Fig. 7.

PcCdc42 protein binds the downstream PcSte20p kinase in the presence of GTP. A: V5-tagged PcCdc42 protein was heterologously expressed in yeast and allowed to interact with Pneumocystis protein extract. PcCdc42 bound complexes were immunoprecipitated with anti-V5 agarose. Precipitated proteins were separated by SDS-PAGE and transferred, and the membranes were cut in half. The presence of PcCdc42 and PcSte20 proteins was detected by Western blotting with synthetic peptide antibodies recognizing each of these proteins. B: to specifically assess the role of GTP in these interactions, both PcCdc42 and PcSte20 were expressed in E. coli and purified. Binding was assessed following preloading (or no preloading) the PcCdc42 with GTP, complexes were immunoprecipitated with PcSte20, and the presence of both PcCdc42 and PcSte20 was assessed by immunoblotting specifically for each of these molecules. C: finally, to determine whether PcCdc42 protein can interact equally with PcSte20 kinase derived from cysts and trophic forms, total protein from isolated cyst and trophic forms was extracted, and equal amounts of protein were immunoprecipitated with an anti-PcSte20-specific antibody, separated by SDS-PAGE, and transferred to nitrocellulose. The membrane was then Western blotted with a pan-specific cdc42 antibody revealing that PcCdc42 found in cysts and trophic forms interact equally with PcSte20 in these separate Pneumocystis populations.