Enhanced export of beta-galactosidase fusion proteins in prlF mutants is Lon dependent.

We have used fusions of the outer membrane protein LamB to beta-galactosidase (encoded by lacZ) to study the protein export process. This LamB-LacZ hybrid protein blocks export when synthesized at high levels, as evidenced by inducer (maltose) sensitivity, a phenomenon termed LacZ hybrid jamming. The prlF1 mutation relieves LacZ hybrid jamming and allows localization of the fusion protein to a noncytoplasmic compartment. prlF1 and similar alleles are gain-of-function mutations. Null mutations in this gene confer no obvious phenotypes. Extragenic suppressors of a gain-of-function prlF allele have been isolated in order to understand how this gene product affects the export process. The suppressors are all lon null mutations, and they are epistatic to all prlF phenotypes tested. Lon protease activity has been measured in prlF1 cells and shown to be increased. However, the synthesis of Lon is not increased in a prlF1 background, suggesting a previously unidentified mechanism of Lon activation. Further analysis reveals that prlF1 activates degradation of cytoplasmically localized precursors in a Lon protease-dependent manner. It is proposed that accumulation of precursors during conditions of hybrid protein jamming titrates an essential export component(s), possibly a chaperone. Increased Lon-dependent precursor degradation would free this component, thus allowing increased protein export under jamming conditions.     

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.     

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.     

Analysis of the Escherichia coli Alp phenotype: heat shock induction in ssrA mutants

The major phenotypes of lon mutations, UV sensitivity and overproduction of capsule, are due to the stabilization of two substrates, SulA and RcsA. Inactivation of transfer mRNA (tmRNA) (encoded by ssrA), coupled with a multicopy kanamycin resistance determinant, suppressed both lon phenotypes and restored the rapid degradation of SulA. This novel protease activity was named Alp but was never identified further. We report here the identification, mapping, and characterization of a chromosomal mutation, faa (for function affecting Alp), that leads to full suppression of a Deltalon ssrA::cat host and thus bypasses the requirement for multicopy Kan(r); faa and ssrA mutants are additive in their ability to suppress lon mutants. The faa mutation was mapped to the C terminus of dnaJ(G232); dnaJ null mutants have similar effects. The identification of a lon suppressor in dnaJ suggested the possible involvement of heat shock. We find that ssrA mutants alone significantly induce the heat shock response. The suppression of UV sensitivity, both in the original Alp strain and in faa mutants, is reversed by mutations in clpY, encoding a subunit of the heat shock-induced ClpYQ protease that is known to degrade SulA. However, capsule synthesis is not restored by clpY mutants, probably because less RcsA accumulates in the Alp strain and because the RcsA that does accumulate is inactive. Both ssrA effects are partially relieved by ssrA derivatives encoding protease-resistant tags, implicating ribosome stalling as the primary defect. Thus, ssrA and faa each suppress two lon mutant phenotypes but by somewhat different mechanisms, with heat shock induction playing a major role.     

Secretion of LamB-LacZ by the signal recognition particle pathway of Escherichia coli

LamB-LacZ fusion proteins have classically been used in studies of the general secretion pathway of Escherichia coli. Here we describe how increasing signal sequence hydrophobicity routes LamB-LacZ Hyb42-1 to the signal recognition particle (SRP) pathway. Secretion of this hydrophobic fusion variant (H*LamB-LacZ) was reduced in the absence of fully functional Ffh and Ffs, and the translocator jamming caused by Hyb42-1 was prevented by efficient delivery of the fusion to the periplasm. Finally, we found that in the absence of the ribosome-associated chaperone, trigger factor (Tig), LamB-LacZ localized to the periplasm in a SecA-dependent, SRP-independent fashion. Collectively, our results provide compelling in vivo evidence that there is an SRP-dependent cotranslational targeting mechanism in E. coli and argue against a role for trigger factor in pathway discrimination.     

The Escherichia coli cell division mutation ftsM1 is in serU.

