Null mutations in a Nudix gene, ygdP, implicate an alarmone response in a novel suppression of hybrid jamming

Induction of the toxic LamB-LacZ protein fusion, Hyb42-1, leads to a lethal generalized protein export defect. The prlF1 suppressor causes hyperactivation of the cytoplasmic Lon protease and relieves the inducer sensitivity of Hyb42-1. Since prlF1 does not cause a detectable change in the stability or level of the hybrid protein, we conducted a suppressor screen, seeking factors genetically downstream of lon with prlF1-like phenotypes. Two independent insertions in the ygdP open reading frame relieve the toxicity of the fusion protein and share two additional properties with prlF1: cold sensitivity and the ability to suppress the temperature sensitivity of a degP null mutation. Despite these similarities, ygdP does not appear to act in the same genetic pathway as prlF1 and lon, suggesting a fundamental link between the phenotypes. We speculate that the common properties of the suppressors relate to secretion defects. The ygdP gene (also known as nudH) has been shown to encode a Nudix protein that acts as a dinucleotide oligophosphate (alarmone) hydrolase. Our results suggest that loss of ygdP function leads to the induction of an alarmone-mediated response that affects secretion. Using an epitope-tagged ygdP construct, we present evidence that this response is sensitive to secretion-related stress and is regulated by differential proteolysis of YgdP in a self-limiting manner.     

Increased expression of the bifunctional protein PrlF suppresses overproduction lethality associated with exported beta-galactosidase hybrid proteins in Escherichia coli.

We have cloned and determined the nucleotide sequence of the prlF gene. An open reading frame predicting a 111-amino-acid protein (Mr 12,351) with an acidic carboxy terminus was identified. The DNA sequence preceding this open reading frame revealed a putative promoter and a ribosome-binding site. The nucleotide sequence of the prlF1 mutation revealed a 7-base-pair duplication resulting in a slightly smaller predicted gene product of Mr 12,009 that lacked the acidic carboxy terminus. Maxicell analysis of prlF and prlF1 subclones identified peptides of sizes similar to those predicted by the nucleotide sequences. The prlF sequence was shown to be expressed in vivo by both maxicell analysis and construction of a prlF-lacZ fusion. Two kanamycin resistance insertions within the prlF open reading frame were introduced into the chromosome, replacing the wild-type gene. In contrast to the prlF1 mutation, these insertions had no detectable effect on cell growth or on the beta-galactosidase activity or maltose sensitivity (two sensitive indicators of hybrid protein export) conferred by the lamB-lacZ42-1 gene fusion. Overproduction of the wild-type prlF gene product from a plasmid carrying an active hybrid promoter, however, conferred a prlF1 phenotype. In addition, both the prlF1 mutation and both kanamycin resistance insertions increased the beta-galactosidase activity of a prlF-lacZ fusion. These results suggest that prlF is autoregulated and that overproduction of the prlF gene product increases the export efficiency of beta-galactosidase hybrid proteins from the cytoplasm.     

Mutation prlF1 relieves the lethality associated with export of beta-galactosidase hybrid proteins in Escherichia coli.

The 42-1 lamB-lacZ gene fusion confers a conditionally lethal, export-dependent phenotype known as maltose sensitivity. A maltose-resistant mutant showing decreased beta-galactosidase activity of the hybrid protein, designated prlF1 (protein localization), was unlinked to the lamB-lacZ fusion. This mutation mapped at 70 min on the Escherichia coli linkage map and conferred maltose resistance, a 30-fold reduction in beta-galactosidase activity, and a 30% decrease in cellular growth rate at 30 degrees C that was independent of the presence of a gene fusion. prlF1 also decreased the beta-galactosidase activity and relieved the maltose sensitivity conferred by fusions of lacZ to the gene specifying the periplasmic maltose-binding protein, malE. The decrease in beta-galactosidase activity, however, was specific for exported hybrid proteins. When export of the hybrid protein was blocked by a signal sequence mutation, prlF1 decreased the beta-galactosidase activity only 2.5-fold. Similarly, prlF1 did not affect the beta-galactosidase activity of fusions of lacZ to a gene specifying a nonexported protein, malK.     

