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

Abstract

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.

A single point mutation leads to an instability of the hetero-octameric structure of yeast phosphofructokinase

“…Different lines of evidence favour the hypothesis of the remaining subunit exerting a catalytic function in i o that escapes detection after disruption of the cells [13,19]. However, enzyme activity could be regained in itro under conditions where both subunits from single-deletion mutants could assemble to a stable octameric structure [28].…”

“…The poor Western-blot signal was at least partly due to high intracellular degradation and\or aggregation (accompanied by precipitation) of misfolded and non-assembled subunits. The latter view is supported by the observation that in single-deletion mutants the amount of precipitated phosphofructokinase was found to be significantly higher than in wild-type strains [28]. It is noteworthy, however, that the pellet of mutants in the present study did not contain significant amounts of the mutated subunit that was lacking in the extract (results not shown).…”

“…These phenotypes indicate that not only the formation of the hetero-octameric structure in conjunction with the corresponding wild-type subunit was impaired, but also the correct folding of each subunit per se. Different lines of evidence indicate that each wild-type subunit can serve a catalytic function in i o which cannot be detected by assays in itro [19,28]. This function is also abolished in the mutant alleles, where Asp or Lys replaces the Ala residue on either subunit.…”

6‐phosphofructokinase from Pichia pastoris: purification, kinetic and molecular characterization of the enzyme

“…in the cell-free extract. Single deletion mutants of S. cerevisiae carrying only one type of the two genes can apparently also generate Pfk activity in vivo, since they are able to grow on glucose (Klinder et al, 1998). However, the enzyme was found catalytically active only in the intact cell.…”

“…However, the enzyme was found catalytically active only in the intact cell. After disruption of the cells, the enzyme inactivates rapidly and forms aggregates, as observed in extracts of single deletion mutants (Klinder et al, 1998;Arvanitidis and Heinisch, 1994).…”