The ftsM1 mutation is believed to be in a gene implicated in the regulation of cell division in Escherichia coli because it displayed the lon mutation phenotypes. In this study, we show that this mutation is located in serU, a gene which codes for tRNA(Ser)2, and has the phenotypes of the serU allele supH. Both ftsM1 and supH suppressed the leuB6 and ilvD145 missense mutations, and both conferred temperature and UV light irradiation sensitivity to the harboring cells. Cells which carried the ftsM1 mutation or the supH suppressor had very low colony-forming abilities on salt-free L agar, and this phenotype was almost completely abolished by the presence of plasmids bearing the ftsZ+ gene. Furthermore, sensitivity of the mutant cells to UV irradiation was also markedly diminished when they carried a ftsZ+-bearing plasmid. These results suggest that supH-containing cells have reduced FtsZ activities, in accordance with their displaying the phenotypes of the lon mutant cells. The possibility that ftsM1 (supH) is functionally involved in the biosynthesis of a specific protein which affects cell division is discussed.     

A snc1 endocytosis mutant: phenotypic analysis and suppression by overproduction of dihydrosphingosine phosphate lyase

The v-SNARE proteins Snc1p and Snc2p are required for fusion of secretory vesicles with the plasma membrane in yeast. Mutation of a methionine-based sorting signal in the cytoplasmic domain of either Sncp inhibits Sncp endocytosis and prevents recycling of Sncp to the Golgi after exocytosis. snc1-M43A mutant yeast have reduced growth and secretion rates and accumulate post-Golgi secretory vesicles and fragmented vacuoles. However, cells continue to grow and secrete for several hours after de novo Snc2-M42A synthesis is repressed. DPL1, the structural gene for dihydrosphingosine phosphate lyase, was selected as a high copy number snc1-M43A suppressor. Because DPL1 also partially suppresses the growth and secretion phenotypes of a snc deletion, we propose that enhanced degradation of dihydrosphingosine-1-phosphate allows an alternative protein to replace Sncp as the secretory vesicle v-SNARE.     

An extraintestinal, pathogenic isolate of Escherichia coli (O4/K54/H5) can produce a group 1 capsule which is divergently regulated from its constitutively produced group 2, K54 capsular polysaccharide.

We are studying an O4/K54/H5 Escherichia coli bacteremic isolate (CP9) as a model pathogen for extraintestinal infection. Its group 2, K54 capsular polysaccharide is an important virulence determinant and confers serum resistance. In this study the effect of the group 1 capsule regulators, RcsA, RcsB, and Lon protease, on the regulation of CP9's capsular polysaccharides was assessed. It was established that in the presence of multicopy rcsA or with disruption of lon, CP9 can be induced to produce a group 1 capsule. RcsA, RcsB, and Lon are present in this K54 background and regulate group 1 capsule expression in a fashion similar to that described for K-12 strains. Two independent group 2 capsule gene protein fusions (cl1.29::TnphoA and cl1.137::TnphoA) were used to evaluate the effects of these regulators on group 2 K54 capsule production. Disruption of lon resulted in 1.9-fold (TR293 [cl1.29::TnphoA lon-146]) and 3.4-fold (TR1373 [cl1.137::TnphoA lon-146]) decreases in fusion activity at 28 degrees C, relative to the baseline level. However, decreases in fusion activity at 42 degrees C were only 1.2- and 1.4-fold, respectively. Inactivation of both lon and rcsA or lon and rcsB restored fusion activity to baseline levels at 28 degrees C, but only a partial restoration of activity was seen at higher temperatures. To assess whether these differences in fusion activity reflected a functional change in capsule production, the effects of 80% normal human serum (NHS) were tested against CP9 and TR93 (lon-146). Since the group 2 K54 capsule protects against the bactericidal activity of 80% NHS, a decrease in its production results in an increase in serum sensitivity. Viable counts of CP9 increased 10-fold in 80% NHS over 3 h at 28 degrees C, as expected. In contrast to CP9, TR93 (lon-146) incurred a 10-fold loss in viability under the same conditions. The levels of RcsA are increased in TR93 (lon 146) as consequence of lon disruption; therefore, these results in conjunction with the cl1::TnphoA protein fusion data establish RcsA as a negative regulator of the group 2 K54 capsular polysaccharide. Furthermore, these results also suggest existence of another Lon-sensitive negative regulator of group 2 K54 capsule production, which is active higher temperatures.     

Overproduction of NlpE, a new outer membrane lipoprotein, suppresses the toxicity of periplasmic LacZ by activation of the Cpx signal transduction pathway.

