The Correlation between Cd2+ Sensitivity and Cd2+ Uptake in the Strains of Saccharomyces cerevisiae

The sensitivity of twelve strains of Saccharomyces cerevisiaeto Cd²⁺ was examined in correlation with the uptake of Cd²⁺.Strains of S. cerevisiae were grouped into three categoriesdepending on the sensitivity of cells grown on agar-plates containingvarious concentrations of Cd²⁺. 1) The sensitive group did notgrow in 0.1 mM Cd²⁺. 2) The sub-tolerant group was capable ofgrowth at 0.3 min Cd²⁺, but not at 0.4 mM Cd²⁺. 3) The tolerantgroup was capable of growth at 0.4 mM Cd²⁺ or higher. In thesestrain groups the increase in sensitivity to Cd²⁺ was associatedwith an increase in the activity of Cd²⁺ absorption. ¹ This study is dedicated to the late president J. Ashida ofEhime University. (Received November 25, 1982; Accepted February 14, 1983)     

Energetics of copper trafficking between the Atx1 metallochaperone and the intracellular copper transporter, Ccc2

The Atx1 metallochaperone protein is a cytoplasmic Cu(I) receptor that functions in intracellular copper trafficking pathways in plants, microbes, and humans. A key physiological partner of the Saccharomyces cerevisiae Atx1 is Ccc2, a cation transporting P-type ATPase located in secretory vesicles. Here, we show that Atx1 donates its metal ion cargo to the first N-terminal Atx1-like domain of Ccc2 in a direct and reversible manner. The thermodynamic gradient for metal transfer is shallow (K(exchange) = 1.4 +/- 0.2), establishing that vectorial delivery of copper by Atx1 is not based on a higher copper affinity of the target domain. Instead, Atx1 allows rapid metal transfer to its partner. This equilibrium is unaffected by a 50-fold excess of the Cu(I) competitor, glutathione, indicating that Atx1 also protects Cu(I) from nonspecific reactions. Mechanistically, we propose that a low activation barrier for transfer between partners results from complementary electrostatic forces that ultimately orient the metal-binding loops of Atx1 and Ccc2 for formation of copper-bridged intermediates. These thermodynamic and kinetic considerations suggest that copper trafficking proteins overcome the extraordinary copper chelation capacity of the eukaryotic cytoplasm by catalyzing the rate of copper transfer between physiological partners. In this sense, metallochaperones work like enzymes, carefully tailoring energetic barriers along specific reaction pathways but not others.     

Prokaryotic diversity of the Saccharomyces cerevisiae Atx1p-mediated copper pathway

MOTIVATION: Several genes involved in the cellular import of copper and its subsequent incorporation into the high-affinity iron transport complex in Saccharomyces cerevisiae are known to be conserved between eukaryotes and prokaryotes. However, the degree to which these genes share their functional context as members of the same pathway in the prokaryotic domain is less clear. RESULTS: The co-occurrence of gene families involved in Atx1p-mediated copper transport in the genomes and operon structures of 80 non-redundant prokaryotes was investigated. For this purpose, we developed a Web tool (SHOPS) to display the operon context for a given set of proteins. In total, a set of 43 putative operons was identified. These were found to be involved in a variety of pathways and indicate a large diversity in the functional context of the individual gene family members. AVAILABILITY: The SHOPS tool can be found at http://www.bioinformatics.med.uu.nl/shops supplemental data are available at http://humgen.med.uu.nl/publications/vanbakel/pathway/     

Characterization of the binding interface between the copper chaperone Atx1 and the first cytosolic domain of Ccc2 ATPase

