OCCURRENCE OF GTP-BINDING PROTEINS IN THE ASCOMYCETE SACCOBOLUS-PLATENSIS

We have previously shown that in the ascomycete Saccobolus platensis t he presence of cyclic AMP can overcome the requirement for light to pr oduce apothecia. With the aim to gain insight into the phototransducti on mechanism the existence of G protein-like molecules was investigate d. By using the GTP analog [S-35]GTP gamma S we detected the existence of GTP-binding proteins; the dissociation constant for the binding of the nucleotide was calculated (K-d = 52 nM); the binding was effectiv ely competed by GTP and GDP beta S and to a lesser extent by ATP. We a lso demonstrated by SDS-PAGE the existence of proteins of 48, 41, 26, and 15 kDa that are substrates of the cholera toxin-dependent ADP-ribo sylation reaction. Proteins resolved by SDS-PAGE were identified by We stern blotting with antisera specific for vertebrate G protein subunit s; the antibody anti-alpha(common) revealed two main bands at 48 and 4 1 kDa; the AS/7 antibody (anti-alpha(t), -alpha(i1), and -alpha(i2)) l ed to the identification of one band at 41 kDa; the antibody anti-beta subunit revealed a polipeptide band at around 37 kDa. The antibody an ti-alpha(common) also abolished the binding of [S-35]GTP gamma S to cr ude membranes. The results presented in this paper provide evidence fo r the existence in S. platensis of proteins that proved to have a beha vior similar to that of mammalian G proteins. (C) 1994 Academic Press, Inc.