ASSEMBLY OF PHOSPHOFRUCTOKINASE-1 FROM SACCHAROMYCES-CEREVISIAE IN EXTRACTS OF SINGLE-DELETION MUTANTS

Phosphofructokinase-1 from Saccharomyces cerevisiae is an octameric en zyme comprising two non-identical subunits, alpha and beta, which are encoded by the unlinked genes PFK1 and PFK2. In this paper, assembly a nd reactivation of the enzyme have been studied in cell-free extracts of single-deletion mutants. In contrast to the previously described la ck of phosphofructokinase-1 activity in cell-free extracts of these mu tants, 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 no t stable after cell disruption. Upon mixing of separately prepared cel l-free extracts of both deletion mutants very low activity could be me asured. About 40% of the wild-type activity was regained when both mut ants were mixed prior to disruption. The reactivation rate could be sl ightly increased by addition of ATP and fructose 6-phosphate and was f ound to be a function of the growth state, particularly of the beta-su bunit-carrying cells. The individual subunits did not interact with Ci bacron Blue F3G-A, a biomimetic ligand of phosphofructokinase-1. After reassembly of both subunits in vitro a strong affinity of the reconst ituted phosphofructokinase-1 to the dye-ligand was observed. The inabi lity of the subunits to reconstitute under certain conditions seems to result from alterations of the intracellular environment following di sruption. These changes give rise to induce an unproductive side react ion like self-aggregation of the subunits. Because reconstitution of p hosphofructokinase-1 from S. cerevisiae behaves in a similar way to th at of hemoglobin and luciferase, we would speculate a general mechanis m for assembly of oligomeric proteins in vivo. (C) 1998 John Wiley & S ons, Ltd.