appear to be impaired. We conclude that this periplasmic secY mutation causes unbalanced early and late functions in translocation, compromising the translocase's ability to catalyze multiple rounds of reactions.     

Yeast gene which suppresses the defect in protein export of a secY mutant of E. coli

To find factors participating in protein translocation in yeast, we screened a yeast genomic library for genes which, when introduced into Escherichia coli, suppressed secY24, a temperature sensitive mutation of an essential integral membrane protein (SecY) required for protein export. We isolated and characterized a gene (YSY6) which improved the translocation of the OmpA protein in mutant strain IQ85(secY24). It could also suppress another mutant [rplO215(Am)], in which the level of expression of the SecY protein is decreased at high-temperature. The YSY6 gene encodes a small amphiphilic peptide consisting of 65 amino acids, which can be expressed in E. coli cells.     

The deleterious effect of an insertion sequence removing the last twenty percent of the essential Escherichia coli rpsA gene is due to mRNA destabilization, not protein truncation

Ribosomal protein S1, the product of the essential rpsA gene, consists of six imperfect repeats of the same motif. Besides playing a critical role in translation initiation on most mRNAs, S1 also specifically autoregulates the translation of its own messenger. ssyF29 is a viable rpsA allele that carries an IS10R insertion within the coding sequence, resulting in a protein lacking the last motif (S1DeltaC). The growth of ssyF29 cells is slower than that of wild-type cells. Moreover, translation of a reporter rpsA-lacZ fusion is specifically stimulated, suggesting that the last motif is necessary for autoregulation. However, in ssyF29 cells the rpsA mRNA is also strongly destabilized; this destabilization, by causing S1DeltaC shortage, might also explain the observed slow-growth and autoregulation defect. To fix this ambiguity, we have introduced an early stop codon in the rpsA chromosomal gene, resulting in the synthesis of the S1DeltaC protein without an IS10R insertion (rpsADeltaC allele). rpsADeltaC cells grow much faster than their ssyF29 counterparts; moreover, in these cells S1 autoregulation and mRNA stability are normal. In vitro, the S1DeltaC protein binds mRNAs (including its own) almost as avidly as wild-type S1. These results demonstrate that the last S1 motif is dispensable for translation and autoregulation: the defects seen with ssyF29 cells reflect an IS10R-mediated destabilization of the rpsA mRNA, probably due to facilitated exonucleolytic degradation.     

Genetic analysis of SecY: additional export-defective mutations and factors affecting their phenotypes

A number of secY mutants of Escherichia coli showing protein export defects were isolated by a combination of localized mutagenesis and secA-lacZ screening. Most of them were cold sensitive and contained single base substitutions in secY leading to amino acid replacements in various parts of the SecY protein, mainly in the cytoplasmic and the transmembrane domains. A temperature-sensitive mutant with an export defect had the same base substitution as secY24, which was characterized previously. Many cold-sensitive secY mutants exhibited rapid responses to temperature lowering but their apparent defects varied at the permissive temperature. Others exhibited delayed responses to the temperature shift. Some secY mutations, including secY39, interfered with protein export when expressed from a multicopy plasmid, even in the presence of wild-type secY on the chromosome. Such dominant negative mutations, including secY ^–d l, which was studied previously, were all located in either cytoplasmic domain 5 or 6, which is consistent with our previous proposal that the C-terminal region of SecY is important for its function as a protein translocator. We also studied the phenotypes of strains in which one of the secY mutations was combined with the components of the SecD operon. Overexpression of SecD partially suppressed the secY39 mutation, while overexpression of secF exacerbated the export defects of secY122 and secY125 mutations. Overexpression of yajC, located within the SecD operon, suppressed sec Y ^–d1. Although yajC itself proved to be dispensable, its disruption impaired the growth of the secY39 mutant at 42°C. These observations suggest that SecY interacts with SecD, SecF, and the product of yajC.     

SecY and SecA interact to allow SecA insertion and protein translocation across the Escherichia coli plasma membrane.

SecA, the preprotein-driving ATPase in Escherichia coli, was shown previously to insert deeply into the plasma membrane in the presence of ATP and a preprotein; this movement of SecA was proposed to be mechanistically coupled with preprotein translocation. We now address the role played by SecY, the central subunit of the membrane-embedded heterotrimeric complex, in the SecA insertion reaction. We identified a secY mutation (secY205), affecting the most carboxyterminal cytoplasmic domain, that did not allow ATP and preprotein-dependent productive SecA insertion, while allowing idling insertion without the preprotein. Thus, the secY205 mutation might affect the SecYEG 'channel' structure in accepting the preprotein-SecA complex or its opening by the complex. We isolated secA mutations that allele-specifically suppressed the secY205 translocation defect in vivo. One mutant protein, SecA36, with an amino acid alteration near the high-affinity ATP-binding site, was purified and suppressed the in vitro translocation defect of the inverted membrane vesicles carrying the SecY205 protein. The SecA36 protein could also insert into the mutant membrane vesicles in vitro. These results provide genetic evidence that SecA and SecY specifically interact, and show that SecY plays an essential role in insertion of SecA in response to a preprotein and ATP and suggest that SecA drives protein translocation by inserting into the membrane in vivo.     

Identification of five new essential genes involved in the synthesis of a secreted protein in Escherichia coli.

