Utp9p Facilitates Msn5p-mediated Nuclear Reexport of Retrograded tRNAs in Saccharomyces cerevisiae

Utp9p is a nucleolar protein that is part of a subcomplex containing several U3 snoRNA-associated proteins including Utp8p, which is a protein that shuttles aminoacyl-tRNAs from the nucleolus to the nuclear tRNA export receptors Los1p and Msn5p in Saccharomyces cerevisiae. Here we show that Utp9p is also an intranuclear component of the Msn5p-mediated nuclear tRNA export pathway. Depletion of Utp9p caused nuclear accumulation of mature tRNAs derived from intron-containing precursors, but not tRNAs made from intronless pre-tRNAs. Utp9p binds tRNA directly and saturably, and copurifies with Utp8p, Gsp1p, and Msn5p, but not with Los1p or aminoacyl-tRNA synthetases. Utp9p interacts directly with Utp8p, Gsp1p, and Msn5p in vitro. Furthermore, Gsp1p forms a complex with Msn5p and Utp9p in a tRNA-dependent manner. However, Utp9p does not shuttle between the nucleus and the cytoplasm. Because tRNA splicing occurs in the cytoplasm and the spliced tRNAs are retrograded back to the nucleus, we propose that Utp9p facilitates nuclear reexport of retrograded tRNAs. Moreover, the data suggest that Utp9p together with Utp8p translocate aminoacyl-tRNAs from the nucleolus to Msn5p and assist with formation of the Msn5p-tRNA-Gsp1p-GTP export complex.     

过氧化氢诱导酿酒酵母细胞膜透性和组成的变化

以下简述了过氧化氢(H2O2)作为一种信号分子诱导并调节酿酒酵母(Saccharomyces cerevisiae)细胞膜的变化。H2O2是一种强氧化剂,可以跨膜扩散进入细胞中,形成跨膜梯度;当外源H2O2达到亚致死剂量时,酿酒酵母的细胞膜透性和流动性降低,产生跨膜梯度,从而限制H2O2向细胞内的扩散速率,保护细胞免受氧化胁迫的伤害。研究表明,由H2O2引起的膜透性和流动性的变化与膜的组成有关:当酵母细胞对H2O2产生适应时,与膜组成和微区域变化有关的几个基因的表达发生了改变。膜组成的变化和微区域的调整还可能与H2O2依赖的信号途径有关,即以H2O2为信号分子,调节膜的变化并赋予细胞对氧化压力更高的适应性,但这种信号分子的具体传递途径及机制还需要进一步研究。     

Decrease of H2O2 plasma membrane permeability during adaptation to H2O2 in Saccharomyces cerevisiae

Contrary to what is widely believed, recent published results show that H2O2 does not freely diffuse across biomembranes. The fast removal of H2O2 by antioxidant enzymes is able to generate a gradient if H2O2 is produced in a different compartment from that containing the enzymes (Antunes, F., and Cadenas, E. (2000) FEBS Lett. 475, 121-126). In this work, we extended these studies and tested whether an active regulation of biomembranes permeability characteristics is part of the cell response to oxidative stress. Using Saccharomyces cerevisiae as a model, we showed that: (a) H2O2 gradients across the plasma membrane are formed upon exposure to external H2O2; (b) there is a correlation between the magnitude of the gradients and the resistance to H2O2; (c) there is not a correlation between the intracellular capacity to remove H2O2 and the resistance to H2O2; (d) the plasma membrane permeability to H2O2 decreases by a factor of two upon acquisition of resistance to this agent by pre-exposing cells either to nonlethal doses of H2O2 or to cycloheximide, an inhibitor of protein synthesis; and (e) erg3Delta and erg6Delta mutants, which have impaired ergosterol biosynthesis pathways, show higher plasma membrane permeability to H2O2 and are more sensitive to H2O2. Altogether, the regulation of the plasma membrane permeability to H2O2 emerged as a new mechanism by which cells respond and adapt to H2O2. The consequences of the results to cellular redox compartmentalization and to the origin and evolution of the eukaryotic cell are discussed.     

