Importance of mitochondria in survival of Cryptococcus neoformans under low oxygen conditions and tolerance to cobalt chloride

Cryptococcus neoformans is an environmental fungal pathogen that requires atmospheric levels of oxygen for optimal growth. For the fungus to be able to establish an infection, it must adapt to the low oxygen concentrations in the host environment compared to its natural habitat. In order to investigate the oxygen sensing mechanism in C. neoformans, we screened T-DNA insertional mutants for hypoxia-mimetic cobalt chloride (CoCl(2))-sensitive mutants. All the CoCl(2)-sensitive mutants had a growth defect under low oxygen conditions at 37 degrees C. The majority of mutants are compromised in their mitochondrial function, which is reflected by their reduced rate of respiration. Some of the mutants are also defective in mitochondrial membrane permeability, suggesting the importance of an intact respiratory system for survival under both high concentrations of CoCl(2) as well as low oxygen conditions. In addition, the mutants tend to accumulate intracellular reactive oxygen species (ROS), and all mutants show sensitivity to various ROS generating chemicals. Gene expression analysis revealed the involvement of several pathways in response to cobalt chloride. Our findings indicate cobalt chloride sensitivity and/or sensitivity to low oxygen conditions are linked to mitochondrial function, sterol and iron homeostasis, ubiquitination, and the ability of cells to respond to ROS. These findings imply that multiple pathways are involved in oxygen sensing in C. neoformans.     

酿酒酵母组氨酸转运蛋白Hip1p的泛素化水平对组氨酸利用的影响

【目的】为了研究泛素化对组氨酸转运及调控的影响。【方法】应用泛素化位点预测及定点突变等技术手段,Hip1p的3个潜在的泛素化位点K30、K42和K52被突变。这些突变的Hip1p被克隆到泛素化检测质粒中,检测泛素化位点突变对Hip1p的泛素化水平的影响。同时这些突变对细胞生长及组氨酸利用的影响也进一步做了检测。【结果】Hip1p的3个赖氨酸位点K30、K42和K52突变能有效降低其泛素化水平。同时,双重突变对其泛素化水平有明显的协同作用,并进一步影响了细胞生长和组氨酸利用。【结论】泛素化水平调控能有效调节组氨酸代谢,引起细胞对组氨酸利用的改变,为进一步研究氨基酸转运蛋白的调控机制提供了重要的依据。     

A transporter in the endoplasmic reticulum of Schizosaccharomyces pombe cells mediates zinc storage and differentially affects transition metal tolerance

The cation diffusion facilitator (CDF) family represents a class of ubiquitous metal transporters. Inactivation of a CDF in Schizosaccharomyces pombe, Zhf, causes drastically different effects on the tolerance toward various metals. A deletion mutant is Zn(2+)/Co(2+)-hypersensitive yet displays significantly enhanced Cd(2+) and Ni(2+) tolerance. Accumulation of zinc, cobalt, and cadmium is reduced in mutant cells. Non-vacuolar zinc content, as measured by analytical electron microscopy, is lower in zhf(-) cells compared with wild-type cells in the presence of elevated Zn(2+) concentrations. The protective effect against cadmium toxicity is independent of the phytochelatin detoxification pathway. Phytochelatin synthase-deficient cells show extremely enhanced (about 200-fold) cadmium tolerance when zhf is disrupted. Immunogold labeling indicates endoplasmic reticulum (ER) localization of Zhf. Electron spectroscopic imaging shows that accumulation of zinc coincides with Zhf localization, demonstrating a major role of the ER for metal storage and the involvement of Zhf in cellular zinc homeostasis. Also, these observations indicate that Cd(2+) ions exert their toxic effects on cellular metabolism in the ER rather than in the cytosol.     

