TcNST2 encodes a Golgi-localized UDP-galactose transporter in Trypanosoma cruzi.

Survival and infectivity of trypanosomatids rely on cell-surface and secreted glycoconjugates, many of which contain a variable number of galactose residues. Incorporation of galactose to proteins and lipids occurs along the secretory pathway from UDP-galactose (UDP-Gal). Before being used in glycosylation reactions, however, this activated sugar donor must first be transported across the endoplasmic reticulum and Golgi membranes by a specific nucleotide sugar transporter (NST). In this study, we identified an UDP-Gal transporter (named TcNST2 and encoded by the TcCLB.504085.60 gene) from Trypanosoma cruzi, the etiological agent of Chagas disease. TcNST2 was identified by heterologous expression of selected putative nucleotide sugar transporters in a mutant Chinese Hamster Ovary cell line. TcNST2 mRNA levels were detected in all T. cruzi life-cycle forms, with an increase in expression in axenic amastigotes. Confocal microscope analysis indicated that the transporter is specifically localized to the Golgi apparatus. A three-dimensional model of TcNST2 suggested an overall structural conservation as compared with members of the metabolite transporter superfamily and also suggested specific features that could be related to its activity. The identification of this transporter is an important step toward a better understanding of glycoconjugate biosynthesis and the role NSTs play in this process in trypanosomatids.

Functional expression of the maize mitochondrial URF13 down-regulates galactose-induced GAL1 gene expression in Saccharomyces cerevisiae.

Genes for the enzymes that metabolize galactose in Saccharomyces cerevisiae are strongly induced by galactose and tightly repressed by glucose. Because glucose also represses mitochondrial activity, we examined if derepression of the GAL1 galactokinase gene requires physiologically active mitochondria. The effect of mitochondria on the expression of GAL1 was analyzed by a novel approach in which the activity of the organelles was altered by functional expression of URF13, a mitochondrial protein unique to the Texas-type cytoplasmic male sterility phenotype in maize. Mitochondrial targeting and functional expression of the URF13 protein in yeast result in a decrease of the mitochondrial membrane potential similar to those observed in cells treated with mitochondrial inhibitors such as antimycin A or sodium azide. Activation of URF13 in galactose-induced cells results in the inhibition of GAL1 expression in the absence of repressing concentrations of glucose. Our data reveal the existence of a regulatory pathway that connects the derepression of the GAL1 gene with mitochondrial activity.

Oxygen- and glucose-dependent expression of Trhxt1, a putative glucose transporter gene of Trichoderma reesei.

The filamentous fungus Trichoderma reesei is adapted to nutrient-poor environments, in which it uses extracellular cellulases to obtain glucose from the available cellulose biomass. We have isolated and characterized Trhxt1, a putative glucose transporter gene, as judged by the glucose accumulation phenotype of a DeltaTrhxt1 mutant. This gene is repressed at high glucose concentrations and expressed at micromolar levels and in the absence of glucose. The gene is also induced during the growth of T. reesei on cellulose when the glucose concentration generated from the hydrolysis of cellulose present in the culture medium is in the micromolar range. We also show that oxygen availability controls the expression of the Trxht1 gene. In this regard, the gene is down-regulated by hypoxia and also by the inhibition of the flow of electrons through the respiratory chain using antimycin A. Intriguingly, anoxia but not hypoxia strongly induces the expression of the gene in the presence of an otherwise repressive concentration of glucose. These results indicate that although the absence of repressing concentrations of glucose and an active respiratory chain are required for Trhxt1 expression under normoxic conditions these physiological processes have no effect on the expression of this gene under an anoxic state. Thus, our results highlight the presence of a novel coordinated interaction between oxygen and the regulatory circuit for glucose repression under anoxic conditions.

Transcriptional response of the obligatory aerobe Trichoderma reesei to hypoxia and transient anoxia: implications for energy production and survival in the absence of oxygen.

Oxygen is essential for the survival of obligatorily aerobic eukaryotic microorganisms, such as the multicellular fungus Trichoderma reesei. However, the molecular basis for the inability of such cells to survive for extended periods under anoxic conditions is not fully understood. Using cDNA microarray analysis, we show that changes in oxygen availability have a drastic effect on gene expression in T. reesei. The expression levels of 392 (19.6%) out of 2000 genes examined changed significantly in response to hypoxia, transient anoxia, and reoxygenation. In addition to modulating many genes with no previously assigned function, cells respond to hypoxia by readjusting the balance of expression between genes required for energy production and consumption, and altering the expression of genes involved in protective mechanisms and signaling pathways. Moreover, we show that transient anoxia strongly represses genes for enzymes that are critical for glycolysis, and are essential for energy production under anaerobic conditions. Our study thus reveals crucial differences between the facultative anaerobe Saccharomyces cerevisiae and T. reesei with regard to the oxygen-dependent transcriptional control of the glycolytic pathway, which can account for the differential survival of the two species in the absence of oxygen.

Elucidation of the metabolic fate of glucose in the filamentous fungus Trichoderma reesei using expressed sequence tag (EST) analysis and cDNA microarrays.

Despite the intense interest in the metabolic regulation and evolution of the ATP-producing pathways, the long standing question of why most multicellular microorganisms metabolize glucose by respiration rather than fermentation remains unanswered. One such microorganism is the cellulolytic fungus Trichoderma reesei (Hypocrea jecorina). Using EST analysis and cDNA microarrays, we find that in T. reesei expression of the genes encoding the enzymes of the tricarboxylic acid cycle and the proteins of the electron transport chain is programmed in a way that favors the oxidation of pyruvate via the tricarboxylic acid cycle rather than its reduction to ethanol by fermentation. Moreover, the results indicate that acetaldehyde may be channeled into acetate rather than ethanol, thus preventing the regeneration of NAD(+), a pivotal product required for anaerobic metabolism. The studies also point out that the regulatory machinery controlled by glucose was most probably the target of evolutionary pressure that directed the flow of metabolites into respiratory metabolism rather than fermentation. This finding has significant implications for the development of metabolically engineered cellulolytic microorganisms for fuel production from cellulose biomass.