Bub1p Kinase Activates the Saccharomyces cerevisiae Spindle Assembly Checkpoint

Saccharomyces cerevisiae BUB1 encodes a protein kinase required for spindle assembly checkpoint function. In the presence of spindle damage, BUB1 is required to prevent cell cycle progression into anaphase. We have identified a dominantly acting BUB1 allele that appears to activate the spindle assembly checkpoint pathway in cells with undamaged spindles. High-level expression of BUB1-5 did not cause detectable spindle damage, yet it delayed yeast cells in mitosis at a stage following bipolar spindle assembly but prior to anaphase spindle elongation. Delayed cells possessed a G₂ DNA content and elevated Clb2p mitotic cyclin levels. Unlike cells delayed in mitosis by spindle damage or MPS1 kinase overexpression, hyperphosphorylated forms of the Mad1p checkpoint protein did not accumulate. Similar to cells overexpressing MPS1, the BUB1-5 delay was dependent upon the functions of the other checkpoint genes, including BUB2 and BUB3 and MAD1, MAD2, and MAD3. We found that the mitotic delay caused by BUB1-5 or MPS1 overexpression was interdependent upon the function of the other. This suggests that the Bub1p and Mps1p kinases act together at an early step in generating the spindle damage signal.     

Std1p (Msn3p) positively regulates the Snf1 kinase in Saccharomyces cerevisiae

The Snf1 protein kinase of the glucose signaling pathway in Saccharomyces cerevisiae is regulated by an autoinhibitory interaction between the regulatory and catalytic domains of Snf1p. Transitions between the autoinhibited and active states are controlled by an upstream kinase and the Reg1p-Glc7p protein phosphatase 1. Previous studies suggested that Snf1 kinase activity is also modulated by Std1p (Msn3p), which interacts physically with Snf1p and also interacts with glucose sensors. Here we address the relationship between Std1p and the Snf1 kinase. Two-hybrid assays showed that Std1p interacts with the catalytic domain of Snf1p, and analysis of mutant kinases suggested that this interaction is incompatible with the autoinhibitory interaction of the regulatory and catalytic domains. Overexpression of Std1p increased the two-hybrid interaction of Snf1p with its activating subunit Snf4p, which is diagnostic of an open, uninhibited conformation of the kinase complex. Overexpression of Std1p elevated Snf1 kinase activity in both in vitro and in vivo assays. These findings suggest that Std1p stimulates the Snf1 kinase by an interaction with the catalytic domain that antagonizes autoinhibition and promotes an active conformation of the kinase.     

Overexpression of Bop3 confers resistance to methylmercury in Saccharomyces cerevisiae through interaction with other proteins such as Fkh1, Rts1, and Msn2

We found that overexpression of Bop3, a protein of unknown function, confers resistance to methylmercury in Saccharomyces cerevisiae. Bmh2, Fkh1, and Rts1 are proteins that have been previously shown to bind Bop3 by the two-hybrid method. Overexpression of Bmh2 and the homologous protein Bmh1 confers resistance to methylmercury in yeast, but overexpression of either Fkh1 or Rts1 has a minimal effect. However, the increased level of resistance to methylmercury produced by overexpression of Bop3 was smaller in Fhk1-deleted yeast as compared with that of the wild-type strain. In contrast, the degree of resistance was significantly elevated in Rts1-deleted yeast. Msn2 and Msn4 were previously reported as proteins that bind to Bmh1 and Bmh2. Overexpression of Msn2 conferred a much greater sensitivity to methylmercury in yeast, while deletion of the corresponding gene lowered the degree of resistance to methylmercury induced by overexpression of Bop3. These results suggest that multiple proteins are involved in minimizing the toxicity of methylmercury induced by overexpression of Bop3.     

Protein expression profiles in Saccharomyces cerevisiae during apoptosis induced by H2O2

We identified the proteins involved during apoptosis induced by H2O2 in Saccharomyces cerevisiae, and analyzed the global protein pattern by 2-DE. We analyzed classical parameters of apoptosis such as chromatin condensation, DNA fragmentation, and morphology changes of cells. Exposure of yeast cells to nonphysiological doses of peroxides decreases the expression (or increases degradation) of enzymes involved in protection against oxidative stress. This leads the yeast cells to a reduction of their antioxidant defense and makes the cells more prone to apoptosis. In our data the down expression of peroxiredoxin II and GST I, could induce a perturbation of mitochondrial function with an alteration of permeability of the membrane leading to the mitochondria-mediated apoptosis. Moreover, we identified a new spot of a classical glycolytic enzyme: the glyceraldehyde 3-phosphate dehydrogenase during apoptosis. It is known that GAPDH is an extremely abundant glycolytic enzyme with multiple functions and that its overexpression is evident during apoptosis induced by a variety of stimuli. Our results confirm that it is a major intracellular messenger mediating apoptotic death and that this new spot of GAPDH could be an intracellular sensor of oxidative stress during apoptosis induced by H2O2 in S. cerevisiae.     

