Specific immunodetection of G-proteins in plant cell membranes

Abstract

At present, much is known concerning the biochemistry of heterotrimeric G-proteins in animal cells [l] and lower eukaryotic cells, e.g. Saccharomyces cerevisiae 121 and many examples of Ga, GP and G8 subunits have been identified by cloning and sequencing studies. The a-subunits, for which most examples have been discovered, show a number of highly conserved regions, which are concerned with GTP binding and hydrolysis and other less conserved regions which are responsible for coupling to receptor and effector molecules respectively. Synthetic peptides which correspond in sequence to these regions have been used to generate antisera which are panspecific to Ga subunits (Gac antisera) or specific to the various GQ subtypes, e.g. Gia, Gsa [3,41. In contrast, no G-proteins have yet been isolated from plant cells. However, for some time there has been strong circumstantial evidence indicating their existence and involvement in plant signal transducing pathways. Non-hydrolysable GTP analogues were shown to modulate inositol phospholipid turnover in Acer pseudoplatinus cells [5] and it has been suggested that auxins initiate G-protein coupled events in plant cells [6]. In addition, microsomal membrane and highly resolved plasmalemma preparations from various species including Arabidopsis thaliana [7] and Cucurbita pep0 [8] have been shown to bind GTPVS with high affinity (Kb= 10-SM) and to possess polypeptides of M= 31-37 kDa that crossreacted with Gac antisera. Finally, genes for a Ga subunit and a GP subunit homologs have been cloned and sequenced in A . thaliana [9] and Chlamydamonas rheinhardtii [lo] respectively. In the present communication we report the specific immunodetection of Ga subunits in plant cell membrane preparations and comment on the use of antipeptide antibodies. A number of antisera and affinity purified antibodies directed against the Gac, Gia, Goa and GP sequences [3,4] were used to probe immunoblots of microsomal fraction and plasmalemma membrane proteins prepared from higher plants. Typical positive data from these studies are shown in Fig.1. Microsomal membranes from A. thaliana . and Pisum sativum were shown to possess polypeptides of Mr 37 kDa (tracks 1,3) which were labelled with anti-Gia whilst a 41 kDa protein in Zed mays microsomal membranes was labelled with antiGac antiserum (track 5). Preabsorption of the antisera with the relevant synthetic peptides (tracks 2,4,6) was shown to abolish the immune detection. In addition, the anti-Gac serum was also shown to detect the a-subunit of an authentic G-protein, bovine transducin (Gta tracks 7,8). In addition to the immunological data presented, specific high affinity binding of the GTP analogue, GTPXS was also found in purified plasmalemma from the above species (not shown). The ability of the synthetic peptides to block the immune detection is important since all of the antibodies used displayed crossreactivity of various strengths and against polypeptides of Mr's between 17 and 90 kDa (data not shown). Some of the proteins detected were indeed in the Mr range expected for Ga subunits (37-50 kDa) and GP subunits (35-36 kDa). However, with the exception of the polypeptides indicated in Fig.1, in these cases the immune detection could not be abolished and the crossreactivity must indicate a non-specific labelling or the presence of antibodies that are directed towards epitopes other than those represented by the complete synthetic peptide sequence. 34