Interactions between gene products involved in divalent cation transport in Saccharomyces cerevisiae

The COT1 and ZRC1 genes of Saccharomyces cerevisiae are structurally related dosage-dependent suppressors of metal toxicity. COT1 confers increased tolerance to high levels of cobalt; ZRC1 confers increased tolerance to high levels of zinc. The two genes are not linked and have been mapped; COT1 to chromosome XV and ZRC1 to chromosome XIII. Phenotypes related to metal homeostasis have been examined in strains with varied COT1 and ZRC1 gene doses. Overexpression of COT1 confers tolerance to moderately toxic levels of zinc and ZRC1 confers tolerance to moderately toxic levels of cobalt. Strains that carry null alleles at both loci are viable. The metal-hypersensitive phenotypes of mutations in either gene are largely unaffected by changes in dosage of the other. COT1 and ZRCI function independently in conferring tolerance to their respective metals, yet the uptake of cobalt ions by yeast cells is dependent on the gene dosage of ZRC1 as well as of COT1 Strains that overexpress ZRC1 have increased uptake of cobalt ions, while ZRCI null mutants exhibit decreased cobalt uptake. The defects in cobalt uptake due to mutations at COT1 and ZRC1 are additive, suggesting that the two genes are responsible for the majority of cobalt and zinc uptake in yeast cells. The function of either gene product seems to be more important in metal homeostasis than is the GRR1 gene product, which is also involved in metal metabolism. Mutations in the GRR1 gene have no effect on the cobalt-related phenotypes of strains that have altered gene dosage of either COT1 or ZRC1.     

Interactions between gene products involved in divalent cation transport in Saccharomyces cerevisiae

The COT1 and ZRC1 genes of Saccharomyces cerevisiae are structurally related dosage-dependent suppressors of metal toxicity. COT1 confers increased tolerance to high levels of cobalt; ZRC1 confers increased tolerance to high levels of zinc. The two genes are not linked and have been mapped; COT1 to chromosome XV and ZRC1 to chromosome XIII. Phenotypes related to metal homeostasis have been examined in strains with varied COT1 and ZRC1 gene doses. Overexpression of COT1 confers tolerance to moderately toxic levels of zinc and ZRC1 confers tolerance to moderately toxic levels of cobalt. Strains that carry null alleles at both loci are viable. The metal-hypersensitive phenotypes of mutations in either gene are largely unaffected by changes in dosage of the other. COT1 and ZRCI function independently in conferring tolerance to their respective metals, yet the uptake of cobalt ions by yeast cells is dependent on the gene dosage of ZRC1 as well as of COT1 Strains that overexpress ZRC1 have increased uptake of cobalt ions, while ZRCI null mutants exhibit decreased cobalt uptake. The defects in cobalt uptake due to mutations at COT1 and ZRC1 are additive, suggesting that the two genes are responsible for the majority of cobalt and zinc uptake in yeast cells. The function of either gene product seems to be more important in metal homeostasis than is the GRR1 gene product, which is also involved in metal metabolism. Mutations in the GRR1 gene have no effect on the cobalt-related phenotypes of strains that have altered gene dosage of either COT1 or ZRC1.     

Soil bacteria confer plant salt tolerance by tissue-specific regulation of the sodium transporter HKT1

Elevated sodium (Na(+)) decreases plant growth and, thereby, agricultural productivity. The ion transporter high-affinity K(+) transporter (HKT)1 controls Na(+) import in roots, yet dysfunction or overexpression of HKT1 fails to increase salt tolerance, raising questions as to HKT1's role in regulating Na(+) homeostasis. Here, we report that tissue-specific regulation of HKT1 by the soil bacterium Bacillus subtilis GB03 confers salt tolerance in Arabidopsis thaliana. Under salt stress (100 mM NaCl), GB03 concurrently down- and upregulates HKT1 expression in roots and shoots, respectively, resulting in lower Na(+) accumulation throughout the plant compared with controls. Consistent with HKT1 participation in GB03-induced salt tolerance, GB03 fails to rescue salt-stressed athkt1 mutants from stunted foliar growth and elevated total Na(+) whereas salt-stressed Na(+) export mutants sos3 show GB03-induced salt tolerance with enhanced shoot and root growth as well as reduced total Na(+). These results demonstrate that tissue-specific regulation of HKT1 is critical for managing Na(+) homeostasis in salt-stressed plants, as well as underscore the breadth and sophistication of plant-microbe interactions.     