A novel gain-of-function mutant of the cyclic GMP-dependent protein kinase egl-4 affects multiple physiological processes in Caenorhabditis elegans

cGMP-dependent protein kinases are key intracellular transducers of cell signaling. We identified a novel dominant mutation in the C. elegans egl-4 cGMP-dependent protein kinase (PKG) and show that this mutation causes increased normal gene activity although it is associated with a reduced EGL-4 protein level. Prior phenotypic analyses of this gain-of-function mutant demonstrated a reduced longevity and a reduced feeding behavior when the animals were left unperturbed. We characterize several additional phenotypes caused by increased gene activity of egl-4. These phenotypes include a small body size, reduced locomotion in the presence of food, a pale intestine, increased intestinal fat storage, and a decreased propensity to form dauer larvae. The multiple phenotypes of egl-4 dominant mutants are consistent with an instructive signaling role of PKG to control many aspects of animal physiology. This is among the first reported gain-of-function mutations in this enzyme of central physiological importance. In a genetic screen we have identified extragenic suppressors of this gain-of-function mutant. Thus, this mutant promises to be a useful tool for identifying downstream targets of PKG.     

Beta-galactosidase is inactivated by intermolecular disulfide bonds and is toxic when secreted to the periplasm of Escherichia coli.

The wild-type LamB-LacZ hybrid protein inhibits the export machinery upon induction when assayed by biochemical and genetic techniques, a phenotype referred to as hybrid protein jamming. This hybrid protein also renders cells sensitive to growth in the presence of the inducer maltose, presumably because of the jamming. We constructed a new version of this fusion by adding alkaline phosphatase, encoded by phoA, to the C terminus of the LamB-LacZ hybrid protein. This tripartite protein, LamB-LacZ-PhoA, is as toxic to cells as the hybrid LamB-LacZ; however, it does not jam at temperatures greater than 33 degrees C. Extreme C-terminal sequences of LacZ function as a critical folding domain and are therefore responsible for stabilizing the LacZ structure. To determine if this region of LacZ is important for jamming, we recombined a late nonsense mutation (X90) onto the hybrid construct. We found the toxicity of this new hybrid, LamB-LacZX90, to be nearly identical to that of the full-length protein, but it also does not jam the secretion machinery. This suggests that jamming is caused by LacZ folding. We found no inhibition of secretion in the tripartite and X90 fusion strains at 37 degrees C, suggesting that the toxicity of the new fusions is novel. Under these conditions, the tripartite and X90 fusion proteins form disulfide-bonded aggregates with high molecular weights in the periplasm. Accordingly, we believe that LacZ disrupts some essential function(s) in the periplasm.     

A new Escherichia coli gene,fdrA, identified by suppression analysis of dominant negative FtsH mutations

An Escherichia coli membrane protein, FtsH, has been implicated in several cellular processes, including integration of membrane proteins, translocation of secreted proteins, and degradation of some unstable proteins. However, how it takes part in such diverse cellular events is largely unknown. We previously isolated dominant negative ftsH mutations and proposed that FtsH functions in association with some other cellular factor(s). To test this proposal we isolated multicopy suppressors of dominant negative ftsH mutations. One of the multicopy suppressor clones contained an N-terminally truncated version of a new gene that was designated fdrA. The FdrA fragment suppressed both of the phenotypes — increased abnormal translocation of a normally cytoplasmic domain of a model membrane protein and retardation of protein export — caused by dominant negative FtsH proteins. The intact fdrA gene (11.9 min on the chromosome) directed the synthesis of a 60 kDa protein in vitro.     

