A Deubiquitylating Complex Required for Neosynthesis of a Yeast Mitochondrial ATP Synthase Subunit

The ubiquitin system is known to be involved in maintaining the integrity of mitochondria, but little is known about the role of deubiquitylating (DUB) enzymes in such functions. Budding yeast cells deleted for UBP13 and its close homolog UBP9 displayed a high incidence of petite colonies and slow respiratory growth at 37°C. Both Ubp9 and Ubp13 interacted directly with Duf1 (DUB-associated factor 1), a WD40 motif-containing protein. Duf1 activates the DUB activity of recombinant Ubp9 and Ubp13 in vitro and deletion of DUF1 resulted in the same respiratory phenotype as the deletion of both UBP9 and UBP13. We show that the mitochondrial defects of these mutants resulted from a strong decrease at 37°C in the de novo biosynthesis of Atp9, a membrane-bound component of ATP synthase encoded by mitochondrial DNA. The defect appears at the level of ATP9 mRNA translation, while its maturation remained unchanged in the mutants. This study describes a new role of the ubiquitin system in mitochondrial biogenesis

Genes Differentially Expressed in Conidia and Hyphae of Aspergillus fumigatus upon Exposure to Human Neutrophils

Aspergillus fumigatus is the most common etiologic agent of invasive aspergillosis in immunocompromised patients. Several studies have addressed the mechanism involved in host defense but only few have investigated the pathogen's response to attack by the host cells. To our knowledge, this is the first study that investigates the genes differentially expressed in conidia vs hyphae of A. fumigatus in response to neutrophils from healthy donors as well as from those with chronic granulomatous disease (CGD) which are defective in the production of reactive oxygen species.Transcriptional profiles of conidia and hyphae exposed to neutrophils, either from normal donors or from CGD patients, were obtained by using the genome-wide microarray. Upon exposure to either normal or CGD neutrophils, 244 genes were up-regulated in conidia but not in hyphae. Several of these genes are involved in the degradation of fatty acids, peroxisome function and the glyoxylate cycle which suggests that conidia exposed to neutrophils reprogram their metabolism to adjust to the host environment. In addition, the mRNA levels of four genes encoding proteins putatively involved in iron/copper assimilation were found to be higher in conidia and hyphae exposed to normal neutrophils compared to those exposed to CGD neutrophils. Deletants in several of the differentially expressed genes showed phenotypes related to the proposed functions, i.e. deletants of genes involved in fatty acid catabolism showed defective growth on fatty acids and the deletants of iron/copper assimilation showed higher sensitivity to the oxidative agent menadione. None of these deletants, however, showed reduced resistance to neutrophil attack.This work reveals the complex response of the fungus to leukocytes, one of the major host factors involved in antifungal defense, and identifies fungal genes that may be involved in establishing or prolonging infections in humans

Proteomic Analysis Reveals That Iron Availability Alters the Metabolic Status of the Pathogenic Fungus Paracoccidioides brasiliensis

Paracoccidioides brasiliensis is a thermodimorphic fungus and the causative agent of paracoccidioidomycosis (PCM). The ability of P. brasiliensis to uptake nutrients is fundamental for growth, but a reduction in the availability of iron and other nutrients is a host defense mechanism many pathogenic fungi must overcome. Thus, fungal mechanisms that scavenge iron from host may contribute to P. brasiliensis virulence. In order to better understand how P. brasiliensis adapts to iron starvation in the host we compared the two-dimensional (2D) gel protein profile of yeast cells during iron starvation to that of iron rich condition. Protein spots were selected for comparative analysis based on the protein staining intensity as determined by image analysis. A total of 1752 protein spots were selected for comparison, and a total of 274 out of the 1752 protein spots were determined to have changed significantly in abundance due to iron depletion. Ninety six of the 274 proteins were grouped into the following functional categories; energy, metabolism, cell rescue, virulence, cell cycle, protein synthesis, protein fate, transcription, cellular communication, and cell fate. A correlation between protein and transcript levels was also discovered using quantitative RT-PCR analysis from RNA obtained from P. brasiliensis under iron restricting conditions and from yeast cells isolated from infected mouse spleens. In addition, western blot analysis and enzyme activity assays validated the differential regulation of proteins identified by 2-D gel analysis. We observed an increase in glycolytic pathway protein regulation while tricarboxylic acid cycle, glyoxylate and methylcitrate cycles, and electron transport chain proteins decreased in abundance under iron limiting conditions. These data suggest a remodeling of P. brasiliensis metabolism by prioritizing iron independent pathways