Genome-wide screening for genes associated with FK506 sensitivity in fission yeast

We have been studying calcineurin signal transduction pathway in fission yeast Schizosaccharomyces pombe (S. pombe) by developing a genetic screen for mutants that show hypersensitivity to the immunosuppressive calcineurin inhibitor FK506 (tacrolimus). In the present study, to identify nonessential genes that are functionally related to the calcineurin signaling pathway, we performed a genome-wide screen of 3004 haploid deletion strains and confirmed 72 deletion strains to be FK506 sensitive. These 72 genes are classified into nine functional groups to include membrane trafficking (16 genes), signal transduction (10 genes), ubiquitination (8 genes), chromatin remodeling (6 genes), cytokinesis (4 genes), ribosomal protein (3 genes), RNA binding protein (3 genes), and a variety of other known functions (17 genes) or still unknown functions (5 genes) in the biological system. In our previous screening of FK506-sensitive mutants we isolated several membrane-trafficking mutants showing defective cell wall integrity. Here, we further examined the vacuolar fusion, the v-SNARE synaptobrevin Syb1 localization, and the sensitivity to the β-glucan synthase inhibitor micafungin in these 72 FK506-sensitive strains. Results showed that 25 deletion strains exhibited abnormal vacuole fusion, 19 deletion strains exhibited Syb1 mislocalization, and 14 deletion strains exhibited both abnormal vacuole fusion and Syb1 mislocalization, while 42 deletion strains showed both normal vacuole fusion and Syb1 localization. Likewise, 16 deletion strains showed sensitivity to micafungin. Altogether, our present study indicates that calcineurin mediates a plethora of physiological processes in fission yeast, and that calcineurin is extensively involved in cross-talk between signaling pathways.     

HOL1 mutations confer novel ion transport in Saccharomyces cerevisiae.

Saccharomyces cerevisiae histidine auxotrophs are unable to use L-histidinol as a source of histidine even when they have a functional histidinol dehydrogenase. Mutations in the hol1 gene permit growth of His- cells on histidinol by enhancing the ability of cells to take up histidinol from the medium. Second-site mutations linked to HOL1-1 further increase histidinol uptake. HOL1 double mutants and, to a lesser extent, HOL1-1 single mutants show hypersensitivity to specific cations added to the growth medium, including Na+, Li+, Cs+, Be2+, guanidinium ion, and histidinol, but not K+, Rb+, Ca2+, or Mg2+. The Na(+)-hypersensitive phenotype is correlated with increased uptake and accumulation of this ion. The HOL1-1-101 gene was cloned and used to generate a viable haploid strain containing a hol1 deletion mutation (hol1 delta). The uptake of cations, the dominance of the mutant alleles, and the relative inability of hol1 delta cells to take up histidinol or Na+ suggest that hol1 encodes an ion transporter. The novel pattern of ion transport conferred by HOL1-1 and HOL1-1-101 mutants may be explained by reduced selectivity for the permeant ions.     

Involvement of histidine-rich domain of ZIP family transporter TjZNT1 in metal ion specificity.

The Zrt/Irt-like protein (ZIP) family generally contributes to metal homeostasis by regulating cation transport into the cytoplasm. Most ZIP members have a long variable loop between transmembrane domains III and IV, and these loops are predicted to be located in the cytoplasm. The loops contain a histidine-rich domain (HRD) postulated to serve as a metal ion binding site; however, its role has not yet been determined. We previously determined that deletion of the HRD did not affect the Ni tolerance ability of TjZNT1-a ZIP transporter that confers high Ni tolerance to yeast. In this study, we investigated the effect of HRD deletion on the ion transport ability of TjZNT1. The deletion of HRD increased the specificity for Zn2+, but not for Cd2+. In addition, we confirmed subcellular localizations of TjZNT1 and HRD-deleted mutants by green fluorescence protein (GFP)-fused proteins, indicating that the deletion of HRD did not affect the localization of TjZNT1. From these results, we propose that the HRD could be involved in the ion specificity of TjZNT1.     

