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1.
Immunofluorescence of the microtubular skeleton in growing and drug-treated yeast protoplasts 总被引:3,自引:0,他引:3
The microtubular system in growing protoplasts of Saccharomyces uvarum was visualized by immunofluorescence using the monoclonal antitubulin antibody TU 01. We confirmed the coexistence of regular spindle configuration and extensive cytoplasmic networks in growing protoplasts and also observed a distinct distortion of cytoplasmic microtubules in association with wall removal. After a short period for recovery of protoplasts in nutrient medium a restitution of cytoplasmic microtubules and their resumed contact with the protoplast surface was observed. Treatment of growing protoplasts with nocodazole resulted in the disappearance of spindle and cytoplasmic microtubules in the relevant fraction of the protoplast population. In carbendazime (MBC)-arrested protoplasts spindle microtubules were absent but cytoplasmic microtubules associated with spindle pole bodies were clearly visible. Microtubule reassembly on spindle pole bodies occurred within 30 min after washing out nocodazole as well as carbendazime. The approach using protoplasts suggests a simple way in which the differential effect of antimicrotubule agents can be experimentally tested and the microtubule organizing activity of yeast protoplasts visualized at the population level. 相似文献
2.
Mark Rinnerthaler Renata Lejskova Tomas Grousl Vendula Stradalova Gino Heeren Klaus Richter Lore Breitenbach-Koller Jan Malinsky Jiri Hasek Michael Breitenbach 《PloS one》2013,8(10)
As we have shown previously, yeast Mmi1 protein translocates from the cytoplasm to the outer surface of mitochondria when vegetatively growing yeast cells are exposed to oxidative stress. Here we analyzed the effect of heat stress on Mmi1 distribution. We performed domain analyses and found that binding of Mmi1 to mitochondria is mediated by its central alpha-helical domain (V-domain) under all conditions tested. In contrast, the isolated N-terminal flexible loop domain of the protein always displays nuclear localization. Using immunoelectron microscopy we confirmed re-location of Mmi1 to the nucleus and showed association of Mmi1 with intact and heat shock-altered mitochondria. We also show here that mmi1Δ mutant strains are resistant to robust heat shock with respect to clonogenicity of the cells. To elucidate this phenotype we found that the cytosolic Mmi1 holoprotein re-localized to the nucleus even in cells heat-shocked at 40°C. Upon robust heat shock at 46°C, Mmi1 partly co-localized with the proteasome marker Rpn1 in the nuclear region as well as with the cytoplasmic stress granules defined by Rpg1 (eIF3a). We co-localized Mmi1 also with Bre5, Ubp3 and Cdc48 which are involved in the protein de-ubiquitination machinery, protecting protein substrates from proteasomal degradation. A comparison of proteolytic activities of wild type and mmi1Δ cells revealed that Mmi1 appears to be an inhibitor of the proteasome. We conclude that one of the physiological functions of the multifunctional protein module, Mmi1, is likely in regulating degradation and/or protection of proteins thereby indirectly regulating the pathways leading to cell death in stressed cells. 相似文献
3.
Valásek L Hasek J Nielsen KH Hinnebusch AG 《The Journal of biological chemistry》2001,276(46):43351-43360
eIF3j/Hcr1p, a protein associated with eIF3, was shown to bind to, and stabilize, the multifactor complex containing eIFs 1, 2, 3, and 5 and Met-tRNA(i)(Met), whose formation is required for an optimal rate of translation initiation. Here we present evidence that eIF3j/Hcr1p is an RNA binding protein that enhances a late step in 40 S ribosome maturation involving cleavage of the 20 S precursor of 18 S rRNA in the cytoplasm. Immunofluorescence staining shows that eIF3j/Hcr1p is localized predominantly in the cytoplasm. The hcr1Delta mutant exhibits a decreased amount of 40 S subunits, hypersensitivity to paromomycin, and increased levels of 20 S pre-rRNA. Combining the hcr1Delta mutation with drs2Delta or rps0aDelta, deletions of two other genes involved in the same step of 40 S subunit biogenesis, produced a synthetic growth defect. p35, the human ortholog of eIF3j/Hcr1p, partially complemented the slow growth phenotype conferred by hcr1Delta when overexpressed in yeast. heIF3j/p35 was found physically associated with yeast eIF3 and 43 S initiation complexes in vitro and in vivo. Because it did not complement the 40 S biogenesis defect of hcr1Delta, it appears that heIF3j can substitute for eIF3j/Hcr1p only in translation initiation. We conclude that eIF3j/Hcr1p is required for rapid processing of 20 S to 18 S rRNA besides its role in translation initiation, providing an intriguing link between ribosome biogenesis and translation. 相似文献
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5.