The interaction of the copper chaperone Atx1 and the first cytosolic domain of Ccc2 ATPase, Ccc2a, was investigated by NMR in solution. In particular, a solution of Cu(I)-15NAtx1 was titrated with apo-Ccc2a, and, vice versa, a solution of Cu(I)-15NCcc2a was titrated with apo-Atx1. By following the 15N and 1H chemical shifts, a new species is detected in both experiments. This species is the same in both titrations and is in fast exchange with the parent species on the NMR time scale. Nuclear relaxation data are consistent with the formation of an adduct. Judging from the nuclear Overhauser effect spectroscopy patterns, the structure of Cu(I)-15NCcc2a in the presence of apo-Atx1 is not significantly altered, whereas Cu(I)-15NAtx1 in the presence of apo-Ccc2a experiences some changes with respect to both the apoproteins and the Cu(I)-loaded proteins. The structure of the Cu(I)-15NAtx1 moiety in the adduct was obtained from 1137 nuclear Overhauser effects to a final root mean square deviation to the mean structure of 0.76 +/- 0.13 A for the backbone and 1.11 +/- 0.11 A for the heavy atoms. 15N and 1H chemical shifts suggest the regions of interaction that, together with independent information, allow a structural model of the adduct to be proposed. The apo form of Atx1 displays significant mobility in loops 1 and 5, the N-terminal part of helix alpha1, and the C-terminal part of helix alpha2 on the ms-micros time scale. These regions correspond to the metal binding site. Such mobility is largely reduced in the free Cu(I)-Atx1 and in the adduct with apo-Ccc2a. The analogous mobility of Ccc2a in both Cu(I) and apo forms is reduced with respect to Atx1. Such an adduct is relevant as a structural and kinetic model for copper transfer from Atx1 to Ccc2a in physiological conditions.     

Genetic interaction between the ero1-1 and leu2 mutations in Saccharomyces cerevisiae

The conditional ero1-1 mutant, deficient in the ER-localized PDI oxidase Ero1p, is blocked in disulfide bond formation under restrictive conditions, such as high temperature, lack of oxygen, or high concentrations of membrane-permeant thiols. Previous studies of the physiological consequences of the ero1-1 mutation were carried out in a leu2 mutant. The ero1-1 leu2 strain does not grow in standard synthetic complete medium at 30 degrees C, a defect that can be remedied by increasing the L-leucine concentration in the medium or by transforming the ero1-1 leu2 strain with the LEU2 wild-type allele. In addition, the LEU2 gene can partially complement the growth impairment at 37 degrees C of the ero1-1 leu2 mutant. The leucine transporter Bap2p exhibits a dramatic decrease in stability in an ero1-1 strain, which may account for the pronounced leucine demand observed in the ero1-1 leu2 mutant.     

Cd2+- or Hg2+-binding proteins can replace the Cu+-chaperone Atx1 in delivering Cu+ to the secretory pathway in yeast

Copper delivery to Ccc2--the Golgi Cu+-ATPase--was investigated in vivo, replacing the Cu+-chaperone Atx1 by various structural homologues in an atx1-Delta yeast strain. Various proteins, displaying the same ferredoxin-like fold and (M/L)(T/S)CXXC metal-binding motif as Atx1 and known as Cu+-, Cd2+- or Hg2+-binding proteins were able to replace Atx1. Therefore, regardless of their original function, these proteins could all bind copper and transfer it to Ccc2, suggesting that Ccc2 is opportunistic and can interact with many different proteins to gain Cu+. The possible role of electrostatic potential surfaces in the docking of Ccc2 with these Atx1-homologues is discussed.     

Saccharomyces cerevisiae RAP1 binds to telomeric sequences with spatial flexibility

A wide divergence has been detected in the telomeric sequences among budding yeast species. Despite their length and homogeneity differences, all these yeast telomeric sequences show a conserved core which closely matches the consensus RAP1-binding sequence. We demonstrate that the RAP1 protein binds this sequence core, without involving the diverged sequences outside the core. In Saccharomyces castellii and Saccharomyces dairensis specific classes of interspersed variant repeats are present. We show here that a RAP1-binding site is formed in these species by connecting two consecutive 8 bp telomeric repeats. DNase I footprint analyses specify the binding site as the 13 bp sequence CTGGGTGTCTGGG. The RAP1 protein also binds the variant repeats, although with a lowered affinity. However, a split footprint is produced when RAP1 binds a variant repeat where the two half-sites of the binding site are separated by an additional 6 nt. This is probably caused by the intervening sequence looping out of the RAP1-DNA complex. We suggest that the bipartite subdomain structure of the RAP1 protein allows it to remodel telomeric chromatin, a feature which may be of great relevance for telomeric chromatin assembly and structure in vivo.     

