Previously, it was shown that optimal functioning of the Yersinia type III secretion system (T3SS) in cell culture infection assays requires the exoribonuclease polynucleotide phosphorylase (PNPase) and that normal T3SS activity could be restored in the Deltapnp strains by expressing just the approximately 70-aa S1 RNA-binding domain of PNPase. Here, it is shown that the Yersinia Deltapnp strain is less virulent in the mouse compared with the isogenic wild-type strain. To begin to understand what could be limiting T3SS activity in the absence of PNPase, T3SS-encoding transcripts and proteins in the YersiniaDeltapnp strains were analyzed. Surprisingly, it was found that the Deltapnp Yersinia strains possessed enhanced levels of T3SS-encoding transcripts and proteins compared with the wild-type strains. We then found that an S1 variant containing a disruption in its RNA-binding subdomain was inactive in terms of restoring normal T3SS activity. However, T3SS expression levels did not differ between Deltapnp strains expressing active and inactive S1 proteins, further showing that T3SS activity and expression levels, at least as related to PNPase and its S1 domain, are not linked. The results suggest that PNPase affects the expression and activity of the T3SS by distinct mechanisms and that the S1-dependent effect on T3SS activity involves an RNA intermediate.
Yersinia spp. use a type 3 secretion system (T3SS) to directly inject six proteins into macrophages, and any impairment of this process results in a profound reduction in virulence. We previously showed that the exoribonuclease polynucleotide phosphorylase (PNPase) was required for optimal T3SS functioning in Yersinia pseudotuberculosis and Yersinia pestis. Here we report that Y. pseudotuberculosis cells with reduced RNase E activity are likewise impaired in T3SS functioning and that phenotypically they resemble ⌬pnp cells. RNase E does not affect expression levels of the T3SS substrates but instead, like PNPase, regulates a terminal event in the secretion pathway. This similarity, together with the fact that RNase E and PNPase can be readily copurified from Y. pseudotuberculosis cell extracts, suggests that these two RNases regulate T3SS activity through a common mechanism. This is the first report that RNase E activity impacts the T3SS as well as playing a more general role in infectivity.
Adducin is a cytoskeletal protein having regulatory roles that involve actin filaments, functions that are inhibited by phosphorylation of adducin by protein kinase C. Adducin is hyperphosphorylated in nervous system tissue in patients with the neurodegenerative disease amyotrophic lateral sclerosis, and mice lacking β-adducin have impaired synaptic plasticity and learning. We have found that Drosophila adducin, encoded by hu-li tai shao (hts), is localized to the post-synaptic larval neuromuscular junction (NMJ) in a complex with the scaffolding protein Discs large (Dlg), a regulator of synaptic plasticity during growth of the NMJ. hts mutant NMJs are underdeveloped, whereas over-expression of Hts promotes Dlg phosphorylation, delocalizes Dlg away from the NMJ, and causes NMJ overgrowth. Dlg is a component of septate junctions at the lateral membrane of epithelial cells, and we show that Hts regulates Dlg localization in the amnioserosa, an embryonic epithelium, and that embryos doubly mutant for hts and dlg exhibit defects in epithelial morphogenesis. The phosphorylation of Dlg by the kinases PAR-1 and CaMKII has been shown to disrupt Dlg targeting to the NMJ and we present evidence that Hts regulates Dlg targeting to the NMJ in muscle and the lateral membrane of epithelial cells by controlling the protein levels of PAR-1 and CaMKII, and consequently the extent of Dlg phosphorylation.
Budding yeast divides asymmetrically, giving rise to a mother cell that progressively ages and a daughter cell with full lifespan. It is generally assumed that mother cells retain damaged, lifespan limiting materials ("aging factors") through asymmetric division. However, the identity of these aging factors and the mechanisms through which they limit lifespan remain poorly understood. Using a flow cytometry-based, high-throughput approach, we quantified the asymmetric partitioning of the yeast proteome between mother and daughter cells during cell division, discovering 74 mother-enriched and 60 daughterenriched proteins. While daughter-enriched proteins are biased toward those needed for bud construction and genome maintenance, mother-enriched proteins are biased towards those localized in the plasma membrane and vacuole. Deletion of 23 of the 74 motherenriched proteins leads to lifespan extension, a fraction that is about six times that of the genes picked randomly from the genome. Among these lifespan-extending genes, three are involved in endosomal sorting/endosome to vacuole transport, and three are nitrogen source transporters. Tracking the dynamic expression of specific mother-enriched proteins revealed that their concentration steadily increases in the mother cells as they age, but is kept relatively low in the daughter cells via asymmetric distribution. Our results suggest that some mother-enriched proteins may increase to a concentration that becomes deleterious and lifespan-limiting in aged cells, possibly by upsetting homeostasis or leading to aberrant signaling. Our study provides a comprehensive resource for analyzing asymmetric cell division and aging in yeast, which should also be valuable for understanding similar phenomena in other organisms.aging | asymmetric cell division | proteome C ellular aging and asymmetric cell division are intimately linked. In budding yeast, asymmetric cell division yields a mother cell and a daughter cell that are easily distinguishable under the microscope. Tracking the fate of the mother lineage led to the discovery that individual mother cells have a finite replicative lifespan, defined by the number of daughters a mother cell produced before senescence (1). It is known that although the mother cell ages with each division, their daughters retain the same full lifespan independent of the age of the mother at least until the last few mother cell divisions (2, 3). Thus, the asymmetry in cell division leads to asymmetry of aging.Even in single-celled organisms in which cell division is seemingly morphologically symmetric, such as fission yeast or Escherichia coli, asymmetric partitioning of cellular contents can still occur and have a differential impact on the aging/death fate of the two offspring (4-8). Asymmetric cell division is also a general phenomenon in mammalian cells (e.g., during development or in mitotically active tissues), where cell division typically leads to two cells with distinct fates, often with different replicative potential. It has been argued...
