Presence of GTP-Binding Proteins in the Plasma Membrane of the Phycomyces Sporangiophore

Abstract

Ashktorab, H., and Cohen, R. J. 1994. Presence of GTP-binding proteins in the plasma membrane of the Phycomyces sporangiophore. Experimental Mycology 18, 139-149. When a plasma membrane-enriched fraction isolated from the sporangiophore of the Zygomycete Phycomyces blakesleeanus was subject to immunoblotting, two polypeptide bands reacted with an antibody directed to a conserved sequence of the ∝ subunit of G-proteins; their apparent molecular masses were 40 and 51 kDa. Upon treating the plasma membrane preparation with cholera toxin, bands at 40 and 51 kDa appeared to be ADP-ribosylated but no band appeared with pertussis toxin incubation. Apparent dissociation constants for the binding of GTPγS were determined for plasma membrane from Phycomyces sporangiophore grown in the light (K_D = 39 ± 16 nM) (±SD) and in the dark (K_D = 11 ± 6 nM). GTP served as a strong competitor for binding as did GDP, although somewhat less well, while ATP competed considerably more weakly. Northern analysis of sporangiophore mRNA displayed two bands hybridizing to the Gα2 probes coding for a G_α subunit from Dictyostelium discoideum. Furthermore, Western blotting of plasma membrane revealed several bands containing polypeptides with presumptive covalently attached immunoreactive flavins. (The prevailing evidence from the action spectra of Phycomyces is that the photoreceptor is a flavoprotein residing in the plasma membrane.) Immunoblotting also detected a H⁺ ATPase similar to the plasma membrane enzyme of yeast, corroborating our isolation of plasma membrane and suggesting another possible player in the signal responses of Phycomyces . This is apparently the first evidence for a G-protein in this class of eukaryotes. G-proteins may serve a role in the flavoprotein-mediated phototransduction system of P. blakesleeanus.