(1,3)β-Glucan synthase activity of Neurospora crassa: identification of a substrate-binding protein

Abstract

(1,3)β-Glucan synthase activity from the filamentous Ascomyceet Neurospora crassa was purified 1300-fold to a specific activitiy of 14 000 nmol glucose incorporated/min per mg protein. Hyphae were disrupted and crude membrane fractions obtained by high-speed centrifugation. Membrane fractions with Tergitol NP-40 and a second high-speed particulate fraction was obtained. Enzyme activity was solubilized with (3-((3-cholamidopropyl)dimethylammonio)-1-propanesulfonate and octyl-β- d-glucoside from Tergitol-extracted membrane preprations. Solubilized enzyme activity was purified by product entrapment and recovered by low-speed centrifugation through a layer of sucrose. SDS-PAGE analysis revealed 2 proteins of 165 and 100 kDa as likely candidates for subunits of the (1,3)β0-glucan synthase enzyme complex. 5-Azido-[ 32P]-UDP-glucose was photo-crosslinked to UDP-glucose-binding proteins in each fraction of the purification procedure. Autoradiograms of SDS-PAGE gels revealed a single protein of 165 kDa enriching with enzyme activity and labeling with the substrate analog. Photoincorporation of 5-azido-[ 32P]UDP-glucose by the 165 kDa protein was competed by 0.25 mM UDP-glucose (80%) and TDP-glucose (65%) while ADP-glucose (27%), CDP-glucose (36%), and GDP-glucose (8%) where less effective. These results were similar to in vitro inhibition of enzyme activity by the same compounds. These data strongly suggest that the 165 kDa protein is a substrate-binding subunit of (1,3)β-glucan synthase.