The Yersinia protein kinase A (YpkA) and outer protein J (YopJ) are co-expressed from a single transcript and are injected directly into eukaryotic cells by the plague bacterium Yersinia pestis. When overexpressed in vertebrate or yeast cells, YpkA disrupts the actin-based cytoskeletal system by an unknown mechanism, whereas YopJ obstructs inductive chemokine expression by inhibiting MAPK and NF-B signaling. Previously, we showed that the fission yeast Schizosaccharomyces pombe was sensitive to the kinase activity of YpkA. Here, we screened yeast for cellular processes important for YpkA activity and found that the eIF2␣ kinases mollify the toxicity imparted by the kinase activity of YpkA. Specifically, strains lacking the eIF2␣ kinase Hri2 were particularly sensitive to YpkA. Unexpectedly, the activity of YopJ, which conferred a phenotype consistent with its inhibitory effect on MAPK signaling, was also found to be dependent on Hri2. When expressed in S. pombe, YopJ sensitized cells to osmotic and oxidative stresses through a Hri2-dependent mechanism. However, when co-expressed with YpkA, YopJ protected cells from YpkA-mediated toxicity, and this protection was entirely dependent on Hri2. In contrast, YopJ did not confer protection against the toxic effects of the Yersinia virulence factor YopE. These findings are the first to functionally link YpkA and YopJ and suggest that eIF2␣ kinases, which are critically important in antiviral defenses and protection against environmental stresses, also play a role in bacterial virulence.The plague bacterium Yersinia pestis, as well as the closely related enteric pathogens Yersinia pseudotuberculosis and Yersinia enterocolitica, are relatively resistant to the antimicrobial killing systems of innate immune cells. This property is largely dependent on a membrane-bound type 3 secretion system (T3SS) 2 that injects proteins (here referred to as "effectors") directly into the eukaryotic cell (1, 2). This study is focused on two such effectors: YpkA and YopJ (YopO and YopP in Y. enterocolitica). Although they are encoded by a single transcription unit on the 70-kb extrachromosomal virulence plasmid of Yersinia, these two effectors shape the bacterial host relationship in very different ways. YpkA and its associated kinase activity is necessary for the immediate survival of Yersinia following attachment to host cells; this was suggested by animal infection experiments and confirmed at the cellular level in a function-based "infectivity" assay that measures both survival and growth of macrophage-associated Yersinia (3, 4). In contrast, there are no discernible differences in the infectivity assay between the wild-type and ⌬yopJ mutant strains (5). However, YopJ very efficiently blocks MAPK-and NF-B-mediated signaling pathways and the resulting inductive expression of proinflammatory chemokines; the consequences of YopJ activity during an actual infection is a reduction in the local inflammatory response (6 -10). Therefore, unlike YpkA, which is important for the immediate surviv...
tRNase Z is the endonuclease that is involved in tRNA 3'-end maturation by removal of the 3'-trailer sequences from tRNA precursors. Most eukaryotes examined to date, including the budding yeast Saccharomyces cerevisiae and humans, have a single long form of tRNase Z (tRNase ZL). In contrast, the fission yeast Schizosaccharomyces pombe contains two candidate tRNase ZLs encoded by the essential genes sptrz1+ and sptrz2+. In the present study, we have expressed recombinant SpTrz1p and SpTrz2p in S. pombe. Both recombinant proteins possess precursor tRNA 3'-endonucleolytic activity in vitro. SpTrz1p localizes to the nucleus and has a simian virus 40 NLS (nuclear localization signal)-like NLS at its N-terminus, which contains four consecutive arginine and lysine residues between residues 208 and 211 that are critical for the NLS function. In contrast, SpTrz2p is a mitochondrial protein with an N-terminal MTS (mitochondrial-targeting signal). High-level overexpression of sptrz1+ has no detectable phenotypes. In contrast, strong overexpression of sptrz2+ is lethal in wild-type cells and results in morphological abnormalities, including swollen and round cells, demonstrating that the correct expression level of sptrz2+ is critical. The present study provides evidence for partitioning of tRNase Z function between two different proteins in S. pombe, although we cannot rule out specialized functions for each protein.
The molecular electronegativity-distance vector (MEDV) is employed to describe the chemical structure of bisphenol A analogs and their correlated estrogen activities. The result shows that the constructed models have good predictability and indicates substructures that may influence estrogen activities of chemicals.
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