Nucleo-cytoplasmic interaction between oligomycin-resistant mutations in Saccharomyces cerevisiae

1.A single-gene nuclear mutant of Saccharomyces cerevisiae, isolated as oligomycin-resistant, exhibits in vivo cross-resistance to venturicidin and collateral sensitivity to Synthalin. All three compounds are inhibitors of mitochondrial oxidative phosphorylation. Oligomycin resistance and Synthalin sensitivity are recessive, while venturicidin resistance is dominant. 2. Acytoplasmic mutant, also isolated as oligomycin-resistant, shows collateral sensitivity to both Synthalin and venturicidin. All three traits undergo mitotic segregation in diploids formed by crossing mutant and normal halpoids. 3. A novel nucleocytoplasmic interaction is observed in diploids formed by crossing haploid strains containing the nuclear and the cytoplasmic mutations, respectively. The dominant venturicidin resistance determined by the nuclear gene undergoes mitotic segregation, which results from a suppression of the nuclear phenotype by the cytoplasmic mutation. When a diploid mitotic segregant contains primarily mutant-type mitochondria, venturicidin resistance is completely suppressed. In haploids containing both the nuclear and cytoplasmic mutations, suppression is only partial. 4. Oxidative phosphorylation and ATPase in mitochondrial fractions isolated fromcytoplasmic mutant cells are less sensitive to inhibition by oligomycin than normal, but in vitro sensitivity to venturicidin is not significantly changed. In similar mitochondrial fractions isolated from normal and nuclear mutant cells, no significant differences in sensitivity to either inhibitor are detected. 5. The molecular basis for the nucleocytoplasmic suppression of venturicidin resistance may involve participation of mitochondrial membrane, plasma membrane or both. Either mitochondria can undergo changes in venturicidin sensitivity upon isolation, or the molecular entity which controls access of venturicidin to the mitochondria resides outside of the organelles. 6. Our data establish that aspects of the response in vivo of both venturicidin and Snythalin are controlled by the mitochondrial genome. 7. The nucleocytoplasmic interaction described here is the first example in which a specific restricted mitochondrial mutation modifies the phenotypic expression of a nuclear gene.     

REG1 binds to protein phosphatase type 1 and regulates glucose repression in Saccharomyces cerevisiae.

Protein phosphatase type 1 (PP1) is encoded by GLC7, an essential gene in Saccharomyces cerevisiae. The GLC7 phosphatase is required for glucose repression and appears to function antagonistically to the SNF1 protein kinase. Previously, we characterized a mutation, glc7-T152K, that relieves glucose repression but does not interfere with the function of GLC7 in glycogen metabolism. We proposed that the mutant GLC7T152K phosphatase is defective in its interaction with a regulatory subunit that directs participation of PP1 in the glucose repression mechanism. Here, we present evidence that REG1, a protein required for glucose repression, is one such regulatory subunit. We show that REG1 is physically associated with GLC7. REG1 interacts with GLC7 strongly and specifically in the two-hybrid system, and REG1 and GLC7 fusion proteins co-immunoprecipitate from cell extracts. Moreover, overexpression of a REG1 fusion protein suppresses the glc7-T152K mutant defect in glucose repression. This and other genetic evidence indicate that the two proteins function together in regulating glucose repression. These results suggest that REG1 is a regulatory subunit of PP1 that targets its activity to proteins in the glucose repression regulatory pathway.     

Further studies on the subcellular distribution of Cd2+ in Cd-sensitive and Cd-resistant strains of Saccharomyces cerevisiae

When a Cd-resistant strain (301 N) and a Cd-sensitive strain (101 N) of Saccharomyces cerevisiae were incubated in medium containing Cd2+, a large proportion of the cellular Cd2+ was found in the cytosol of strain 301 N, but not in that of strain 101 N. Approximately 65% of the cellular Cd2+ was released from strain 301 N after treatment with chitosan, which affects cell membrane permeability. About 80% of the cellular Cd2+ released from strain 301 N by chitosan treatment was detected in a 30 000-10 000 molecular weight fraction prepared by ultrafiltration. The distribution of Cd2+ into the cytosol in strain 301 N was inhibited in the presence of cycloheximide. The proportion of cellular Cu2+ or Zn2+ present in the cytosol after incubation with these ions was similar for the two strains (about 40%).     

