The Tlo Proteins Are Stoichiometric Components of Candida albicans Mediator Anchored via the Med3 Subunit

Zhang, Anda; Petrov, Kostadin; Hyun, Emily R.; Liu, Zhongle; Gerber, Scott A.; Myers, Lawrence C.

doi:10.1128/ec.00095-12

Cited by 49 publications

(118 citation statements)

References 51 publications

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“…The absence of Med8 from the large-scale data set is not surprising, since the tryptic peptide generated form the N terminus of Med8, which contains the most likely Cdk8 P sites, is above the molecular mass typically detected by this methodology. To determine if Cdk8 can phosphorylate a Tlo protein family member and Med3, we performed an [ 32 P]ATP kinase assay in which purified Mediator was incubated with a complex of recombinant Tlo␣12 bound to 6ϫHis-tagged Med3 (44). The phosphorylation pattern showed that the recombinant proteins were detected in a dose-dependent manner at the expected sizes (Fig.…”

Section: Resultsmentioning

confidence: 99%

“…The Mediator complex was purified from the high-salt elution fraction of the heparin column by Flag affinity purification through the 6ϫHis-3ϫFlag tag placed on the C terminus of the Med8 (cTTR03) or Ssn3 (yLM256) subunit as previously described (44). Free CDK8 module, containing only Ssn3/Cdk8, Ssn8/CycC, Ssn5/Med12, and Ssn2/Med13, was purified from the heparin flowthrough fraction of yLM255 lysate by Flag purification.…”

Section: Go Term Analysismentioning

confidence: 99%

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Analysis of the Candida albicans Phosphoproteome

et al. 2015

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dCandida albicans is an important human fungal pathogen in both immunocompetent and immunocompromised individuals. C. albicans regulation has been studied in many contexts, including morphological transitions, mating competence, biofilm formation, stress resistance, and cell wall synthesis. Analysis of kinase-and phosphatase-deficient mutants has made it clear that protein phosphorylation plays an important role in the regulation of these pathways. In this study, to further our understanding of phosphorylation in C. albicans regulation, we performed a deep analysis of the phosphoproteome in C. albicans. We identified 19,590 unique peptides that corresponded to 15,906 unique phosphosites on 2,896 proteins. The ratios of serine, threonine, and tyrosine phosphosites were 80.01%, 18.11%, and 1.81%, respectively. The majority of proteins (2,111) contained at least two detected phosphorylation sites. Consistent with findings in other fungi, cytoskeletal proteins were among the most highly phosphorylated proteins, and there were differences in Gene Ontology (GO) terms for proteins with serine and threonine versus tyrosine phosphorylation sites. This large-scale analysis identified phosphosites in protein components of Mediator, an important transcriptional coregulatory protein complex. A targeted analysis of the phosphosites in Mediator complex proteins confirmed the large-scale studies, and further in vitro assays identified a subset of these phosphorylations that were catalyzed by Cdk8 (Ssn3), a kinase within the Mediator complex. These data represent the deepest single analysis of a fungal phosphoproteome and lay the groundwork for future analyses of the C. albicans phosphoproteome and specific phosphoproteins.

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Section: Resultsmentioning

confidence: 99%

Section: Go Term Analysismentioning

confidence: 99%

Analysis of the Candida albicans Phosphoproteome

et al. 2015

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“…Most strains, except where noted, were derived from the SN152 (53) MTLa/a strain. The specific open reading frames (ORFs) of Mediator subunit genes were previously identified (13). Mediator subunit single gene deletions (med5⌬/ ⌬, med9⌬/⌬, med1⌬/⌬, med16⌬/⌬, med3⌬/⌬, med15⌬/⌬, med20⌬/⌬, med12⌬/⌬) were created by using the C. maltosa LEU2 and C. dubliniensis HIS1 fusion PCR cassettes as described by Noble and Johnson (53).…”

Section: Methodsmentioning

confidence: 99%

“…Saccharomyces cerevisiae Mediator (scMediator) has structurally distinct modules referred to as Tail, Middle, Head, and Cdk8 (9)(10)(11)(12). Purification and characterization of Candida albicans Mediator (caMediator) (13) showed that the caMediator complex has a composition and architecture similar to that of scMediator. Certain scMediator subunits are critical for the activation of specific sets of genes (14,15), and the Tail module has been shown to be a direct target of activation domains (16)(17)(18)(19).…”

