scholarly journals CgCYN1, a Plasma Membrane Cystine-specific Transporter of Candida glabrata with Orthologues Prevalent among Pathogenic Yeast and Fungi

2011 ◽  
Vol 286 (22) ◽  
pp. 19714-19723 ◽  
Author(s):  
Amit Kumar Yadav ◽  
Anand Kumar Bachhawat
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Plasma Membrane ◽  
Candida Glabrata ◽  
Histoplasma Capsulatum ◽  
Kinetic Studies ◽  
Sulfur Source ◽  
Amino Acid Permease ◽  
Pathogenic Yeast ◽  
Cystine Transporter ◽  
Energy Dependent

We describe a novel plasma membrane cystine transporter, CgCYN1, from Candida glabrata, the first such transporter to be described from yeast and fungi. C. glabrata met15Δ strains, organic sulfur auxotrophs, were observed to utilize cystine as a sulfur source, and this phenotype was exploited in the discovery of CgCYN1. Heterologous expression of CgCYN1 in Saccharomyces cerevisiae met15Δ strains conferred the ability of S. cerevisiae strains to grow on cystine. Deletion of the CgCYN1 ORF (CAGL0M00154g) in C. glabrata met15Δ strains caused abrogation of growth on cystine with growth being restored when CgCYN1 was reintroduced. The CgCYN1 protein belongs to the amino acid permease family of transporters, with no similarity to known plasma membrane cystine transporters of bacteria and humans, or lysosomal cystine transporters of humans/yeast. Kinetic studies revealed a Km of 18 ± 5 μm for cystine. Cystine uptake was inhibited by cystine, but not by other amino acids, including cysteine. The structurally similar cystathionine, lanthionine, and selenocystine alone inhibited transport, confirming that the transporter was specific for cystine. CgCYN1 localized to the plasma membrane and transport was energy-dependent. Functional orthologues could be demonstrated from other pathogenic yeast like Candida albicans and Histoplasma capsulatum, but were absent in Schizosaccharomyces pombe and S. cerevisiae.

scholarly journals Genome and transcriptome of a pathogenic yeast, Candida nivariensis

2021 ◽  
Author(s):  
Yunfan Fan ◽  
Andrew N Gale ◽  
Anna Bailey ◽  
Kali Barnes ◽  
Kiersten Colotti ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Candida Glabrata ◽  
Nanopore Sequencing ◽  
Cdna Sequencing ◽  
Pathogenic Yeast ◽  
Mitochondrial Sequence ◽  
Dna And Rna ◽  
Oxford Nanopore ◽  
Candida Nivariensis ◽  
Oxford Nanopore Technologies

Abstract We present a highly contiguous genome and transcriptome of the pathogenic yeast, Candida nivariensis. We sequenced both the DNA and RNA of this species using both the Oxford Nanopore Technologies (ONT) and Illumina platforms. We assembled the genome into an 11.8 Mb draft composed of 16 contigs with an N50 of 886 Kb, including a circular mitochondrial sequence of 28 Kb. Using direct RNA nanopore sequencing and Illumina cDNA sequencing, we constructed an annotation of our new assembly, supplemented by lifting over genes from Saccharomyces cerevisiae and Candida glabrata.

scholarly journals Metabolic instability and constitutive endocytosis of STE6, the a-factor transporter of Saccharomyces cerevisiae.

1994 ◽  
Vol 5 (11) ◽  
pp. 1185-1198 ◽  
Author(s):  
C Berkower ◽  
D Loayza ◽  
S Michaelis
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Plasma Membrane ◽  
Sufficient Information ◽  
Plasma Membrane Atpase ◽  
Amino Acid Permease ◽  
Membrane Atpase ◽  
A Cell ◽  
Pattern Characteristic ◽  
General Amino Acid Permease ◽  
Punctate Pattern