Regulation of gene expression on the level of translation and mRNA turnover is widely conserved evolutionarily. We have found that the main mRNA decay enzyme, exoribonuclease Xrn1, accumulates at the plasma membrane-associated eisosomes after glucose exhaustion in a culture of the yeast S. cerevisiae. Eisosomal localization of Xrn1 is not achieved in cells lacking the main component of eisosomes, Pil1, or Sur7, the protein accumulating at the membrane compartment of Can1 (MCC) - the eisosome-organized plasma membrane microdomain. In contrast to the conditions of diauxic shift, when Xrn1 accumulates in processing bodies (P-bodies), or acute heat stress, in which these cytosolic accumulations of Xrn1 associate with eIF3a/Rpg1-containing stress granules, Xrn1 is not accompanied by other mRNA-decay machinery components when it accumulates at eisosomes in post-diauxic cells. It is important that Xrn1 is released from eisosomes after addition of fermentable substrate. We suggest that this spatial segregation of Xrn1 from the rest of the mRNA-decay machinery reflects a general regulatory mechanism, in which the key enzyme is kept separate from the rest of mRNA decay factors in resting cells but ready for immediate use when fermentable nutrients emerge and appropriate metabolism reprogramming is required. In particular, the localization of Xrn1 to the eisosome, together with previously published data, accents the relevance of this plasma membrane-associated compartment as a multipotent regulatory site. 相似文献
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The PkwA protein of the thermophilic actinomycete Thermomonospora curvata has already been reported as the first instance of a WD-40 module-containing protein of prokaryotic origin. This protein is composed of an N-terminal eukaryotic-type protein kinase domain and of seven C-terminal WD-40 repeats. PkwA is a peripheral membrane protein that is linked to the early exponential growth phase of the bacterium. Its intracellular concentrations are extremely low. We have shown that the protein forms high molecular weight complexes and is localized mainly in the tips of the young Thermomonospora vegetative hyphae. 相似文献
8.
Ophioglossum petiolatum . Unlike Angiopteris (Marattiales), which is monoplastidic, Ophioglossum undergoes polyplastidic meiosis like members of the fern-seed plant clade. The meiotic spindle is distinctly multipolar in
origin and is consolidated into a bipolar spindle that is variously twisted and curved to accommodate the large number of
chromosomes. Although a phragmoplast forms after first meiosis, no wall is deposited. Instead, an organelle band consisting
of intermingled plastids and mitochondria is formed in the equatorial region between the dyad domains. Following second meiosis,
a complex of phragmoplasts forms among sister and non-sister nuclei. Cell plates are deposited first between sister nuclei
and then in the region of the organelle band resulting in a tetrad of spores each with a equal allotment of organelles.
Received 30 January 2001/ Accepted in revised form 24 April 2001 相似文献
9.
A flux analysis of glucose metabolism in the filamentous fungus Rhizopus oryzae was achieved using a specific radioactivity curve-matching program, TFLUX. Glycolytic and tricarboxylic acid cycle intermediates labeled through the addition of extracellular [U-14C]glucose were isolated and purified for specific radioactivity determinations. This information, together with pool sizes and the rates of glucose utilization and end product production, provided input for flux maps of the metabolic network under two different experimental conditions. Based upon the flux analysis of this system, a mutant of R. oryzae with higher lactate and lower ethanol yields than the parent was sought for and found. 相似文献
10.
Antigen-mediated macrophage adherence inhibition 总被引:1,自引:0,他引:1
Antigen-mediated macrophage adherence inhibition (MAI) was studied in inbred rats immunized with various transplantation, tumour-specific and protein antigens. A macrophage-rich suspension of peritoneal cells (PC) was obtained from the peritoneal cavity of immunized and control animals by washing. The adherence to glass of PC was specifically inhibited by the addition of the antigens used for sensitization of PC donors or by related (cross-reacting) antigens but not with unrelated antigens. The MAI seems to be due to the direct interaction of the respective antigen with a corresponding PC receptor and not due to the humoral factor released from immune lymphocytes of PC population upon contact with the specific antigen. 相似文献