Complementation of soluble phosphofructokinase activity in yeast mutants

Abstract

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].

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

“…The maximum reactivation rate of this reconstituted phosphofructokinase-1, which was shown to be a hetero-octamer of 800 kDa ( Figure 4B), was 35-40% compared to the wild-type activity if both exponentially grown cells were mixed in equal ratio. These findings partly agree with the results of Gayatri and Maitra (1991) who reconstituted phosphofructokinase-1 activity by mixing cell-free extracts of single point mutants. But in contrast to their results, in the experiments presented here very low activity could be detected when cell-free extracts of single-deletion mutants PFK1pfk2 ( ) and pfk1 PFK2 ( ) were prepared separately and mixed subsequently.…”

“…However, in cell-free extracts of all these mutants phosphofructokinase-1 activity was lacking. After complementation of single-or double-deletion mutants with the corresponding phosphofructokinase-1 gene(s) the extract activity was regained (Huse, 1988;Gayatri and Maitra, 1991). Therefore it is obvious that the individual subunit can function by itself in vivo but the assembly of both subunits is essential for recovery of enzyme activity in vitro.…”