H2O2 induces rapid biophysical and permeability changes in the plasma membrane of Saccharomyces cerevisiae

In Saccharomyces cerevisiae, the diffusion rate of hydrogen peroxide (H2O2) through the plasma membrane decreases during adaptation to H2O2 by means of a mechanism that is still unknown. Here, evidence is presented that during adaptation to H2O2 the anisotropy of the plasma membrane increases. Adaptation to H2O2 was studied at several times (15min up to 90min) by applying the steady-state H2O2 delivery model. For wild-type cells, the steady-state fluorescence anisotropy increased after 30min, or 60min, when using 2-(9-anthroyloxy) stearic acid (2-AS), or diphenylhexatriene (DPH) membrane probe, respectively. Moreover, a 40% decrease in plasma membrane permeability to H2O2 was observed at 15min with a concomitant two-fold increase in catalase activity. Disruption of the ergosterol pathway, by knocking out either ERG3 or ERG6, prevents the changes in anisotropy during H2O2 adaptation. H2O2 diffusion through the plasma membrane in S. cerevisiae cells is not mediated by aquaporins since the H2O2 permeability constant is not altered in the presence of the aquaporin inhibitor mercuric chloride. Altogether, these results indicate that the regulation of the plasma membrane permeability towards H2O2 is mediated by modulation of the biophysical properties of the plasma membrane.     

Modulation of plasma membrane lipid profile and microdomains by H2O2 in Saccharomyces cerevisiae

In Saccharomyces cerevisiae, the rate of hydrogen peroxide (H₂O₂) diffusion through the plasma membrane decreases during adaptation to H₂O₂ by a still unknown mechanism. Here, adaptation to H₂O₂ was observed to modulate rapidly the expression of genes coding for enzymes involved in ergosterol and lipid metabolism. Adaptation to H₂O₂ also alters plasma membrane lipid composition. The main changes were the following: (a) there was a decrease in oleic acid (30%) and in the ratio between unsaturated and saturated long-chain fatty acids; (b) the phosphatidylcholine:phosphatidylethanolamine ratio increased threefold; (c) sterol levels were unaltered but there was an increased heterogeneity of sterol-rich microdomains and increased ordered domains; (d) the levels of the sterol precursor squalene increased twofold, in agreement with ERG1 gene down-regulation; and (e) C26:0 became the major very long chain fatty acid owing to an 80% decrease in 2-hydroxy-C26:0 levels and a 50% decrease in C20:0 levels, probably related to the down-regulation of fatty acid elongation (FAS1, FEN1, SUR4) and ceramide synthase (LIP1, LAC1) genes. Therefore, H₂O₂ leads to a reorganization of the plasma membrane microdomains, which may explain the lower permeability to H₂O₂, and emerges as an important regulator of lipid metabolism and plasma membrane lipid composition.     

Nuclear mutants of Saccharomyces cerevisiae affected in bc1 complex

Three nuclear mutants, affected to various degrees with respect to cytochrome b are described. Detailed genetical study revealed that each mutant strain carried a single gene mutation; in complementation test the three mutations proved to be non-allelic. The measurements of enzymatic activities strongly suggest that the bc1 complex is the target of the mutations.     

Thioredoxins are required for protection against a reductive stress in the yeast Saccharomyces cerevisiae

Thioredoxins are small, highly conserved oxidoreductases that are required to maintain the redox homeostasis of the cell. They have been best characterized for their role as antioxidants in protection against reactive oxygen species. We show here that thioredoxins (TRX1, TRX2) and thioredoxin reductase (TRR1) are also required for protection against a reductive stress induced by exposure to dithiothreitol (DTT). This sensitivity to reducing conditions is not a general property of mutants affected in redox control, as mutants lacking components of the glutathione/glutaredoxin system are unaffected. Furthermore, TRX2 gene expression is induced in response to DTT treatment, indicating that thioredoxins form part of the cellular response to a reductive challenge. Our data indicate that the sensitivity of thioredoxin mutants to reducing stress appears to be a consequence of elevated glutathione levels, which is present predominantly in the reduced form (GSH). The elevated GSH levels also result in a constitutively high unfolded protein response (UPR), indicative of an accumulation of unfolded proteins in the endoplasmic reticulum (ER). However, there does not appear to be a general defect in ER function in thioredoxin mutants, as oxidative protein folding of the model protein carboxypeptidase Y occurs with similar kinetics to the wild-type strain, and trx1 trx2 mutants are unaffected in sensitivity to the glycosylation inhibitor tunicamycin. Furthermore, trr1 mutants are resistant to tunicamycin, consistent with their high UPR. The high UPR seen in trr1 mutants can be abrogated by the GSH-specific reagent 1-chloro-2,4-dinitrobenzene. In summary, thioredoxins are required to maintain redox homeostasis in response to both oxidative and reductive stress conditions.