The salt tolerant yeast Zygosaccharomyces rouxii possesses two plasma-membrane Na+/H+-antiporters (ZrNha1p and ZrSod2-22p) playing different roles in cation homeostasis and cell physiology

Antiporters exporting Na(+) and K(+) in exchange for protons are conserved among yeast species. The only exception so far has been Zygosaccharomyces rouxii, an osmotolerant species closely related to Saccharomyces cerevisiae. Z. rouxii was described as possessing one plasma-membrane antiporter transporting only Na(+) (ZrSod2-22p in the CBS 732(T) type strain). We report the characterization of a second gene, ZrNHA1, encoding a new K(+)(Na(+))/H(+)-antiporter capable of both K(+) and Na(+) export. Synteny analyses suggested that ZrSOD2-22 originated by single duplication of the ZrNHA1 gene. Substrate specificities and transport properties of ZrNha1p and ZrSod2-22p were compared upon heterologous expression in S. cerevisiae, and then directly in Z. rouxii. Deletion mutants and phenotype analyses revealed that ZrSod2-22 antiporter is important for Na(+) detoxification, probably together with ZrEna1 ATPase; ZrNha1p is indispensable to maintain potassium homeostasis and ZrEna1p is not, in contrast to the situation in S. cerevisiae, involved in this function.     

Cloning,Expression, and Functional Characterization of Secondary Amino Acid Transporters of Lactococcus lactis

Fourteen genes encoding putative secondary amino acid transporters were identified in the genomes of Lactococcus lactis subsp. cremoris strains MG1363 and SK11 and L. lactis subsp. lactis strains IL1403 and KF147, 12 of which were common to all four strains. Amino acid uptake in L. lactis cells overexpressing the genes revealed transporters specific for histidine, lysine, arginine, agmatine, putrescine, aromatic amino acids, acidic amino acids, serine, and branched-chain amino acids. Substrate specificities were demonstrated by inhibition profiles determined in the presence of excesses of the other amino acids. Four knockout mutants, lacking the lysine transporter LysP, the histidine transporter HisP (formerly LysQ), the acidic amino acid transporter AcaP (YlcA), or the aromatic amino acid transporter FywP (YsjA), were constructed. The LysP, HisP, and FywP deletion mutants showed drastically decreased rates of uptake of the corresponding substrates at low concentrations. The same was observed for the AcaP mutant with aspartate but not with glutamate. In rich M17 medium, the deletion of none of the transporters affected growth. In contrast, the deletion of the HisP, AcaP, and FywP transporters did affect growth in a defined medium with free amino acids as the sole amino acid source. HisP was essential at low histidine concentrations, and AcaP was essential in the absence of glutamine. FywP appeared to play a role in retaining intracellularly synthesized aromatic amino acids when these were not added to the medium. Finally, HisP, AcaP, and FywP did not play a role in the excretion of accumulated histidine, glutamate, or phenylalanine, respectively, indicating the involvement of other transporters.     

Inhibition of proteasome activity is involved in cobalt-induced apoptosis of human alveolar macrophages

Inhalation of particulate cobalt has been known to induce interstitial lung disease. There is growing evidence that apoptosis plays a crucial role in physiological and pathological settings and that the ubiquitin-proteasome system is involved in the regulation of apoptosis. Cadmium, the same transitional heavy metal as cobalt, has been reported to accumulate ubiquitinated proteins in neuronal cells. On the basis of these findings, we hypothesized that cobalt would induce apoptosis in the lung by disturbance of the ubiquitin-proteasome pathway. To evaluate this, we exposed U-937 cells and human alveolar macrophages (AMs) to cobalt chloride (CoCl(2)) and examined their apoptosis by DNA fragmentation assay, 4',6-diamidino-2'-phenylindol dihydrochloride staining, and Western blot analysis. CoCl(2) induced apoptosis and accumulated ubiquitinated proteins. Exposure to CoCl(2) inhibited proteasome activity in U-937 cells. Cobalt-induced apoptosis was mediated via mitochondrial pathway because CoCl(2) released cytochrome c from mitochondria. These results suggest that cobalt-induced apoptosis of AMs may be one of the mechanisms for cobalt-induced lung injury and that the accumulation of ubiquitinated proteins might be involved in this apoptotic process.     