The LamB-LacZ-PhoA tripartite fusion protein is secreted to the periplasm, where it exerts a toxicity of unknown origin during high-level synthesis in the presence of the inducer maltose, a phenotype referred to as maltose sensitivity. We selected multicopy suppressors of this toxicity that allow growth of the tripartite fusion strains in the presence of maltose. Mapping and subclone analysis of the suppressor locus identified a previously uncharacterized chromosomal region at 4.7 min that is responsible for suppression. DNA sequence analysis revealed a new gene with the potential to code for a protein of 236 amino acids with a predicted molecular mass of 25,829 Da. The gene product contains an amino-terminal signal sequence to direct the protein for secretion and a consensus lipoprotein modification sequence. As predicted from the sequence, the suppressor protein is labeled with [3H]palmitate and is localized to the outer membrane. Accordingly, the gene has been named nlpE (for new lipoprotein E). Increased expression of NlpE suppresses the maltose sensitivity of tripartite fusion strains and also the extracytoplasmic toxicities conferred by a mutant outer membrane protein, LamBA23D. Suppression occurs by activation of the Cpx two-component signal transduction pathway. This pathway controls the expression of the periplasmic protease DegP and other factors that can combat certain types of extracytoplasmic stress.     

Escherichia coli expression and processing of human interleukin-1 beta fused to signal peptides

Escherichia coli expression, processing, and secretion of human interleukin-1 beta (IL-1 beta) fused to the signal peptide of E. coli OmpA or PhoA protein were studied. With fusion to either signal sequence, high-level expression was observed and the products accumulated to about 20% of total cell protein. In the fusion to OmpA leader sequence, more than 50% of the product has the OmpA signal peptide removed precisely. The majority of the processed material is not released by osmotic shock. On the other hand, very little of the material from the fusion to PhoA has the PhoA signal peptide removed. Use of the host with a mutation in prlA or prlF, variation of temperature for cell growth, and alteration of the amino acid residues around the cleavage site do not facilitate processing of the PhoA signal peptide. These results suggest that some component in the PhoA signal peptide, interacting with the Il-1 beta sequence, is interfering with the processing of the signal peptide.     

Soluble Epstein-Barr virus glycoproteins gH, gL, and gp42 form a 1:1:1 stable complex that acts like soluble gp42 in B-cell fusion but not in epithelial cell fusion

Epstein-Barr virus (EBV) is a herpesvirus that infects cells by fusing its lipid envelope with the target cell membrane. The fusion process requires the actions of viral glycoproteins gH, gL, and gB for entry into epithelial cells and additionally requires gp42 for entry into B cells. To further study the roles of these membrane-associated glycoproteins, purified soluble forms of gp42, gH, and gL were expressed that lack the membrane-spanning regions. The soluble gH/gL protein complex binds to soluble gp42 with high affinity, forming a stable heterotrimer with 1:1:1 stoichiometry, and this complex is not formed by an N-terminally truncated variant of gp42. The effects of adding soluble gp42, gH/gL, and gH/gL/gp42 were examined with a virus-free cell-cell fusion assay. The results demonstrate that, in contrast to gp42, membrane fusion does not proceed with secreted gH/gL. The addition of soluble gH/gL does not inhibit or enhance B-cell or epithelial cell fusion when membrane-bound gH/gL, gB, and gp42 are present. However, the soluble gH/gL/gp42 complex does activate membrane fusion with B cells, similarly to soluble gp42, but it does not inhibit fusion with epithelial cells, as observed for gp42 alone. A gp42 peptide, derived from an N-terminal segment involved in gH/gL interactions, binds to soluble gH/gL and inhibits EBV-mediated epithelial cell fusion, mimicking gp42. These observations reveal distinct functional requirements for gH/gL and gp42 complexes in EBV-mediated membrane fusion.     

