Isolation, characterization, and inactivation of the APA1 gene encoding yeast diadenosine 5',5'''-P1,P4-tetraphosphate phosphorylase

Plateau, Pierre; Fromant, Michel; Schmitter, Jean‐Marie; Buhler, Jean‐Marie; Blanquet, Sylvain

doi:10.1128/jb.171.12.6437-6445.1989

Cited by 33 publications

(38 citation statements)

References 59 publications

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“…In eukaryotic cells, some adenosine polyphosphate species may be signal nucleotides (reviewed by Jakubowski, 1986;Plateau et al, 1989). However, US differed in HPLC retention time from known adenosine PolYPhosPhates (APPPPA, APPPPPA, APPPPPPA, PAPP, PPAPP) (Fig.…”

Section: Changes In the Nucleotide Pool During Growth On Agar Mediamentioning

confidence: 99%

A relaxed (rel) mutant of Streptomyces coelicolor A3(2) with a missing ribosomal protein lacks the ability to accumulate ppGpp, A-factor and prodigiosin

Ochi¹

1990

Journal of General Microbiology

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A relaxed (ref) mutant was found among 70 thiopeptin-resistant isolates of Streptomyces coeficofor A3(2) which arose spontaneously. The ability of the ref mutant to accumulate ppGpp during Casamino acid deprivation was reduced 10-fold compared to the wild-type. Analysis of the ribosomal proteins by two-dimensional PAGE revealed that the mutant lacked a ribosomal protein, tentatively designated ST-L11. It was therefore classified as a vefC mutant. The mutant was defective in producing A-factor and the pigmented antibiotic prodigiosin, in both liquid and agar cultures, but produced agarase normally. Production of actinorhodin, another pigmented antibiotic, was also abnormal; it appeared suddenly in agar cultures after 10 d incubation. Although aerial mycelium still formed, its appearance was markedly delayed. Whereas liquid cultures of the parent strain accumulated ppGpp, agar cultures accumulated only trace amounts. Instead, a substance characterized only as an unidentified HPLC peak accumulated intracellularly in the late growth phase, just before aerial mycelium formation and antibiotic production. This substance did not accumulate in mutant cells. It was found in S. fividans 66 and S.parvufus, but not in seven other Streptomyces species tested. The significance of these observations, and the relationship of the mutant to earlier ref isolates of Streptomyces is discussed.

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Section: Changes In the Nucleotide Pool During Growth On Agar Mediamentioning

confidence: 99%

A relaxed (rel) mutant of Streptomyces coelicolor A3(2) with a missing ribosomal protein lacks the ability to accumulate ppGpp, A-factor and prodigiosin

Ochi¹

1990

Journal of General Microbiology

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“…Phosphorolysis of Ap4A in the presence of Pi and a divalent cation yields ADP and ATP (18). About 80 to 85% of the endogenous Ap4A is catabolized by Ap4A phosphorylase 1 (29,30). The APAI gene encoding this enzyme has been cloned and sequenced (22,30) and physically mapped on chromosome III (16,22).…”

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“…About 80 to 85% of the endogenous Ap4A is catabolized by Ap4A phosphorylase 1 (29,30). The APAI gene encoding this enzyme has been cloned and sequenced (22,30) and physically mapped on chromosome III (16,22). (We had previously used the designation DTP for the gene encoding Ap4A phosphorylase I [22] (20).…”

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“…In Saccharomyces cerevisiae, Ap4A is degraded by two similar phosphorylases (29,30). Phosphorolysis of Ap4A in the presence of Pi and a divalent cation yields ADP and ATP (18).…”

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“…The residual activity is due to Ap4A phosphorylase II (29,30 (22). The yeast cells had been grown in a rich medium containing 3% glucose and 2% NZamineA to 6 x 107 to 7 x 107 cells per ml (late log phase).…”

