Purification, characterization and exit routes of two acid phosphatases secreted by Botrytis cinerea

Abstract

The two forms of acid phosphatase secreted by Botrytis cinerea were purified, characterized and found to possess fundamentally different properties. The phosphate-repressible acid phosphatase was a monomer of clearly defined molecular weight (55–56 kDa), with a polypeptide component of 43 kDa. The enzyme was secreted through the hyphal, apex and accumulated in the culture fluid. Its high affinity for a wide range of substrates rendered it suitable for a putative role as an extracellular phosphate-scavenging enzyme. Secretion of a constitutive acid phosphatase occurred at low levels in phosphate-enriched culture; at phosphate starvation, the activity of this enzyme was enhanced ten-fold in the culture fluid and two-fold in the cell wall. The constitutive enzyme had strikingly different properties from those of the repressible form. It consisted of a 93 kDa polypeptide which was heavily glycosylated such that the secreted protein displayed size heterogeneity of 140–200 kDa by SDS-polyacrylamide gel electrophoresis and 320–450 kDa by native Sephacryl S-300 chromatography. Irrespective of the phosphate status, most of the secreted constitutive enzyme activity was retained by the cell wall. These results were consistent with ultrastructural histochemical observations which suggested that this enzyme form was secreted by a novel pathway via the fusion of vacuoles with the plasma membrane in mature hyphal segments.