The Physcomitrella patens GP 1 homologue is located at protonemal cell junctions

Abstract

This paper describes characteristics of GTP-binding proteins in the moss Physcomitrella patens, taking into account recent criticisms of artefacts arising in G-protein analyses. The binding of guanosine-5'-O-thiotriphosphate (GTPγS) to microsomal fraction membranes was shown, including controls demonstrating effective competition by GTP, but only partial competition by ATP, ADP and CTP. These controls distinguish GTP-binding by heterotrimeric G-proteins from that due to the nucleoside diphosphate kinase catalytic cycle. The GTPγS was bound with high affinity, with apparent K d of 16 nM and B max of 26.9 nmol mg -1 protein, which is in line with that observed for other systems. Immunoblot analysis of microsomal membrane fraction proteins probed with antibodies raised against recombinant Arabidopsis GPα1 protein revealed a strongly cross-reacting protein of 37 kDa and more weakly cross-reacting proteins of 45 kDa and 27 kDa. A similar analysis probing with anti-peptide antibodies directed against the N-terminal region of nucleoside diphosphate kinase (NDPK) revealed a strong band of 17 kDa. The molecular weights of the major bands for both Gpα1 and NDPK agree with size estimations for these proteins in other organisms and include controls shown to be essential for demonstrating specificity of the cross-reaction. Mosses develop from spores to produce a branched network of filaments called protonemata. Each protonema is a filament of single cells such that cellular communication is via the cross-wall. Immunofluorescence microscopy of intact Physcomitrella patens protonemal tissue exposed to FITC-labelled anti-GPα1 antibody, located the moss GPα1 protein homologue to the protonemal cross-walls.