Cnh1 Na(+) /H(+) antiporter and Ena1 Na(+) -ATPase play different roles in cation homeostasis and cell physiology of Candida glabrata

Yeasts tightly regulate their intracellular concentrations of alkali metal cations. In Saccharomyces cerevisiae, the Nha1 Na(+) /H(+) -antiporter and Ena1 Na(+) -ATPase, mediate the efflux of toxic sodium and surplus potassium. We report the characterization of Candida glabrata CgCnh1 and CgEna1 homologues. Their substrate specificity and transport properties were compared upon expression in S. cerevisiae, and their function characterized directly in C. glabrata. The CgCnh1 antiporter and the CgEna1 ATPase transport both potassium and sodium when expressed in S. cerevisiae. CgEna1p fully complements the lack of S. cerevisiae own Na(+) -ATPases but the activity of the CgCnh1 antiporter is lower than that of ScNha1p. Candida glabrata deletion mutants and analyses of their phenotypes revealed that though both transporters have a broad substrate specificity, their function in C. glabrata cells is not the same. Their differing physiological roles are also reflected in their regulation of expression, CgENA1 is highly upregulated by an increased osmotic pressure or sodium concentration, whereas CgCNH1 is expressed constitutively. The Cnh1 antiporter is involved in the regulation of potassium content and the Ena1 ATPase in sodium detoxification of C. glabrata cells. This situation differs from S. cerevisiae, where the Nha1 antiporter and Ena ATPases both participate together in Na(+) detoxification and in the regulation of K(+) homeostasis.     

Genome-Wide Analysis of Host Factors in Nodavirus RNA Replication

Flock House virus (FHV), the best studied of the animal nodaviruses, has been used as a model for positive-strand RNA virus research. As one approach to identify host genes that affect FHV RNA replication, we performed a genome-wide analysis using a yeast single gene deletion library and a modified, reporter gene-expressing FHV derivative. A total of 4,491 yeast deletion mutants were tested for their ability to support FHV replication. Candidates for host genes modulating FHV replication were selected based on the initial genome-wide reporter gene assay and validated in repeated Northern blot assays for their ability to support wild type FHV RNA1 replication. Overall, 65 deletion strains were confirmed to show significant changes in the replication of both FHV genomic RNA1 and sub-genomic RNA3 with a false discovery rate of 5%. Among them, eight genes support FHV replication, since their deletion significantly reduced viral RNA accumulation, while 57 genes limit FHV replication, since their deletion increased FHV RNA accumulation. Of the gene products implicated in affecting FHV replication, three are localized to mitochondria, where FHV RNA replication occurs, 16 normally reside in the nucleus and may have indirect roles in FHV replication, and the remaining 46 are in the cytoplasm, with functions enriched in translation, RNA processing and trafficking.     

The role of the Parkinson's disease gene PARK9 in essential cellular pathways and the manganese homeostasis network in yeast

YPK9 (Yeast PARK9; also known as YOR291W) is a non-essential yeast gene predicted by sequence to encode a transmembrane P-type transport ATPase. However, its substrate specificity is unknown. Mutations in the human homolog of YPK9, ATP13A2/PARK9, have been linked to genetic forms of early onset parkinsonism. We previously described a strong genetic interaction between Ypk9 and another Parkinson's disease (PD) protein α-synuclein in multiple model systems, and a role for Ypk9 in manganese detoxification in yeast. In humans, environmental exposure to toxic levels of manganese causes a syndrome similar to PD and is thus an environmental risk factor for the disease. How manganese contributes to neurodegeneration is poorly understood. Here we describe multiple genome-wide screens in yeast aimed at defining the cellular function of Ypk9 and the mechanisms by which it protects cells from manganese toxicity. In physiological conditions, we found that Ypk9 genetically interacts with essential genes involved in cellular trafficking and the cell cycle. Deletion of Ypk9 sensitizes yeast cells to exposure to excess manganese. Using a library of non-essential gene deletions, we screened for additional genes involved in tolerance to excess manganese exposure, discovering several novel pathways involved in manganese homeostasis. We defined the dependence of the deletion strain phenotypes in the presence of manganese on Ypk9, and found that Ypk9 deletion modifies the manganese tolerance of only a subset of strains. These results confirm a role for Ypk9 in manganese homeostasis and illuminates cellular pathways and biological processes in which Ypk9 likely functions.     

