The small GTP-binding protein Rho is expressed differentially during sporegermination of Phycomyces blakesleeanus

Evidence has been obtained for the presence of the small 22 kDa GTP-binding Rho protein in dormant spores of Phycomyces blakesleeanus. Immunoblotting with a polyclonal antibody against RhoA revealed a soluble and membrane-ass ociated 22 kDa protein. When [P-32]ADP-ribosylated by Clostridium botulinum C3 exotoxin the protein had a pi of 5.7, a value similar to that reported for other RhoA proteins. The 22 kDa protein was expressed at all stages of growth investigated, but radiolabelling of the [P-32]ADP-ribosylated protei n increased when tube-formation occurred and decreased as the hyphae branch ed. Localization of RhoA during spore germination studied by immunofluoresc ence microscopy revealed the presence of RhoA in the cell membrane of the s pore. When the spore started to swell, RhoA was observed as patches in the cell membrane which become concentrated in the neck region of the site of t he protuberation tube, but this protein was never observed at the point of growth of the hyphal tip. The above results suggest that RhoA associated wi th one or more membrane proteins could participate in the molecular mechani sm involved in maintaining cell integrity during the extrusion of the germ- tube of P. blakesleeanus.