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A paradoxical increase of a metabolite upon increased expression of its catabolic enzyme: the case of diadenosine tetraphosphate (Ap4A) and Ap4A phosphorylase I in Saccharomyces cerevisiae

et al. 1991

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The APA1 gene in Saccharomyces cerevisiae encodes Ap4A phosphorylase I, the catabolic enzyme for diadenosine 5',5"'-P1,P4-tetraphosphate (Ap4A). APA1 has been inserted into a multicopy plasmid and into a centromeric plasmid with a GAL1 promoter. Enhanced expression of APA1 via the plasmids resulted in 10- and 90-fold increases in Ap4A phosphorylase activity, respectively, as assayed in vitro. However, the intracellular concentration of Ap4A exhibited increases of 2- and 15-fold, respectively, from the two different plasmids. Intracellular Ap4A increased 3- to 20-fold during growth on galactose of a transformant with APA1 under the control of the GAL1 promoter. Intracellular adenosine 5'-P1-tetraphospho-P4-5"'-guanosine (Ap4G) and diguanosine 5',5"'-P1,P4-tetraphosphate (Gp4G) also increased in the transformant under these conditions. The chromosomal locus of APA1 has been disrupted in a haploid strain. The Ap4A phosphorylase activity decreased by 80% and the intracellular Ap4A concentration increased by a factor of five in the null mutant. These results with the null mutant agree with previous results reported by Plateau et al. (P. Plateau, M. Fromant, J.-M. Schmitter, J.-M. Buhler, and S. Blancquet, J. Bacteriol. 171:6437-6445, 1989). The paradoxical increase in Ap4A upon enhanced expression of APA1 indicates that the metabolic consequences of altered gene expression may be more complex than indicated solely by assay of enzymatic activity of the gene product.

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Multiple Copies ofPBS2,MHP1 orLRE1 Produce Glucanase Resistance and Other Cell Wall Effects inSaccharomyces cerevisiae

Lai¹,

Silverman²,

Gaughran³

et al. 1997

Yeast

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Five sequences were isolated by selection for multiple copy plasmids that conferred resistance to laminarinase, an enzyme that specifically degrades cell wall (1-3) glucan linkages. Strains carrying three of these plasmids showed alterations in cell wall glucan labelling. One of these plasmids carried PBS2, a previously identified, non-essential gene which produces a variety of phenotypes and encodes a mitogen-activated protein kinase kinase analogue (Boguslawski and Polazzi, 1987). Cells carrying PBS2 at multiple copy show a small decrease in cell wall (1-6) glucans. Measurements of (1-3) glucan synthase activity in multi-copy PBS2 cells showed an approximate 30-45% increase in enzyme specific activity while a pbs2 disruption strain showed a decrease in glucan synthase activity of approximately 45% relative to control. A pbs2 disruption strain was laminarinase super-sensitive and supersensitive to K 1 killer toxin while a strain carrying PBS2 at multiple copy was resistant to killer toxin. A second plasmid carried a portion of the MHP1 gene which has been reported to encode a microtubule-interacting protein (Irminger-Finger et al., 1996). The MHP1 gene product is a predicted 1398 amino acid protein and only approximately 80% of the amino portion of this protein is required for laminarinase resistance. Cells carrying the amino portion of MHP1 at multiple copy show a decrease in high molecular weight cell wall (1-6) glucans and were killer toxin resistant while a disruption strain was viable and killer toxin super-sensitive. Cells carrying this plasmid showed decreased levels of high molecular weight (1-6) glucans and increased glucan synthase activity. The laminarinase resistance conferred by the third plasmid mapped to the previously uncharacterized YCL051W open reading frame and this gene was therefore named LRE1 (laminarinase resistance). The LRE1 gene encodes a non-essential 604 amino acid hydrophilic protein. Unexpectedly, cells carrying LRE1 at multiple copy show no alteration in cell wall glucans or glucan synthase activity. Subcloning experiments demonstrated that the production of these cell wall effects requires the presence of both LRE1 and YCL052C (PBN1), a second open reading frame present on the original plasmid. Cells carrying multiple copies of PBN1 alone show no significant alterations in cell wall glucans or glucan synthase activity, indicating that these effects require the presence of multiple copies of both genes.

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Isolation, characterization, and inactivation of the APA1 gene encoding yeast diadenosine 5',5'''-P1,P4-tetraphosphate phosphorylase

Cited by 33 publications

References 59 publications

A relaxed (rel) mutant of Streptomyces coelicolor A3(2) with a missing ribosomal protein lacks the ability to accumulate ppGpp, A-factor and prodigiosin

A relaxed (rel) mutant of Streptomyces coelicolor A3(2) with a missing ribosomal protein lacks the ability to accumulate ppGpp, A-factor and prodigiosin

A paradoxical increase of a metabolite upon increased expression of its catabolic enzyme: the case of diadenosine tetraphosphate (Ap4A) and Ap4A phosphorylase I in Saccharomyces cerevisiae

Multiple Copies ofPBS2,MHP1 orLRE1 Produce Glucanase Resistance and Other Cell Wall Effects inSaccharomyces cerevisiae

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