To define additional components of the export machinery of Escherichia coli, I have isolated extragenic suppressors of a mutant [secA(Ts)] that is temperature sensitive for growth and secretion at 37 degrees C. Suppressors that restored growth at 37 degrees C, but that rendered the cell cold sensitive for growth at 28 degrees C, were obtained. The suppressor mutations fall into at least seven loci, two of which (prlA and secC) have been previously implicated in protein secretion. The five remaining loci (ssaD, ssaE, ssaF, ssaG, and ssaH) have been mapped by P1 transduction and appear to define new genes in E. coli. All of the suppressor mutations allow both enhanced growth and protein secretion of the secA(Ts) mutant at 37 degrees C, but not 42 degrees C, indicating a continued requirement for SecA protein. Strains carrying solely the cold-sensitive mutations show reduced levels of certain periplasmic proteins when grown at low temperatures. In at least one case, that of maltose-binding protein, this defect is at the level of synthesis of the protein. Since mutants in any of seven genes as well as secA amber mutants halt or reduce the synthesis of an exported protein, it appears that E. coli may possess a general and complex mechanism for coupling protein synthesis and secretion.     

Mutational analysis of transmembrane regions 3 and 4 of SecY, a central component of protein translocase

The SecYEG heterotrimeric membrane protein complex functions as a channel for protein translocation across the Escherichia coli cytoplasmic membrane. SecY is the central subunit of the SecYEG complex and contains 10 transmembrane segments (TM1 to TM10). Previous mutation studies suggested that TM3 and TM4 are particularly important for SecY function. To further characterize TM3 and TM4, we introduced a series of cysteine-scanning mutations into these segments. With one exception (an unstable product), all the mutant proteins complemented the cold-sensitive growth defect of the secY39 mutant. A combination of this secY mutation and the secG deletion resulted in synthetic lethality, and the TM3 and TM4 SecY cysteine substitution mutations were examined for their ability to complement this lethality. Although they were all positive for complementation, some of the complemented cells exhibited significant retardation of protein export. The substitution-sensitive residues in TM3 can be aligned to one side of the alpha-helix, and those in TM4 revealed a tendency for residues closer to the cytosolic side of the membrane to be more severely affected. Disulfide cross-linking experiments identified a specific contact point for TM3 and SecG TM2 as well as for TM4 and SecG TM1. Thus, although TM3 and TM4 do not contain any single residue that is absolutely required, they include functionally important helix surfaces and specific contact points with SecG. These results are discussed in light of the structural information available for the SecY complex.     

Temperature-sensitive sec mutants of Escherichia coli: inhibition of protein export at the permissive temperature.

Phenotypes of secY and secA temperature-sensitive mutants at permissive (low) temperature have been examined. The secY24 mutant was found to be extremely susceptible to export inhibition by a basal-level synthesis of the MalE-LacZ 72-47 hybrid protein or to overproduction of a normal secretory protein such as maltose-binding protein or beta-lactamase. Comparison of this phenotype of secY24 with those of the secY100 and secA51 mutants under similar conditions suggested that MalE-LacZ protein and overproduced secretory protein do not nonspecifically enhance the partial secretion defect but act synergistically with secY24 to inhibit protein export.     

Targeting of signal sequenceless proteins for export in Escherichia coli with altered protein translocase.

Most extracytoplasmic proteins are synthesized with an N-terminal signal sequence that targets them to the export apparatus. Escherichia coli prlA mutants (altered in the secY gene) are able to export cell envelope proteins lacking any signal sequence. In order to understand how such proteins are targeted for export, we isolated mutations in a signal sequenceless version of alkaline phosphatase that block its export in a prlA mutant. The mutations introduce basic amino acyl residues near the N-terminus of alkaline phosphatase. These changes do not disrupt an N-terminal export signal in this protein since the first 25 amino acids can be removed without affecting its export competence. These findings suggest that signal sequenceless alkaline phosphatase does not contain a discrete domain that targets it for export and may be targeted simply because it remains unfolded in the cytoplasm. We propose that basic amino acids near the N-terminus of a signal sequenceless protein affect its insertion into the translocation apparatus after it has been targeted for export. These findings allow the formulation of a model for the entry of proteins into the membrane-embedded export machinery.     

Genetic Interactions at the Fla10 Locus: Suppressors and Synthetic Phenotypes That Affect the Cell Cycle and Flagellar Function in Chlamydomonas Reinhardtii

Through the isolation of suppressors of temperature-sensitive flagellar assembly mutations at the FLA10 locus of Chlamydomonas reinhardtii, we have identified six other genes involved in flagellar assembly. Mutations at these suppressor loci, termed SUF1-SUF6, display allele specificity with respect to which fla10- mutant alleles they suppress. An additional mutation, apm1-122, which confers resistance to the plant herbicides amiprophos-methyl and oryzalin, was also found to interact with mutations at the FLA10 locus. The apm1-122 mutation in combination with three fla10- mutant alleles results in synthetic cold-sensitive cell division defects, and in combination with an additional pseudo-wild-type fla10- allele yields a synthetic temperature-sensitive flagellar motility phenotype. Based upon the genetic interactions of these loci, we propose that the FLA10 gene product interacts with multiple components of the flagellar apparatus and plays a role both in flagellar assembly and in the cell cycle.     

A SecE mutation that modulates SecY-SecE translocase assembly,identified as a specific suppressor of SecY defects

The SecY39(Cs) (cold-sensitive) alteration of Arg357 results in a defect of translocation initiation. As a means to dissect the Sec translocation machinery, we isolated mutations that act as suppressors of the secY39 defect. A specific secE mutation, designated secE105, was thus isolated. This mutation proved to be identical with the prlG2 mutation and to suppress a number of cold-sensitive secY mutations. However, other prlG mutations did not effectively suppress the secY defects. Evidence indicates that the Ser105-to-Pro alteration in the C-terminal transmembrane segment of SecE weakens SecY-SecE association. In vitro analyses showed that the SecE(S105P) alteration preferentially stimulates the initial phase of translocation. It is suggested that the S105P alteration affects the SecYEG channel such that it is more prone to open and to accept the translocation initiation domain of a preprotein molecule.