Occurrence of GTP-binding proteins in the ascomycete Saccobolus platensis

Abstract

We have previously shown that in the ascomycete Saccobolus platensis the presence of cyclic AMP can overcome the requirement for light to produce apothecia. With the aim to gain insight into the phototransduction mechanism the existence of G protein-like molecules was investigated. By using the GTP analog [³⁵S]GTP-γS 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 effectively competed by GTP and GDPPS and to a lesser extent by ATP. We also demonstrated by SDS-PAGE the existence of proteins of 48, 41, 26, and 15 kDa that are substrates of the cholera toxin-dependent ADP-ribosylation reaction. Proteins resolved by SDS-PAGE were identified by Western blotting with antisera specific for vertebrate G protein subunits; the antibody anti-α_common revealed two main bands at 48 and 41 kDa; the AS/7 antibody (anti-α_t, −α₁, and −a_i2) led to the identification of one band at 41 kDa; the antibody anti-β subunit revealed a polipeptide band at around 37 kDa. The antibody anti-α_common also abolished the binding of [³⁵S]GTP-γS to crude membranes. The results presented in this paper provide evidence for the existence in S. platensis of proteins that proved to have a behavior similar to that of mammalian G proteins.