Tobacco mosaic virus 126-kDa protein increases the susceptibility of Nicotiana tabacum to other viruses and its dosage affects virus-induced gene silencing

The Tobacco mosaic virus (TMV) 126-kDa protein is a suppressor of RNA silencing previously shown to delay the silencing of transgenes in Nicotiana tabacum and N. benthamiana. Here, we demonstrate that expression of a 126-kDa protein-green fluorescent protein (GFP) fusion (126-GFP) in N. tabacum increases susceptibility to a broad assortment of viruses, including Alfalfa mosaic virus, Brome mosaic virus, Tobacco rattle virus (TRV), and Potato virus X. Given its ability to enhance TRV infection in tobacco, we tested the effect of 126-GFP expression on TRV-mediated virus-induced gene silencing (VIGS) and demonstrate that this protein can enhance silencing phenotypes. To explain these results, we examined the poorly understood effect of suppressor dosage on the VIGS response and demonstrated that enhanced VIGS corresponds to the presence of low levels of suppressor protein. A mutant version of the 126-kDa protein, inhibited in its ability to suppress silencing, had a minimal effect on VIGS, suggesting that the suppressor activity of the 126-kDa protein is indeed responsible for the observed dosage effects. These findings illustrate the sensitivity of host plants to relatively small changes in suppressor dosage and have implications for those interested in enhancing silencing phenotypes in tobacco and other species through VIGS.     

PRMT3 is a distinct member of the protein arginine N-methyltransferase family. Conferral of substrate specificity by a zinc-finger domain

S-Adenosyl-l-methionine-dependent protein arginine N-methyltransferases (PRMTs) catalyze the methylation of arginine residues within a variety of proteins. At least four distinct mammalian family members have now been described, including PRMT1, PRMT3, CARM1/PRMT4, and JBP1/PRMT5. To more fully define the physiological role of PRMT3, we characterized its unique putative zinc-finger domain and how it can affect its enzymatic activity. Here we show that PRMT3 does contain a single zinc-finger domain in its amino terminus. Although the zinc-liganded form of this domain is not required for methylation of an artificial substrate such as the glutathione S-transferase-fibrillarin amino-terminal fusion protein (GST-GAR), it is required for the enzyme to recognize RNA-associated substrates in RAT1 cell extracts. The recombinant form of PRMT3 is inhibited by high concentrations of ZnCl(2) as well as N-ethylmaleimide, reagents that can modify cysteine sulfhydryl groups. We found that we could distinguish PRMT family members by their sensitivity to these reagents; JBP1/PRMT5 and Hsl7 methyltransferases were inhibited in a similar manner as PRMT3, whereas Rmt1, PRMT1, and CARM1/PRMT4 were not affected. We were also able to define differences in these enzymes by their sensitivity to inhibition by Tris and free arginine. Finally, we found that the treatment of RAT1 cell extracts with N-ethylmaleimide leads to a loss of the major PRMT1-associated activity that was immune to inhibition under the same conditions as a GST fusion protein. These results suggest that native forms of PRMTs can have different properties than their GST-catalytic chain fusion protein counterparts, which may lack associated noncatalytic subunits.     

SseF, a type III effector protein from the mammalian pathogen Salmonella enterica, requires resistance-gene-mediated signalling to activate cell death in the model plant Nicotiana benthamiana

Type III effector proteins (T3Es) of many Gram-negative pathogenic bacteria manipulate highly conserved cellular processes, indicating conservation in virulence mechanisms during the infection of hosts of divergent evolutionary origin. In order to identify conserved effector functions, we used a cross-kingdom approach in which we expressed selected T3Es from the mammalian pathogen Salmonella enterica in leaves of Nicotiana benthamiana and searched for possible virulence or avirulence phenotypes. We show that the T3E SseF of S. enterica triggers hypersensitive response (HR)-like symptoms, a hallmark of effector-triggered immunity in plants, either when transiently expressed in leaves of N. benthamiana by Agrobacterium tumefaciens infiltration or when delivered by Xanthomonas campestris pv vesicatoria (Xcv) through the type III secretion system. The ability of SseF to elicit HR-like symptoms was lost upon silencing of suppressor of G2 allele of skp1 (SGT1), indicating that the S. enterica T3E is probably recognized by an R protein in N. benthamiana. Xcv translocating an AvrRpt2-SseF fusion protein was restricted in multiplication within leaves of N. benthamiana. Bacterial growth was not impaired but symptom development was rather accelerated in a compatible interaction with susceptible pepper (Capsicum annuum) plants. We conclude that the S. enterica T3E SseF is probably recognized by the plant immune system in N. benthamiana, resulting in effector-triggered immunity.     