In vitro interaction between Saccharomyces cerevisiae CDC25 and RAS2 proteins.

In Saccharomyces cerevisiae the CDC25 protein is a positive regulator of RAS/cAMP pathway [1-4], enhancing the GDP-releasing rate of RAS2 protein [5]. In this work we have tried to detect a direct interaction between CDC25 and RAS2 gene products. The results indicate that both the whole RAS2 protein and a truncated version that lacks approximately 25 C-terminal residues interact specifically with the CDC25 protein. On the contrary, a derivative of RAS2 that lacks the 112 C-terminal residues as well as the p21TI-ras is not able to bind the CDC25 protein in our assay conditions. The 310 C-terminal aminoacids of CDC25 bind RAS2 while a C-terminus deletion within this aminoacid stretch abolishes the binding. The possible physiological significance of these findings is discussed.     

Genetic interaction between the Ras-CAMP pathway and the Dis2s1/Glc7 protein phosphatase in Saccharomyces cerevisiae

The Saccharomyces cerevisiae DIS2S1/GLC7 gene encodes a type 1 protein phosphatase indispensable for cell proliferation. We found that introduction of a multicopy DIS2S1 plasmid impaired growth of cells with reduced activity of the cAMP-dependent protein kinase. In order to understand further the interaction between the two enzymes, a temperature-sensitive mutation in the DIS2S1 gene was isolated. The mutant accumulated less glycogen than wild type at the permissive temperature, indicating that activity of the Dis2s1 protein phosphatase is attenuated by the mutation. Furthermore, the dis2s1 ^ts mutation was shown to be suppressed by a multicopy plasmid harboring PDE2, a gene for cAMP phosphodiesterase. These results indicate that the Ras-cAMP pathway interacts genetically with the DIS2S1/GLC7 gene.     

Phospholipase C binds to the receptor-like GPR1 protein and controls pseudohyphal differentiation in Saccharomyces cerevisiae.

The hormone receptor-like protein Gpr1p physically interacts with phosphatidylinositol-specific phospholipase C (Plc1p) and with the Galpha protein Gpa2p, as shown by two-hybrid assays and co-immune precipitation of epitope-tagged proteins. Plc1p binds to Gpr1p in either the presence or absence of Gpa2, whereas the Gpr1p/Gpa2p association depends on the presence of Plc1p. Genetic interactions between the null mutations plc1Delta, gpr1Delta, gpa2Delta, and ras2Delta suggest that Plc1p acts together with Gpr1p and Gpa2p in a growth control pathway operating in parallel to the Ras2p function. Diploid cells lacking Gpr1p, Plc1p, or Gpa2p fail to form pseudohyphae upon nitrogen depletion, and the filamentation defect of gpr1Delta and plc1Delta strains is rescued by activating a mitogen-activated protein kinase pathway via STE11-4 or by activating a cAMP pathway via overexpressed Tpk2p. Plc1p is also required for efficient expression of the FG(TyA)::lacZ reporter gene under nitrogen depletion. In conclusion, we have identified two physically interacting proteins, Gpr1p and Plc1p, as novel components of a nitrogen signaling pathway controlling the developmental switch from yeast-like to pseudohyphal growth. Our data suggest that phospholipase C modulates the interaction of the putative nutrient sensor Gpr1p with the Galpha protein Gpa2p as a downstream effector of filamentation control.     

Changes in 45Ca and 109Cd uptake, membrane potential and cell pH in Saccharomyces cerevisiae provoked by Cd2+

The effect of Cd2+ poisoning of Saccharomyces cerevisiae on 45Ca, 109Cd and [14C]tetraphenylphosphonium (TPP) uptake and cell pH was examined. At Cd2+ concentrations that produced substantial K+ efflux the rates of uptake of 45Ca, 109Cd and [14C]TPP increased progressively during incubation of the cells with Cd2+, and the cell pH was lowered concomitantly. The initial rates of uptake of the divalent cations and of TPP were increased in cells pre-loaded with Cd2+, which shows that stimulation of the ion fluxes was exerted by the Cd2+ that accumulated in the cells. The distribution ratio of TPP between cells and medium, however, was decreased by Cd2+. Although hyperpolarization of the cell membrane by Cd2+ cannot be excluded, it is argued that Cd2+ primarily stimulated divalent cation uptake by increasing the cation permeability of the cell membrane allowing the cations to enter the cells more easily.     

