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Journal of Experimental Botany, Vol 49, 1113-1118, Copyright © 1998 by Oxford University Press

ARTICLES

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

J Hutton, C Knight and P Millner
School of Biology, University of Leeds, Leeds LS2 9JT, UK; School of Biochemistry and Molecular Biology, University of Leeds, Leeds LS2 9JT, UK; Corresponding author; e-mail: c.d.knight@leeds.ac.uk

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 (GTPS) 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 GTPS was bound with high affinity, with apparent Kd of 16 nM and Bmax 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 Gp1 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 GP1 and NDPK agree with the 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-GP1 antibody, located the moss GP1 protein homologue to the protonemal crosswalls.Keywords: G-proteins, GP1, Physcomitrella patens, moss, plant signal transduction.
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S. M. Assmann
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