Mutations in the regulatory subunit of soluble phosphofructokinase from yeast

European Journal of Biochemistry

1991

We describe here the genetic and biochemical analyses of two classes of mutations in the soluble phosphofructokinase (PFK I) of Saccharomyces cerevisiae : those leading to the loss of activity and those giving rise to a kinetically altered enzyme. Complementation and allele-testing between these two classes of mutants show that loss of enzyme activity in vitro can come about not only by mutations in the catalytic subunit but also in the regulatory subunit. Also, a mutation in the catalytic subunit can give rise to an enzyme altered in its kinetic properties in a manner phenomenologically similar to that caused by a mutation in the regulatory subunit.The results of the complementation studies in diploids suggest that, in spite of their distinct functions, both the subunits are essential for activity to be detected in vitro. This is confirmed by the reconstitution of an active PFK I enzyme by mixing cell-free extracts of two complementing parents, each of which lacks the enzyme activity. PFK activity appears in the mixture, reaching a maximum value of 60-100% of that of the diploid in 15-30 min at 24°C. Unlike the catalytic subunit which exists in various multimeric states in cell-free extracts of the mutant bearing only this subunit, the regulatory subunit exists largely as a monomer in a mutant devoid of the catalytic subunit. The reconstituted enzyme, however, is indistinguishable from that of the wild type, as analysed by sedimentation studies and Western blot analysis, demonstrating that only the heteromeric complex of the two subunits is active, while neither of the individual subunits displays activity in vitro.The yeast, Saccharomyces cerevisiae, possesses two distinct phosphofructokinases: a soluble form (PFK I) and a particulate form (PFK 11) (for a recent review see [I]). The presence of either of these isozymes is sufficient for growth on glucose [2]. The soluble phosphofructokinase is an octamer with a native molecular mass of about 800 kDa and is composed of four CI subunits and four p subunits [l]. Analyses of mutants (reviewed in [3]) and molecular cloning of the genes involved [4] have shown that PFKI and PFK2 are the structural determinants of the p and CI subunits, respectively (for nomenclature see Materials and Methods). The gene PFK2 is known to be shared between the soluble and the particulate forms of phosphofructokinase [5].

Section: Characterisation Qf Thc Mutunt Dfy70mentioning

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Section: Strains Used and Nomenclaturementioning

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Section: Characterisation Qf Thc Mutunt Dfy70mentioning

confidence: 99%

“…other hand, invariably gave rise to only an alteration of the regulatory properties of the enzyme [7].…”

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confidence: 99%

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confidence: 99%

See 3 more Smart Citations

Complementation of soluble phosphofructokinase activity in yeast mutants

Gayatri

European Journal of Biochemistry

1991

Mutations in phosphofructokinases alter the control characteristics of glycolysis in vivo in Saccharomyces cerevisiae

Lloyd

James

1992

Yeast

Ethanol and CO2 production from glucose by non-proliferating suspensions of aerobically-grown, glucose-derepressed wild-type Saccharomyces cerevisiae is inhibited by O2; monitoring by mass spectrometry provides a direct method for measurement of the Pasteur effect. Under aerobic conditions, that part of the CO2 evolved equivalent to the O2 consumed, is produced by respiration: subtraction of this respiratory CO2 from the total gives CO2 produced by aerobic glycolysis. Pasteur quotients (anaerobic CO2/aerobic glycolytic CO2) were within the range 1.2 to 3.0. The Pasteur effect was not observed in the presence of carbonyl cyanide m-chlorophenylhydrazone, an uncoupler of mitochondrial energy metabolism, or in a rho degree cytoplasmic petite mutant. A 'non-allosteric' mutant with an altered regulatory subunit of phosphofructokinase showed no Pasteur effect. Strains bearing a nonsense mutation pfk1 in the catalytic subunit of soluble phosphofructokinase (PFKI) also showed no Pasteur effect; the residual fermentative activity of this strain was dependent on PFKII, the particulate phosphofructokinase. A double mutant lacking both PFKI and glucose-6-phosphate dehydrogenase showed similar characteristics to those of the single pfk1 mutant; this indicates that the hexose monophosphate shunt is not acting to bypass the phosphofructokinase block. A 'hyper-allosteric' mutant altered in the regulatory subunit encoded by the gene PFK2 showed characteristics of glucose fermentation and ethanol oxidation very similar to those of wild-type organisms. These results indicate that either of the two phosphofructokinases can carry out glycolysis.

Analysis of PFK3—A gene involved in particulate phosphofructokinase synthesis reveals additional functions of TPS2 in Saccharomyces cerevisiae

Sur

Lobo

1994

Yeast

The pfk3 mutation of Saccharomyces cerevisiae causes glucose-negativity in a pfk1 genetic background, the mutant is temperature-sensitive for growth and homozygous diploids do not sporulate. It fails to accumulate trehalose, and has an altered glycogen accumulation profile under glucose-starvation conditions. pfk3-6, one of the alleles of pfk3, has an altered morphology, forming long chain-like structures at 36 degrees C. The PFK3 gene was cloned by complementation of the mutant phenotypes. Integrative transformation demonstrated that the complementing fragment encoded the authentic PFK3 gene. The disruption of the gene does not affect viability. Like the EMS-induced pfk3 mutant, the disruptants are temperature-sensitive and in a pfk1 genetic background are also glucose-negative. The PFK3 transcript is induced by heat-shock. Partial DNA sequence shows that PFK3 is identical to TPS2 (De Virgilio et al., 1993). We demonstrate that, apart from being a structural determinant of trehalose 6-phosphate phosphatase, PFK3 (TPS2) is required for PFKII synthesis and normal regulation of S. cerevisiae response to nutrient and thermal stresses.