利用转录调控因子Bas1p和Bas2p协同作用提高酿酒酵母cAMP产量的研究

【目的】研究转录调控因子Bas1p和Bas2p协同作用对重组酿酒酵母(Saccharomyces cerevisiae)胞外c AMP产生的影响,初步优化发酵培养基。【方法】通过共整合表达策略,在c AMP产生菌酿酒酵母G5中超表达Bas1p和Bas2p,摇瓶发酵实验考察了Bas1p和Bas2p协同作用对菌株生长及胞外c AMP产生的影响,进一步考察了酵母粉和蛋白胨含量及前体物腺嘌呤对菌株生长和c AMP产生的影响。【结果】超表达Bas1p和Bas2p使菌株在1×YP培养基中发酵120 h时的c AMP产量较出发菌株提高51.4%,达到2 253.8μmol/L;将1×YP中的酵母粉和蛋白胨含量翻倍(即2×YP培养基)发酵120 h时的c AMP产量提高至4 450.4μmol/L;在2×YP培养基中添加0.5 g/L浓度的腺嘌呤时,c AMP产量进一步提高至5 314.3μmol/L。【结论】强化Bas1p和Bas2p的协同作用及相应地优化培养基组分有助于酿酒酵母胞外c AMP生产。     

In vivo bipartite interaction between the Hsp40 Sis1 and Hsp70 in Saccharomyces cerevisiae

The essential Hsp40, Sis1, is a J-protein cochaperone for the Ssa class of Hsp70's of Saccharomyces cerevisiae. Sis1 is required for the maintenance of the prion [RNQ(+)], as Sis1 lacking its 55-amino-acid glycine-rich region (G/F) does not maintain [RNQ(+)]. We report that overexpression of Sis1DeltaG/F in an otherwise wild-type strain had a negative effect on both cell growth and [RNQ(+)] maintenance, while overexpression of wild-type Sis1 did not. Overexpression of the related Hsp40 Ydj1 lacking its G/F region did not cause inhibition of growth, indicating that this dominant effect of Sis1DeltaG/F is not a characteristic shared by all Hsp40's. Analysis of small deletions within the SIS1 G/F region indicated that the observed dominant effects were caused by the absence of sequences known to be important for Sis1's unique cellular functions. These inhibitory effects of Sis1DeltaG/F were obviated by alterations in the N-terminal J-domain of Sis1 that affect interaction with Ssa's ATPase domain. In addition, a genetic screen designed to isolate additional mutations that relieved these inhibitory effects identified two residues in Sis1's carboxy-terminal domain. These alterations disrupted the interaction of Sis1 with the 10-kD carboxy-terminal regulatory domain of Ssa1, indicating that Sis1 has a bipartite interaction with Ssa in vivo.     

Mapping the interaction of Snf1 with TORC1 in Saccharomyces cerevisiae

Nutrient sensing and coordination of metabolic pathways are crucial functions for all living cells, but details of the coordination under different environmental conditions remain elusive. We therefore undertook a systems biology approach to investigate the interactions between the Snf1 and the target of rapamycin complex 1 (TORC1) in Saccharomyces cerevisiae. We show that Snf1 regulates a much broader range of biological processes compared with TORC1 under both glucose‐ and ammonium‐limited conditions. We also find that Snf1 has a role in upregulating the NADP⁺‐dependent glutamate dehydrogenase (encoded by GDH3) under derepressing condition, and therefore may also have a role in ammonium assimilation and amino‐acid biosynthesis, which can be considered as a convergence of Snf1 and TORC1 pathways. In addition to the accepted role of Snf1 in regulating fatty acid (FA) metabolism, we show that TORC1 also regulates FA metabolism, likely through modulating the peroxisome and β‐oxidation. Finally, we conclude that direct interactions between Snf1 and TORC1 pathways are unlikely under nutrient‐limited conditions and propose that TORC1 is repressed in a manner that is independent of Snf1.