α-Factor inhibition of the rate of cell passage through the “start” step of cell division in Saccharomyces cerevisiae yeast: Estimation of the division delay per α-factor·receptor complex

Abstract

A highly sensitive, kinetically unambiguous assay for α-factor-induced delay of cell passage through the “start” step of cell division in yeast is presented. The assay employs perfusion with periodic microscopy to monitor the bud emergence kinetics on the 20% of cells within an exponentially growing population which exist prior to the α-factor execution point of start. The t 1 2 for cell passage through start by this population of cells is 31 min in the absence of α-factor. The inhibition constant, K I , represents the α-factor concentration which produces a 50% inhibition of this rate and is equal to 2×10 −10 M. A second assay for maximal cell division arrest by α-factor on whole populations of cells is presented. This assay shows a maximum cell division arrest time of 125±5 h at saturating α-factor, and a K 50 (that is, an α-factor concentration which produces a half-maximal response) of 2.5×10 −8 M. Both assays were performed in the effective absence of α-factor inactivation. Values of the dissociation constant K D and total number of receptors per cell which specifically mediate cell division arrest or delay were estimated to be 2.5×10 −8 M and 10 4, respectively. These estimates, along with the quantitative dose-response data for division arrest which are presented here, are consistent with each receptor·α-factor complex which is present on the cell at equilibrium producing a 43±10 s delay of cell passage through start. Surprisingly, this number is constant within twofold over the entire range of cellular division arrest responses to α-factor, that is, from a 1.9-fold inhibition of the rate of cell passage through start at 0.17 n M α-factor to a 125±5 h maximum arrest at saturating α-factor concentrations of >170 n M. The possible significance of this observation toward the mechanism of α-factor-induced cell division arrest is discussed.