Effect of fusicoccin on protoplasts isolated from suspension-cultured sugar beet cells: Induction of H+ transport and binding to receptors

The effect of fusicoccin (FC) on proton transport in suspension-cultured sugar beet cells was investigated. The stimulation of proton excretion from cells by FC was most pronounced during the logarithmic phase of culture growth. Protoplasts derived from these cells remained sensitive to FC. Cytoplasm alkalinization in protoplasts and the acidification of the external medium induced by FC in protoplast and cell suspensions were studied as functions of FC concentration. Acidification of the external medium by suspension-cultured cells in the presence of FC proceeded linearly with time (within a 120-min lag period), and the rate of acidification rose with FC concentration. In protoplast suspensions, the value of external pH reached a steady-state level 80 min after FC addition, this pH value being a function of FC concentration. The value of cytoplasmic pH increased up to 0.3 units under the action of FC. The effective concentration of FC inducing half-maximum changes in the three parameters measured was about 0.1 mu M. In parallel experiments, the kinetics and concentration dependence of [H-3]-dihydro-FC binding by protoplasts were examined, and the decrease in the number of FC-binding sites was monitored after protoplast disruption by osmotic shock. It is presumed that [3H]-dihydro-FC cannot penetrate protoplasts and becomes bound at the outer surface of the cell membrane with FC-binding sites of two types having dissociation constants of 1 nM and 0.2 mu M After disintegration of the protoplasts, low-affinity sites were rapidly inactivated, whereas slower inactivation of high-affinity binding sites was observed. The results are discussed in relation to a possible role of high- and low-affinity binding sites in the mechanism of FC action.