STE6, a member of the ATP binding cassette (ABC) transporter superfamily, is a membrane protein required for the export of the a-factor mating pheromone in Saccharomyces cerevisiae. To initiate a study of the intracellular trafficking of STE6, we have examined its half-life and localization. We report here that STE6 is metabolically unstable in a wild-type strain, and that this instability is blocked in a pep4 mutant, suggesting that degradation of STE6 occurs in the vacuole and is dependent upon vacuolar proteases. In agreement with a model whereby STE6 is routed to the vacuole via endocytosis from the plasma membrane, we show that degradation of STE6 is substantially reduced at nonpermissive temperature in mutants defective in delivery of proteins to the plasma membrane (sec6) or in endocytosis (end3 and end4). Whereas STE6 appears to undergo constitutive internalization from the plasma membrane, as do the pheromone receptors STE2 and STE3, we show that two other proteins, the plasma membrane ATPase (PMA1) and the general amino acid permease (GAP1), are significantly more stable than STE6, indicating that rapid turnover in the vacuole is not a fate common to all plasma membrane proteins in yeast. Investigation of STE6 partial molecules (half- and quarter-molecules) indicates that both halves of STE6 contain sufficient information to mediate internalization. Examination of STE6 localization by indirect immunofluorescence indicates that STE6 is found in a punctate, possibly vesicular, intracellular pattern, distinct from the rim-staining pattern characteristic of PMA1. The punctate pattern is consistent with the view that most of the STE6 molecules present in a cell at any given moment could be en route either to or from the plasma membrane. In a pep4 mutant, STE6 is concentrated in the vacuole, providing further evidence that the vacuole is the site of STE6 degradation, while in an end4 mutant STE6 exhibits rim-staining, indicating that it can accumulate in the plasma membrane when internalization is blocked. Taken together, the results presented here suggest that STE6 first travels to the plasma membrane and subsequently undergoes endocytosis and degradation in the vacuole, with perhaps only a transient residence at the plasma membrane; an alternative model, in which STE6 circumvents the plasma membrane, is also discussed.

scholarly journals Different Ubiquitin Signals Act at the Golgi and Plasma Membrane to Direct GAP1 Trafficking

2008 ◽  
Vol 19 (7) ◽  
pp. 2962-2972 ◽  
Author(s):  
April L. Risinger ◽  
Chris A. Kaiser
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Amino Acids ◽  
Plasma Membrane ◽  
Amino Acid ◽  
Protein Sorting ◽  
High Capacity ◽  
Amino Acid Permease ◽  
Acid Addition ◽  
Amino Acid Levels ◽  
General Amino Acid Permease

The high capacity general amino acid permease, Gap1p, in Saccharomyces cerevisiae is distributed between the plasma membrane and internal compartments according to availability of amino acids. When internal amino acid levels are low, Gap1p is localized to the plasma membrane where it imports available amino acids from the medium. When sufficient amino acids are imported, Gap1p at the plasma membrane is endocytosed and newly synthesized Gap1p is delivered to the vacuole; both sorting steps require Gap1p ubiquitination. Although it has been suggested that identical trans-acting factors and Gap1p ubiquitin acceptor sites are involved in both processes, we define unique requirements for each of the ubiquitin-mediated sorting steps involved in delivery of Gap1p to the vacuole upon amino acid addition. Our finding that distinct ubiquitin-mediated sorting steps employ unique trans-acting factors, ubiquitination sites on Gap1p, and types of ubiquitination demonstrates a previously unrecognized level of specificity in ubiquitin-mediated protein sorting.

scholarly journals Inner Kinetochore of the Pathogenic Yeast Candida glabrata

2004 ◽  
Vol 3 (5) ◽  
pp. 1154-1163 ◽  
Author(s):  
Tanja Stoyan ◽  
John Carbon
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Candida Albicans ◽  
Chromatin Immunoprecipitation ◽  
Candida Glabrata ◽  
Hybrid Analysis ◽  
Pathogenic Yeast ◽  
Closely Related Species ◽  
Species Specific ◽  
Two Hybrid ◽  
Sequence Identities

ABSTRACT The human pathogenic yeast Candida glabrata is the second most common Candida pathogen after Candida albicans, causing both bloodstream and mucosal infections. The centromere (CEN) DNA of C. glabrata (CgCEN), although structurally very similar to that of Saccharomyces cerevisiae, is not functional in S. cerevisiae. To further examine the structure of the C. glabrata inner kinetochore, we isolated several C. glabrata homologs of S. cerevisiae inner kinetochore protein genes, namely, genes for components of the CBF3 complex (Ndc10p, Cep3p, and Ctf13p) and genes for the proteins Mif2p and Cse4p. The amino acid sequence identities of these proteins were 32 to 49% relative to S. cerevisiae. CgNDC10, CgCEP3, and CgCTF13 are required for growth in C. glabrata and are specifically found at CgCEN, as demonstrated by chromatin immunoprecipitation experiments. Cross-complementation experiments revealed that the isolated genes, with the exception of CgCSE4, are species specific and cannot functionally substitute for the corresponding genes in S. cerevisiae deletion strains. Likewise, the S. cerevisiae CBF3 genes NDC10, CEP3, and CTF13 cannot functionally replace their homologs in C. glabrata CBF3 deletion strains. Two-hybrid analysis revealed several interactions between these proteins, all of which were previously reported for the inner kinetochore proteins of S. cerevisiae. Our findings indicate that although many of the inner kinetochore components have evolved considerably between the two closely related species, the organization of the C. glabrata inner kinetochore is similar to that in S. cerevisiae.