A new Escherichia coli gene, fdrA, identified by suppression analysis of dominant negative FtsH mutations

An Escherichia coli membrane protein, FtsH, has been implicated in several cellular processes, including integration of membrane proteins, translocation of secreted proteins, and degradation of some unstable proteins. However, how it takes part in such diverse cellular events is largely unknown. We previously isolated dominant negative ftsH mutations and proposed that FtsH functions in association with some other cellular factor(s). To test this proposal we isolated multicopy suppressors of dominant negative ftsH mutations. One of the multicopy suppressor clones contained an N-terminally truncated version of a new gene that was designated fdrA. The FdrA fragment suppressed both of the phenotypes — increased abnormal translocation of a normally cytoplasmic domain of a model membrane protein and retardation of protein export — caused by dominant negative FtsH proteins. The intact fdrA gene (11.9 min on the chromosome) directed the synthesis of a 60 kDa protein in vitro.     

The use of extragenic suppressors to define genes involved in protein export in Escherichia coli

Summary The secA gene codes for a membrane component involved in protein export in E. coli. In order to define other genes whose products play such a role, we have characterized extragenic suppressors of a secA(Ts) mutation. These suppressors fall into at least three genetic loci. One such locus is the prlA gene, previously identified by mutations which suppress signal sequence mutants. Thus, this approach may allow the identification of new genes involved in the export process.     

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.     

Differential effects of mutations in the protein and RNA moieties of RNase P on the efficiency of suppression by various tRNA suppressors

We have studied the efficiency of suppression by tRNA suppressors in vivo in strains of Escherichia coli that harbor a mutation in the rnpA gene, the gene for the protein component (C5) of RNase P, and in strains that carry several different alleles of the rnpB gene, the gene for the RNA component (M1) of RNase P. Depending on the genetic background, different efficiencies of suppression by the various tRNA suppressors were observed. Thus, mutations in rnpA have separable and distinct effects from mutations in rnpB on the processing of tRNA precursors by RNase P. In addition, the efficiency of suppression by several derivatives of E. coli tRNA(Tyr) Su3 changed as the genetic background was altered.     

Suppressor analysis suggests a multistep, cyclic mechanism for protein secretion in Escherichia coli.

The sec/prl gene products catalyze the translocation of precursor proteins from the cytoplasm of Escherichia coli. Recessive, conditionally lethal mutant alleles of these genes (sec mutations) cause a generalized defect in protein secretion; dominant suppressor mutant alleles (prl mutations) restore export of precursor proteins with altered signal sequences. In prl strains, a precursor protein with a defective signal sequence can be selectively targeted to the suppressor gene product. When a precursor LacZ hybrid protein is used, the targeted prl protein is inactivated by the large, toxic hybrid molecule, a result termed suppressor-directed inactivation (SDI). Using SDI, two different secretion-related complexes can be generated: a pretranslocation complex that contains a hybrid protein with an unprocessed signal sequence, and a translocation complex in which the hybrid protein is jammed in transmembrane orientation with the signal sequence cleaved. Additional Sec proteins that are contained within, and thus sequestered by, each of these complexes can be identified when their functional levels are lowered using the conditional lethal sec mutations. Results of this genetic analysis suggest a multistep pathway for protein secretion in which the translocation machinery assembles on demand.     

A novel class of secA alleles that exert a signal-sequence-dependent effect on protein export in Escherichia coli

The murine plasminogen activator inhibitor 2 (PAI2) signal sequence inefficiently promotes the export of E. coli alkaline phosphatase (AP). High-level expression of PAI2::AP chimeric proteins from the arabinose P(BAD) promoter is toxic and confers an Ara(S) phenotype. Most Ara(R) suppressors map to secA, as determined by sequencing 21 independent alleles. Mutations occur throughout the gene, including both nucleotide binding domains (NBDI and NBDII) and the putative signal sequence binding domain (SSBD). Using malE and phoA signal sequence mutants, we showed that the vast majority of these secA suppressors exhibit weak Sec phenotypes. Eight of these secA mutations were further characterized in detail. Phenotypically, these eight suppressors can be divided into three groups, each localized to one domain of SecA. Most mutations allow near-normal levels of wild-type preprotein export, but they enhance the secretion defect conferred by signal sequence mutations. Interestingly, one group exerts a selective effect on the export of PAI2::AP when compared to that of AP. In conclusion, this novel class of secA mutations, selected as suppressors of a toxic signal sequence, differs from the classical secA (prlD) mutations, selected as suppressors of defective signal sequences, although both types of mutations affect signal sequence recognition.     

