Sugar uptake by protoplasts of the ectomycorrhizal fungus, Amanita muse aria (L. ex fr.) Hooker

Abstract

Protoplasts have been isolated from the ectomycorrhizal fungus, Amanita muscaria, for the first time. Yields were about 108 protoplasts per g fr. wt of mycelial suspension cultures treated with a combination of Novozym 234, chitinase and cellulase Onozuka R-10, and osmotically supplemented by 0-55 M sorbitol or 035 M KC1. Silicon oil filtration was used to investigate the uptake of metabolites by protoplasts using labelled compounds. About 66% of the initial protoplast population passed the oil layer after 50 s of centrifugation. Both glucose (as well as 3-O-methylglucose) and fructose were taken up by the protoplasts but not sucrose and mannitol. The Km for glucose (fructose) uptake was 1.25 mM (11.3), Vmax was 18 (30) pmoles (106 protoplasts min)-1 . Uptake of fructose was strongly inhibited by glucose but not vice versa; uptake of both was not inhibited by sucrose. The much higher affinity for glucose than for fructose suggests that glucose should be the main carbohydrate source for this fungus. Treatment of protoplasts with 10 μM antimycin A lowered cellular ATP/ADP ratios to well below 1 and significantly inhibited uptake of sugars.