scholarly journals Copper Acts Synergistically with Fluconazole in Candida glabrata by Compromising Drug Efflux, Sterol Metabolism, and Zinc Homeostasis

2021 ◽  
Author(s):  
Ana Gaspar-Cordeiro ◽  
Catarina Amaral ◽  
Vania Pobre ◽  
Wilson Antunes ◽  
Ana Petronilho ◽  
...  
Keyword(s):  
Cell Wall ◽  
Plasma Membrane ◽  
Candida Glabrata ◽  
Zinc Homeostasis ◽  
Ergosterol Biosynthesis ◽  
Gene Encoding ◽  
Pathogenic Yeast ◽  
Transcription Regulator ◽  
Opportunistic Pathogenic ◽  
Irregular Cell

The synergistic combinations of drugs are promising strategies to boost the effectiveness of current antifungals and thus prevent the emergence of resistance. In this work, we show that copper and the antifungal fluconazole act synergistically against Candida glabrata, an opportunistic pathogenic yeast intrinsically tolerant to fluconazole. Analyses of the transcriptomic profile of C. glabrata after the combination of copper and fluconazole showed that the expression of the multidrug transporter gene CDR1 was decreased, suggesting that fluconazole efflux could be affected. In agreement, we observed that copper inhibits the transactivation of Pdr1, the transcription regulator of multidrug transporters, and leads to the intracellular accumulation of fluconazole. Copper also decreases the transcriptional induction of ergosterol biosynthesis (ERG) genes by fluconazole, which culminates in the accumulation of toxic sterols. Co-treatment of cells with copper and fluconazole should affect the function of proteins located in the plasma membrane, as several ultrastructural alterations, including irregular cell wall and plasma membrane and loss of cell wall integrity, were observed. Finally, we show that the combination of copper and fluconazole downregulates the expression of the gene encoding the zinc-responsive transcription regulator Zap1, which possibly, together with the membrane transporters malfunction, generates zinc depletion. Supplementation with zinc reverts the toxic effect of combining copper with fluconazole, underscoring the importance of this metal in the observed synergistic effect. Overall, this work, while unveiling the molecular basis that supports the use of copper to enhance the effectiveness of fluconazole, paves the way for the development of new metal-based antifungal strategies.

scholarly journals Physiological Regulation of Membrane Protein Sorting Late in the Secretory Pathway of Saccharomyces cerevisiae

1997 ◽  
Vol 137 (7) ◽  
pp. 1469-1482 ◽  
Author(s):  
Kevin J. Roberg ◽  
Neil Rowley ◽  
Chris A. Kaiser
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Plasma Membrane ◽  
Mammalian Cells ◽  
Secretory Pathway ◽  
Protein Sorting ◽  
Yeast Cells ◽  
Amino Acid Permease ◽  
Physiological Regulation ◽  
Extracellular Signals ◽  
Nitrogen Sensing

In mammalian cells, extracellular signals can regulate the delivery of particular proteins to the plasma membrane. We have discovered a novel example of regulated protein sorting in the late secretory pathway of Saccharomyces cerevisiae. In yeast cells grown on either ammonia or urea medium, the general amino acid permease (Gap1p) is transported from the Golgi complex to the plasma membrane, whereas, in cells grown on glutamate medium, Gap1p is transported from the Golgi to the vacuole. We have also found that sorting of Gap1p in the Golgi is controlled by SEC13, a gene previously shown to encode a component of the COPII vesicle coat. In sec13 mutants grown on ammonia, Gap1p is transported from the Golgi to the vacuole, instead of to the plasma membrane. Deletion of PEP12, a gene required for vesicular transport from the Golgi to the prevacuolar compartment, counteracts the effect of the sec13 mutation and partially restores Gap1p transport to the plasma membrane. Together, these studies demonstrate that both a nitrogen-sensing mechanism and Sec13p control Gap1p transport from the Golgi to the plasma membrane.

scholarly journals Genome and transcriptome of a pathogenic yeast, Candida nivariensis

2021 ◽  
Author(s):  
Yunfan Fan ◽  
Andrew N Gale ◽  
Anna Bailey ◽  
Kali Barnes ◽  
Kiersten Colotti ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Candida Glabrata ◽  
Nanopore Sequencing ◽  
Cdna Sequencing ◽  
Pathogenic Yeast ◽  
Mitochondrial Sequence ◽  
Dna And Rna ◽  
Oxford Nanopore ◽  
Candida Nivariensis ◽  
Oxford Nanopore Technologies