PrlA (SecY) and PrlG (SecE) interact directly and function sequentially during protein translocation in E. coli

Three strategies for genetic analysis show that two inner membrane components of the export machinery, PrlA (SecY) and PrlG (SecE), interact directly while catalyzing the translocation of secreted proteins across the cytoplasmic membrane of E. coli. The first, suppressor-directed inactivation (SDI), exploits the specific interaction between dominant prl suppressors of signal sequence mutations and mutant LacZ hybrid proteins. The second, Sec titration, extends SDI to allow the identification of various Sec proteins that are present in the translocation complex. The third uses the synthetic lethality of certain double-mutant strains to infer physical interactions between gene products. Biochemical data obtained with SDI strains allow the identification of two different secretory intermediates and indicate that PrlG functions before PrlA in the secretion pathway.     

Improving the stability of a foreign protein in the periplasmic space of Escherichia coli

An efficient expression/export vector comprising the entire phoS (phosphate binding protein) gene fused to a synthetic gene encoding the human growth hormone releasing factor (mhGRF) has recently been constructed [1]. The hybrid protein (PhoS-mhGRF) was exported to the periplasmic space. However, in this location proteolytic degradation occurred at the C-terminal region. Phenylmethylsulfonyl fluoride (PMSF) increased the stability of the hybrid protein indicating that a serine protease may be involved in the proteolytic cleavage. The correct export and subsequent degradation of the recombinant protein in the periplasmic space were demonstrated in situ using double immunogold labeling on ultrathin sections. Using a phoS-based expression/export vector in the presence of PMSF, 2-4 mg of hybrid protein per liter of culture could be obtained.     

Identification and Characterization of Genes That Interact with Lin-12 in Caenorhabditis Elegans

We identified and characterized 14 extragenic mutations that suppressed the dominant egg-laying defect of certain lin-12 gain-of-function mutations. These suppressors defined seven genes: sup-17, lag-2, sel-4, sel-5, sel-6, sel-7 and sel-8. Mutations in six of the genes are recessive suppressors, whereas the two mutations that define the seventh gene, lag-2, are semi-dominant suppressors. These suppressor mutations were able to suppress other lin-12 gain-of-function mutations. The suppressor mutations arose at a very low frequency per gene, 10-50 times below the typical loss-of-function mutation frequency. The suppressor mutations in sup-17 and lag-2 were shown to be rare non-null alleles, and we present evidence that null mutations in these two genes cause lethality. Temperature-shift studies for two suppressor genes, sup-17 and lag-2, suggest that both genes act at approximately the same time as lin-12 in specifying a cell fate. Suppressor alleles of six of these genes enhanced a temperature-sensitive loss-of-function allele of glp-1, a gene related to lin-12 in structure and function. Our analysis of these suppressors suggests that the majority of these genes are part of a shared lin-12/glp-1 signal transduction pathway, or act to regulate the expression or stability of lin-12 and glp-1.     

Suppression of defective-sporulation phenotypes by mutations in transcription factor genes of Bacillus subtilis.

Mutations in the Bacillus subtilis major RNA polymerase sigma factor gene (rpoD/crsA47) and a sensory receiver gene (spoOA/rvtA11) are potent intergenic suppressors of several stage 0 sporulation mutations (spoOB, OE, OF & OK). We show here that these suppressors also rescue temperature-sensitive sporulation phenotypes (Spots) caused by mutations in RNA polymerase, ribosomal protein, and protein synthesis elongation factor EF-G genes. The effects of the crsA and rvtA suppressors on RNA polymerase and ribosomal protein spots mutations are similar to those previously described for mutations in another intergenic suppressor gene rev. We have examined the effects of rvtA and crsA mutations on the expression of sporulation-associated membrane proteins, including flagellin and penicillin binding protein 5* (PBP 5*). Both suppressors restored sporulation and synthesis of PBP 5* in several spoO mutants. However, only rvtA restored flagellin synthesis in spoO suppressed backgrounds. The membrane protein phenotypes resulting from the presence of crsA or rvtA suppressors in spoO strains suggests that these suppressors function via distinct molecular mechanisms. The rvtA and crsA mutations are also able to block the ability of ethanol to induce spoO phenocopies at concentrations of ethanol which prevent sporulation in wild type cells. The effects of ethanol on sporulation-associated membrane protein synthesis in wild type and suppressor containing strains have been examined.     