The YrdC protein--a putative ribosome maturation factor

Release factor one (RF1) terminates protein synthesis in response to stop codons UAG and UAA. A mutant allele of RF1 causes temperature sensitive growth at 42 degrees C. We have earlier described the isolation of a suppressor of the temperature sensitive phenotype. The suppressor mutation is a small deletion in the open reading frame yrdC, and we have shown that the DeltayrdC mutation leads to immature 30S subunits and, as a consequence, to fewer translating ribosomes. YrdC is a small conserved protein with a dsRNA-binding surface. Here, we have characterized the YrdC protein. We show that the deletion leads to no production of functional protein, and we have indications that the YrdC protein might be essential in a wild type background. The protein is needed for the maturation of 16S rRNA, even though it does not interact tightly with either of the ribosomal subunits, or the 70S particles. The less effective maturation of rRNA affects the ribosomal feedback control, leading to an increase in expression from P1rrnB. We suggest that the function of the YrdC protein is to keep an rRNA structure needed for proper processing of 16S rRNA, especially at lower temperatures. This activity may require other factor(s). We suggest the gene be renamed rimN, and the mutant allele rimN141.     

A suppressor of temperature-sensitive rna mutations that affect mRNA metabolism in Saccharomyces cerevisiae.

We have isolated a dominant suppressor of rna mutation (SRN1) that relieves the temperature-sensitive inhibition of mRNA synthesis of ribosomal protein genes in the yeast Saccharomyces cerevisiae. The suppressor was selected for its ability to alleviate simultaneously the temperature-sensitive growth phenotypes of rna2 and rna6. Several independently isolated suppressors appeared to be recessive lethal mutations. One suppressor, SRN1, was recovered as viable in haploid strains. SRN1 can suppress rna2, rna3, rna4, rna5, rna6, and rna8 singly or in pairs, although some combinations of rna mutations are less well suppressed than others. The suppressor allows strains with rna mutations to grow at 34 degrees C but is unable to suppress at 37 degrees C; however, SRN1 does not, by itself, prevent growth at 37 degrees C. In addition, SRN1 suppresses the rna1 mutation which affects general mRNA levels and also leads to the accumulation of precursor tRNA for those tRNAs that have intervening sequences. SRN1 can suppress the rna1 mutation as well as the rna1 rna2 double mutation at 34 degrees C. The suppressor does not affect the temperature-sensitive growth of two unrelated temperature-sensitive mutations, cdc4 and cdc7.     

Fus1p interacts with components of the Hog1p mitogen-activated protein kinase and Cdc42p morphogenesis signaling pathways to control cell fusion during yeast mating

Cell fusion in the budding yeast Saccharomyces cerevisiae is a temporally and spatially regulated process that involves degradation of the septum, which is composed of cell wall material, and occurs between conjugating cells within a prezygote, followed by plasma membrane fusion. The plasma membrane protein Fus1p is known to be required for septum degradation during cell fusion, yet its role at the molecular level is not understood. We identified Sho1p, an osmosensor for the HOG MAPK pathway, as a binding partner for Fus1 in a two-hybrid screen. The Sho1p-Fus1p interaction occurs directly and is mediated through the Sho1p-SH3 domain and a proline-rich peptide ligand on the Fus1p COOH-terminal cytoplasmic region. The cell fusion defect associated with fus1Delta mutants is suppressed by a sho1Delta deletion allele, suggesting that Fus1p negatively regulates Sho1p signaling to ensure efficient cell fusion. A two-hybrid matrix containing fusion proteins and pheromone response pathway signaling molecules reveals that Fus1p may participate in a complex network of interactions. In particular, the Fus1p cytoplasmic domain interacts with Chs5p, a protein required for secretion of specialized Chs3p-containing vesicles during bud development, and chs5Delta mutants were defective in cell surface localization of Fus1p. The Fus1p cytoplasmic domain also interacts with the activated GTP-bound form of Cdc42p and the Fus1p-SH3 domain interacts with Bni1p, a yeast formin that participates in cell fusion and controls the assembly of actin cables to polarize secretion in response to Cdc42p signaling. Taken together, our results suggest that Fus1p acts as a scaffold for the assembly of a cell surface complex involved in polarized secretion of septum-degrading enzymes and inhibition of HOG pathway signaling to promote cell fusion.