AbstractWe present a highly contiguous genome and transcriptome of the pathogenic yeast, Candida nivariensis. We sequenced both the DNA and RNA of this species using both the Oxford Nanopore Technologies (ONT) and Illumina platforms. We assembled the genome into an 11.8 Mb draft composed of 16 contigs with an N50 of 886 Kb, including a circular mitochondrial sequence of 28 Kb. Using direct RNA nanopore sequencing and Illumina cDNA sequencing, we constructed an annotation of our new assembly, supplemented by lifting over genes from Saccharomyces cerevisiae and Candida glabrata.

scholarly journals The inositol regulon controls viability in Candida glabrata

Microbiology ◽  
2010 ◽  
Vol 156 (2) ◽  
pp. 452-462 ◽  
Author(s):  
Emily K. Bethea ◽  
Billy J. Carver ◽  
Anthony E. Montedonico ◽  
Todd B. Reynolds
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Candida Albicans ◽  
Candida Glabrata ◽  
Transcriptional Repressor ◽  
Transcriptional Activator ◽  
Genetic Data ◽  
Ribosomal Genes ◽  
Essential Genes ◽  
Pathogenic Yeast ◽  
Key Enzyme

Inositol is essential in eukaryotes, and must be imported or synthesized. Inositol biosynthesis in Saccharomyces cerevisiae is controlled by three non-essential genes that make up the inositol regulon: ScINO2 and ScINO4, which together encode a heterodimeric transcriptional activator, and ScOPI1, which encodes a transcriptional repressor. ScOpi1p inhibits the ScIno2-ScIno4p activator in response to extracellular inositol levels. An important gene controlled by the inositol regulon is ScINO1, which encodes inositol-3-phosphate synthase, a key enzyme in inositol biosynthesis. In the pathogenic yeast Candida albicans, homologues of the S. cerevisiae inositol regulon genes are ‘transcriptionally rewired’. Instead of regulating the CaINO1 gene, CaINO2 and CaINO4 regulate ribosomal genes. Another Candida species that is a prevalent cause of infections is Candida glabrata; however, C. glabrata is phylogenetically more closely related to S. cerevisiae than C. albicans. Experiments were designed to determine if C. glabrata homologues of the inositol regulon genes function similarly to S. cerevisiae or are transcriptionally rewired. CgINO2, CgINO4 and CgOPI1 regulate CgINO1 in a manner similar to that observed in S. cerevisiae. However, unlike in S. cerevisiae, CgOPI1 is essential. Genetic data indicate that CgOPI1 is a repressor that affects viability by regulating activation of a target of the inositol regulon.

scholarly journals Constitutive Signal Transduction by Mutant Ssy5p and Ptr3p Components of the SPS Amino Acid Sensor System in Saccharomyces cerevisiae

2005 ◽  
Vol 4 (6) ◽  
pp. 1116-1124 ◽  
Author(s):  
Peter Poulsen ◽  
Boqian Wu ◽  
Richard F. Gaber ◽  
Morten C. Kielland-Brandt
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Amino Acids ◽  
Transcription Factors ◽  
Plasma Membrane ◽  
Amino Acid ◽  
Amino Acid Permease ◽  
Associated Proteins ◽  
Amino Acid Sensing ◽  
Median Effective Concentration ◽  
Mutant Cells

ABSTRACT Amino acids in the environment of Saccharomyces cerevisiae can transcriptionally activate a third of the amino acid permease genes through a signal that originates from the interaction between the extracellular amino acids and an integral plasma membrane protein, Ssy1p. Two plasma membrane-associated proteins, Ptr3p and Ssy5p, participate in the sensing, which results in cleavage of the transcription factors Stp1p and Stp2p, removing 10 kDa of the N terminus of each of them. This confers the transcription factors with the ability to gain access to the nucleus and activate transcription of amino acid permease genes. To extend our understanding of the role of Ptr3p and Ssy5p in this amino acid sensing process, we have isolated constitutive gain-of-function mutants in these two components by using a genetic screening in which potassium uptake is made dependent on amino acid signaling. Mutants which exhibit inducer-independent processing of Stp1p and activation of the amino acid permease gene AGP1 were obtained. For each component of the SPS complex, constitutive signaling by a mutant allele depended on the presence of wild-type alleles of the other two components. Despite the signaling in the absence of inducer, the processing of Stp1p was more complete in the presence of inducer. Dose response assays showed that the median effective concentration for Stp1p processing in the mutant cells was decreased; i.e., a lower inducer concentration is needed for signaling in the mutant cells. These results suggest that the three sensor components interact intimately in a complex rather than in separate reactions and support the notion that the three components function as a complex.

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