Genetic and molecular characterization of the caenorhabditis elegans gene,mel-26, a postmeiotic negative regulator of mei-1, a meiotic-specific spindle component.

We have previously described the gene mei-1, which encodes an essential component of the Caenorhabditis elegans meiotic spindle. When ectopically expressed after the completion of meiosis, mei-1 protein disrupts the function of the mitotic cleavage spindles. In this article, we describe the cloning and the further genetic characterization of mel-26, a postmeiotic negative regulator of mei-1. mel-26 was originally identified by a gain-of-function mutation. We have reverted this mutation to a loss-of-function allele, which has recessive phenotypes identical to the dominant defects of its gain-of-function parent. Both the dominant and recessive mutations of mel-26 result in mei-1 protein ectopically localized in mitotic spindles and centrosomes, leading to small and misoriented cleavage spindles. The loss-of-function mutation was used to clone mel-26 by transformation rescue. As suggested by genetic results indicating that mel-26 is required only maternally, mel-26 mRNA was expressed predominantly in the female germline. The gene encodes a protein that includes the BTB motif, which is thought to play a role in protein-protein interactions.     

Saturation and specificity of the Lon protease of Escherichia coli.

Lon is an ATP-dependent protease of Escherichia coli. The lon mutation has a pleiotropic phenotype: UV sensitivity, mucoidy, deficiency for lysogenization by bacteriophage lambda and P1, and lower efficiency in the degradation of abnormal proteins. All of these phenotypes are correlated with the loss of protease activity. Here we examine the effects of overproduction of one Lon substrate, SulA, and show that it protects two other substrates from degradation. To better understand this protection, we mutagenized the sulA gene and selected for mutants that have partially or totally lost their ability to saturate the Lon protease and thus can no longer protect another substrate. Some of the SulA mutants lost their ability to protect RcsA from degradation but could still protect the O thermosensitive mutant protein (Ots). All of the mutants retained their capacity to induce cell division inhibition. It was also found that deletion of the C-terminal end of SulA affected its activity but did not affect its susceptibility to Lon. We propose that Lon may have more than one specificity for peptide cleavage.     

Folding LacZ in the Periplasm of Escherichia coli

Targeted, translational LacZ fusions provided the initial support for the signal sequence hypothesis in prokaryotes and allowed for selection of the mutations that identified the Sec translocon. Many of these selections relied on the fact that expression of targeted, translational lacZ fusions like malE-lacZ and lamB-lacZ42-1 causes lethal toxicity as folded LacZ jams the translocation pore. However, there is another class of targeted LacZ fusions that do not jam the translocon. These targeted, nonjamming fusions also show toxic phenotypes that may be useful for selecting mutations in genes involved in posttranslocational protein folding and targeting; however, they have not been investigated to the same extent as their jamming counterparts. In fact, it is still unclear whether LacZ can be fully translocated in these fusions. It may be that they simply partition into the inner membrane where they can no longer participate in folding or assembly. In the present study, we systematically characterize the nonjamming fusions and determine their ultimate localization. We report that LacZ can be fully translocated into the periplasm, where it is toxic. We show that this toxicity is likely due to LacZ misfolding and that, in the absence of the periplasmic disulfide bond catalyst DsbA, LacZ folds in the periplasm. Using the novel phenotype of periplasmic β-galactosidase activity, we show that the periplasmic chaperone FkpA contributes to LacZ folding in this nonnative compartment. We propose that targeted, nonjamming LacZ fusions may be used to further study folding and targeting in the periplasm of Escherichia coli.