Genetic evidence for distinct catalytic and regulatory subunits in yeast phosphofructokinase

Lobo, Zita; Maitra, P. K.

doi:10.1016/0014-5793(82)80494-8

Cited by 28 publications

(23 citation statements)

References 9 publications

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“…If the latter takes place at the active site, one can assume that, due to the lack of ATP inhibition in our mutants, this binding is favored. Therefore, the mutant enzymes would be in a conformational state rendering them more resistant, as has already been observed for the original PFK1atp PFK2 mutant (22).…”

Section: Fig 5 Heat Inactivation Of Pfk From Different Mutant Strainsmentioning

confidence: 62%

“…Yeast Pfk Activity-Previously, a pfk1 mutant could be isolated by its failure to grow on glucose as a sole carbon source in a strain already carrying a pfk2 defective allele (22). This mutant showed a phosphofructokinase activity insensitive to allosteric inhibition by ATP when crossed back into a PFK2 wild-type background.…”

Section: Sequencing Of the Point Mutation Leading To Atp-insensitivementioning

confidence: 99%

“…This mutant showed a phosphofructokinase activity insensitive to allosteric inhibition by ATP when crossed back into a PFK2 wild-type background. To localize the mutation, we used PCR to amplify the complete coding region of PFK1 with its flanking sequences from the genome of the mutant strain gTI-4C (22). Two fragments were obtained from the PCR product by digestion with appropriate restriction endonucleases and used for subcloning in conjunction with wild type flanking PFK1 sequences in a yeast multicopy vector ( Fig.…”

Section: Sequencing Of the Point Mutation Leading To Atp-insensitivementioning

confidence: 99%

“…Activities measured in 50 mM potassium phosphate buffer, pH 7.0, with 10 mM MgCl 2 at 1 mM ATP, 2.0 mM fructose 6-phosphate, and 10 M fructose 2,6-bisphosphate were set at 100% at time 0. the subunits (21). Here, we localized a mutation obtained in the PFK1-encoded ␣-subunit by classical genetic screening (22) as abolishing ATP inhibition also in the C-terminal half of the subunit (P728L). The effect of this mutation was confirmed by the results of site-directed in vitro mutagenesis of either PFK1 or PFK2 or both.…”

Section: Fig 5 Heat Inactivation Of Pfk From Different Mutant Strainsmentioning

confidence: 99%

“…For this purpose, we took advantage of a previous work, in which mutants insensitive to ATP inhibition were obtained in a genetic screen and shown to be caused by a defect in the ␣-subunits encoded by PFK1 (Ref. 22; note again that these authors designate this gene encoding the larger Pfk subunits as PFK2). After identifying the altered base, we introduced the equivalent exchange by in vitro mutagenesis in the yeast PFK1 and PFK2 genes.…”

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confidence: 99%

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Single Point Mutations in Either Gene Encoding the Subunits of the Heterooctameric Yeast Phosphofructokinase Abolish Allosteric Inhibition by ATP

Rodicio¹,

Strauß²,

Heinisch³

2000

Journal of Biological Chemistry

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Yeast phosphofructokinase is a heterooctameric enzyme subject to a complex allosteric regulation. A mutation in the PFK1 gene, encoding the larger ␣-subunits, rendering the enzyme insensitive to allosteric inhibition by ATP was found to be caused by an exchange of proline 728 for a leucine residue. By in vitro mutagenesis, we introduced this mutation in either PFK1 or PFK2 and found that the exchange in either subunit drastically reduced the sensitivity of the holoenzyme to ATP inhibition. This was accompanied by a lack of allosteric activation by AMP, fructose 2,6-bisphosphate, or ammonium and an increased resistance to heat inactivation. Yeast cells carrying either one mutation or both in conjunction did not display a strong phenotype when grown on fermentable carbon sources and did not show any significant changes in intermediary metabolites. Growth on non-fermentable carbon sources was clearly impaired. The strain carrying both mutant alleles was more sensitive to Congo Red than the wildtype strain or the single mutants indicating differences in cell wall composition. In addition, we found single pfk null mutants to be less viable than wild type at different storage temperatures and a pfk2 null mutant to be temperature-sensitive for growth at 37°C. The latter mutant was shown to be respiration-dependent for growth on glucose.

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Section: Fig 5 Heat Inactivation Of Pfk From Different Mutant Strainsmentioning

confidence: 62%

Section: Sequencing Of the Point Mutation Leading To Atp-insensitivementioning

confidence: 99%

Section: Sequencing Of the Point Mutation Leading To Atp-insensitivementioning

confidence: 99%

Section: Fig 5 Heat Inactivation Of Pfk From Different Mutant Strainsmentioning

confidence: 99%

mentioning

confidence: 99%

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Single Point Mutations in Either Gene Encoding the Subunits of the Heterooctameric Yeast Phosphofructokinase Abolish Allosteric Inhibition by ATP

Rodicio¹,

Strauß²,

Heinisch³

2000

Journal of Biological Chemistry

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Assembly of phosphofructokinase-1 fromSaccharomyces cerevisiae in extracts of single-deletion mutants

Klinder¹,

Kirchberger²,

Edelmann³

et al. 1998

Yeast

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Phosphofructokinase‐1 from Saccharomyces cerevisiae is an octameric enzyme comprising two non‐identical subunits, α and β, which are encoded by the unlinked genes PFK1 and PFK2. In this paper, assembly and reactivation of the enzyme have been studied in cell‐free extracts of single‐deletion mutants. In contrast to the previously described lack of phosphofructokinase‐1 activity in cell‐free extracts of these mutants, we could measure a temporary enzyme activity immediately after lysis of protoplasts. This result supports the assumption that each of the subunits forms an enzyme structure which is active in vivo but not stable after cell disruption. Upon mixing of separately prepared cell‐free extracts of both deletion mutants very low activity could be measured. About 40% of the wild‐type activity was regained when both mutants were mixed prior to disruption. The reactivation rate could be slightly increased by addition of ATP and fructose 6‐phosphate and was found to be a function of the growth state, particularly of the β‐subunit‐carrying cells. The individual subunits did not interact with Cibacron Blue F3G‐A, a biomimetic ligand of phosphofructokinase‐1. After reassembly of both subunits in vitro a strong affinity of the reconstituted phosphofructokinase‐1 to the dye‐ligand was observed. The inability of the subunits to reconstitute under certain conditions seems to result from alterations of the intracellular environment following disruption. These changes give rise to induce an unproductive side reaction like self‐aggregation of the subunits. Because reconstitution of phosphofructokinase‐1 from S. cerevisiae behaves in a similar way to that of hemoglobin and luciferase, we would speculate a general mechanism for assembly of oligomeric proteins in vivo. © 1998 John Wiley & Sons, Ltd.

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Fructose 2,6‐Bisphosphate

Schaftingen

1987

Advances in Enzymology - And Related Areas of Molecular Biology

171

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Genetic evidence for distinct catalytic and regulatory subunits in yeast phosphofructokinase

Cited by 28 publications

References 9 publications

Single Point Mutations in Either Gene Encoding the Subunits of the Heterooctameric Yeast Phosphofructokinase Abolish Allosteric Inhibition by ATP

Single Point Mutations in Either Gene Encoding the Subunits of the Heterooctameric Yeast Phosphofructokinase Abolish Allosteric Inhibition by ATP

Assembly of phosphofructokinase-1 fromSaccharomyces cerevisiae in extracts of single-deletion mutants

Fructose 2,6‐Bisphosphate

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