Global Transcriptome and Deletome Profiles of Yeast Exposed to Transition Metals

A variety of pathologies are associated with exposure to supraphysiological concentrations of essential metals and to non-essential metals and metalloids. The molecular mechanisms linking metal exposure to human pathologies have not been clearly defined. To address these gaps in our understanding of the molecular biology of transition metals, the genomic effects of exposure to Group IB (copper, silver), IIB (zinc, cadmium, mercury), VIA (chromium), and VB (arsenic) elements on the yeast Saccharomyces cerevisiae were examined. Two comprehensive sets of metal-responsive genomic profiles were generated following exposure to equi-toxic concentrations of metal: one that provides information on the transcriptional changes associated with metal exposure (transcriptome), and a second that provides information on the relationship between the expression of ∼4,700 non-essential genes and sensitivity to metal exposure (deletome). Approximately 22% of the genome was affected by exposure to at least one metal. Principal component and cluster analyses suggest that the chemical properties of the metal are major determinants in defining the expression profile. Furthermore, cells may have developed common or convergent regulatory mechanisms to accommodate metal exposure. The transcriptome and deletome had 22 genes in common, however, comparison between Gene Ontology biological processes for the two gene sets revealed that metal stress adaptation and detoxification categories were commonly enriched. Analysis of the transcriptome and deletome identified several evolutionarily conserved, signal transduction pathways that may be involved in regulating the responses to metal exposure. In this study, we identified genes and cognate signaling pathways that respond to exposure to essential and non-essential metals. In addition, genes that are essential for survival in the presence of these metals were identified. This information will contribute to our understanding of the molecular mechanism by which organisms respond to metal stress, and could lead to an understanding of the connection between environmental stress and signal transduction pathways

Inducible dissociation of SCF(Met30) ubiquitin ligase mediates a rapid transcriptional response to cadmium

Activity of the Met4 transcription factor is antagonized by the SCF(Met30) ubiquitin ligase by degradation-dependent and degradation-independent mechanisms, in minimal and rich nutrient conditions, respectively. In this study, we show that the heavy metal Cd(2+) over-rides both mechanisms to enable rapid Met4-dependent induction of metabolic networks needed for production of the antioxidant and Cd(2+)-chelating agent glutathione. Cd(2+) inhibits SCF(Met30) activity through rapid dissociation of the F-box protein Met30 from the holocomplex. In minimal medium, dissociation of SCF(Met30) complex is sufficient to impair the methionine-induced degradation of Met4. In rich medium, dissociation of the SCF(Met30) complex is accompanied by a deubiquitylation mechanism that rapidly removes inhibitory ubiquitin moieties from Met4. Post-translational control of SCF(Met30) assembly by a physiological stress to allow rapid induction of a protective gene expression program represents a novel mode of regulation in the ubiquitin system

Combinatorial control of diverse metabolic and physiological functions by transcriptional regulators of the yeast sulfur assimilation pathway

Methionine abundance affects diverse cellular functions, including cell division, redox homeostasis, survival under starvation, and oxidative stress response. Regulation of the methionine biosynthetic pathway involves three DNA-binding proteins—Met31p, Met32p, and Cbf1p. We hypothesized that there exists a “division of labor” among these proteins that facilitates coordination of methionine biosynthesis with diverse biological processes. To explore combinatorial control in this regulatory circuit, we deleted CBF1, MET31, and MET32 individually and in combination in a strain lacking methionine synthase. We followed genome-wide gene expression as these strains were starved for methionine. Using a combination of bioinformatic methods, we found that these regulators control genes involved in biological processes downstream of sulfur assimilation; many of these processes had not previously been documented as methionine dependent. We also found that the different factors have overlapping but distinct functions. In particular, Met31p and Met32p are important in regulating methionine metabolism, whereas p functions as a “generalist” transcription factor that is not specific to methionine metabolism. In addition, Met31p and Met32p appear to regulate iron–sulfur cluster biogenesis through direct and indirect mechanisms and have distinguishable target specificities. Finally, CBF1 deletion sometimes has the opposite effect on gene expression from MET31 and MET32 deletion

Gex1 is a yeast glutathione exchanger that interferes with pH and redox homeostasis

Gex1 is a glutathione exchanger mostly found at the vacuolar membrane. The expression of Gex1 and Gex2 is induced under conditions of iron depletion, and Gex1 is regulated principally by the iron transcription factor Aft2. Loss of or high Gex1/2 activity modulates PKA and MAPK signaling events