Two types of Bacillus subtilis tetA(L) deletion strains reveal the physiological importance of TetA(L) in K(+) acquisition as well as in Na(+), alkali, and tetracycline resistance

The chromosomally encoded TetA(L) protein of Bacillus subtilis is a multifunctional tetracycline-metal/H(+) antiporter that also exhibits monovalent cation/H(+) antiport activity and a net K(+) uptake mode. In this study, B. subtilis mutant strains JC112 and JC112C were found to be representative of two phenotypic types of tetA(L) deletion strains that are generated in the same selection. Both strains exhibited increased sensitivity to low tetracycline concentrations as expected. The mutants also had significantly reduced ability to grow in media containing low concentrations of K(+), indicating that the net K(+) uptake mode is of physiological consequence; the deficit in JC112 was greater than in JC112C. JC112 also exhibited (i) greater impairment of Na(+)- or K(+)-dependent growth at pH 8.3 than JC112C and (ii) a greater degree of Co(+2) as well as Na(+) sensitivity. Studies were initiated to explore the possibility of two different patterns of compensatory changes in other ion-translocating transporters in these mutants. Increased expression of two loci has thus far been shown. Increased expression of czcD-trkA, a locus with a proposed involvement in K(+) uptake, occurred in both mutants. The increase was highest in the presence of Co(2+) and was higher in JC112 than in JC112C. Deletion of czcD-trkA resulted in diminished growth of the wild-type and both mutant strains at low [K(+)], supporting a significant role for this locus in K(+) uptake. Expression of yheL, which is a homologue of the Na(+)/H(+) antiporter-encoding nhaC gene from Bacillus firmus OF4, was also increased in both tetA(L) deletion strains, again with higher up-regulation in JC112. The phenotypes resulting from deletion of yheL were consistent with a modest role for YheL in Na(+)-dependent pH homeostasis in the wild type. No major role for YheL was indicated in the mutants in spite of the overexpression. The studies underscore the multiple physiological functions of TetA(L), including tetracycline, Na(+), and alkali resistance and K(+) acquisition. The studies also reveal and begin to detail the complexity of the response to mutational loss of these functions.     

Characterization of a Snorhizobium meliloti ATP-binding cassette histidine transporter also involved in betaine and proline uptake

The symbiotic soil bacterium Sinorhizobium meliloti uses the compatible solutes glycine betaine and proline betaine for both protection against osmotic stress and, at low osmolarities, as an energy source. A PCR strategy based on conserved domains in components of the glycine betaine uptake systems from Escherichia coli (ProU) and Bacillus subtilis (OpuA and OpuC) allowed us to identify a highly homologous ATP-binding cassette (ABC) binding protein-dependent transporter in S. meliloti. This system was encoded by three genes (hutXWV) of an operon which also contained a fourth gene (hutH2) encoding a putative histidase, which is an enzyme involved in the first step of histidine catabolism. Site-directed mutagenesis of the gene encoding the periplasmic binding protein (hutX) and of the gene encoding the cytoplasmic ATPase (hutV) was done to study the substrate specificity of this transporter and its contribution in betaine uptake. These mutants showed a 50% reduction in high-affinity uptake of histidine, proline, and proline betaine and about a 30% reduction in low-affinity glycine betaine transport. When histidine was used as a nitrogen source, a 30% inhibition of growth was observed in hut mutants (hutX and hutH2). Expression analysis of the hut operon determined using a hutX-lacZ fusion revealed induction by histidine, but not by salt stress, suggesting this uptake system has a catabolic role rather than being involved in osmoprotection. To our knowledge, Hut is the first characterized histidine ABC transporter also involved in proline and betaine uptake.