Excess Mannose Limits the Growth of Phosphomannose Isomerase PMI40 Deletion Strain of Saccharomyces cerevisiae

Abstract

Phosphomannose isomerase (PMI40) catalyzes the conversion between fructose 6-phosphate and mannose 6-phosphate and thus connects glycolysis, i.e. energy production and GDP-mannose biosynthesis or cell wall synthesis in Saccharomyces cerevisiae. After PMI40 deletion (pmi ؊ ) the cells were viable only if fed with extracellular mannose and glucose. In an attempt to force the GDP-mannose synthesis in the pmi ؊ strain by increasing the extracellular mannose concentrations, the cells showed significantly reduced growth rates without any alterations in the intracellular GDPmannose levels. To reveal the mechanisms resulting in reduced growth rates, we measured genome-wide gene expression levels, several metabolite concentrations, and selected in vitro enzyme activities in central metabolic pathways. The increasing of the initial mannose concentration led to an increase in the mannose 6-phosphate concentration, which inhibited the activity of the second enzyme in glycolysis, i.e. phosphoglucose isomerase converting glucose 6-phosphate to fructose 6-phosphate. As a result of this limitation, the flux through glycolysis was decreased as was the median expression of the genes involved in glycolysis. The expression levels of RAP1, a transcription factor involved in the regulation of the mRNA levels of several enzymes in glycolysis, as well as those of cell cycle regulators CDC28 and CLN3, decreased concomitantly with the growth rates and expression of many genes encoding for enzymes in glycolysis.Phosphomannose isomerase enzyme (PMIe) 1 catalyzes the interconversion of fructose 6-phosphate (Fru-6-P) in glycolysis to mannose 6-phosphate (Man-6-P) through a mannose pathway. In the eukaryotic model organism, yeast Saccharomyces cerevisiae, phosphomannose isomerase is encoded by the PMI40 gene (1). In a PMI40 deletion strain (pmi Ϫ ), the synthesis of Man-6-P from Fru-6-P is not possible, disabling the growth of such a strain on medium without mannose. The inability to grow, caused by defective glycosylation of a temperature-sensitive pmi40 mutant of S. cerevisiae, and repairing the defects by addition of mannose to the growth medium have been described previously (2). In humans PMIe deficiency is the cause of carbohydrate-deficient glycoprotein syndrome type Ib, but the condition can be successfully treated by mannose administration (3). Man-6-P produced either from Fru-6-P or mannose serves as a precursor for the de novo biosynthesis of GDP-mannose. Man-6-P is converted to mannose 1-phosphate (Man-1-P) by phosphomannomutase encoded by SEC53. Subsequently, Man-1-P is ligated with the guanosine 5-triphosphate molecule (GTP) to form GDP-mannose by Man-1-P guanylyltransferase encoded by PSA1 (4). The de novo formation of the purine ring of GTP, required for the biosynthesis of GDPmannose, starts from ribose 5-phosphate in the pentose phosphate pathway and requires also 3-phosphoglycerate in the glycolysis as a precursor. Taken together, the biosynthesis of GTP is more complex than the mannose pathway (4). GDPmannose is needed in ...

Mapmi gene contributes to stress tolerance and virulence of the entomopathogenic fungus, Metarhizium acridum

“…3B), demonstrating that the glycolysis pathway could not rescue the growth of Mapmi-RNAi strain. Growth of mannose concentration-dependent Mapmi mutants was consistent with loss of cell function in Pmi deletion animal cells (DeRossi et al, 2006) and some fungal cells (Fang et al, 2009;Pitkänen et al, 2004;Wills et al, 2001). Biochemical analysis showed that M-6-P accumulated in pmi deletion mutants when exposed to excess mannose in S. cerevisiae (Pitkänen et al, 2004), while increased intracellular ATP occurred in Escherichia coli (de la Fuente et al, 1986).…”

“…Mannose is involved also in the synthesis of the protective capsule and other structures within fungal cells (Bose et al, 2003;Fang et al, 2009;Pitkänen et al, 2004;Wills et al, 2001). GDP-mannose, a nucleotide sugar described by Cabib and Leloir (1954), has been demonstrated to be the activated donor of mannose for biosynthesis of mannose-containing glycoconjugates (Wills et al, 2001).…”

“…Growth was inactivated in a pmi deletion mutant but restored by adding mannose to the medium (Fang et al, 2009;Payton et al, 1991). However, excess exogenous mannose was toxicy to the pmi-deletion mutant due to the accumulation of intracellular Man-6-P, which inhibited further growth (Fang et al, 2009;Pitkänen et al, 2004). In Aspergillus fumigatus, a pmi mutant strain showed defects in cell wall integrity due to structural changes, had reduced conidiation, and expressed abnormal colony morphology (Fang et al, 2009) essential role of pmi for cell growth, pathogenicity was reduced or lost in pmi inactivated strains (Garami and Ilg, 2001;Smith and Payton, 1994;Wills et al, 2001).…”

Substrate Specificity of the Bacillus licheniformis Lyxose Isomerase YdaE and Its Application in In Vitro Catalysis for Bioproduction of Lyxose and Glucose by Two-Step Isomerization

“…Mannose and fructose are utilized according to the Kluyver rule (20), whereas glucose enters the glycolytic pathway directly. Using mannose as the sole carbon source inhibits the phosphomannose isomerase PMI40, which directs mannose into the glycolytic pathway (26); this constitutes another obstacle to the utilization of mannose for ethanol production.…”

Reducing Spatial Flaws in Oligonucleotide Arrays by Using Neighborhood Information

“…The same approach can be used without replicates, providing that information from several arrays of the same type is available. As an example Figure 4 shows the result of comparing the yeast array GSM29929 (see Figure 2) with yeast arrays from two entirely unrelated experiments: GSM34758 from experiment GSE1938 (see Pitkänen et al (2004) for details) and GSM67551 from experiment GSE3076 (see Guan,et al (2006)). The flaws in GSM29929 that were very obvious in Figure 2 are still apparent (though they are much less apparent).…”