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confidence: 99%

Differential Regulation of White-Opaque Switching by Individual Subunits of Candida albicans Mediator

2013

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The multisubunit eukaryotic Mediator complex integrates diverse positive and negative gene regulatory signals and transmits them to the core transcription machinery. Mutations in individual subunits within the complex can lead to decreased or increased transcription of certain subsets of genes, which are highly specific to the mutated subunit. Recent studies suggest a role for Mediator in epigenetic silencing. Using white-opaque morphological switching in Candida albicans as a model, we have shown that Mediator is required for the stability of both the epigenetic silenced (white) and active (opaque) states of the bistable transcription circuit driven by the master regulator Wor1. Individual deletions of eight C. albicans Mediator subunits have shown that different Mediator subunits have dramatically diverse effects on the directionality, frequency, and environmental induction of epigenetic switching. Among the Mediator deletion mutants analyzed, only Med12 has a steady-state transcriptional effect on the components of the Wor1 circuit that clearly corresponds to its effect on switching. The MED16 and MED9 genes have been found to be among a small subset of genes that are required for the stability of both the white and opaque states. Deletion of the Med3 subunit completely destabilizes the opaque state, even though the Wor1 transcription circuit is intact and can be driven by ectopic expression of Wor1. The highly impaired ability of the med3 deletion mutant to mate, even when Wor1 expression is ectopically induced, reveals that the activation of the Wor1 circuit can be decoupled from the opaque state and one of its primary biological consequences.

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“…We found that the C. albicans TLO gene family is continuing to rapidly evolve, including changes both in the number and relative positions of TLO genes within the genome. TLO genes encode proteins with similarity to Med2, a component of Mediator that regulates transcription by RNA polymerase II; and some Tlo proteins have been confirmed as Med2-like Mediator components (Zhang et al 2012). TLO gene expression is also highly variable due primarily to Sir2-dependent telomere associated silencing (Anderson et al 2014).…”

Section: Intra-species Variation In Candida Albicansmentioning

confidence: 99%

Genetic and phenotypic intra-species variation in Candida albicans

et al. 2014

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Candida albicans is a commensal fungus of the human gastrointestinal tract and a prevalent opportunistic pathogen. To examine diversity within this species, extensive genomic and phenotypic analyses were performed on 21 clinical C. albicans isolates. Genomic variation was evident in the form of polymorphisms, copy number variations, chromosomal inversions, subtelomeric hypervariation, loss of heterozygosity (LOH), and whole or partial chromosome aneuploidies. All 21 strains were diploid, although karyotypic changes were present in eight of the 21 isolates, with multiple strains being trisomic for Chromosome 4 or Chromosome 7. Aneuploid strains exhibited a general fitness defect relative to euploid strains when grown under replete conditions. All strains were also heterozygous, yet multiple, distinct LOH tracts were present in each isolate. Higher overall levels of genome heterozygosity correlated with faster growth rates, consistent with increased overall fitness. Genes with the highest rates of amino acid substitutions included many cell wall proteins, implicating fast evolving changes in cell adhesion and host interactions. One clinical isolate, P94015, presented several striking properties including a novel cellular phenotype, an inability to filament, drug resistance, and decreased virulence. Several of these properties were shown to be due to a homozygous nonsense mutation in the EFG1 gene. Furthermore, loss of EFG1 function resulted in increased fitness of P94015 in a commensal model of infection. Our analysis therefore reveals intra-species genetic and phenotypic differences in C. albicans and delineates a natural mutation that alters the balance between commensalism and pathogenicity.

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The Tlo Proteins Are Stoichiometric Components of Candida albicans Mediator Anchored via the Med3 Subunit

Abstract: ABSTRACTThe amplification of theTLO(fortelomere-associated) genes inCandida albicans, compared to its less pathogenic, close relativeCandida dubliniensis, suggests a role in virulence. Little, however, is known about the function of the Tlo proteins… Show more

Cited by 49 publications

References 51 publications

Analysis of the Candida albicans Phosphoproteome

Analysis of the Candida albicans Phosphoproteome

Differential Regulation of White-Opaque Switching by Individual Subunits of Candida albicans Mediator

Genetic and phenotypic intra-species variation in Candida albicans

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