Domain structure, GTP-hydrolyzing activity and 7S RNA binding of Acidianusambivalens Ffh-homologous protein suggest an SRP-like complex in archaea

In this study we provide, for the first time, experimental evidence that a protein homologous to bacterial Ffh is part of an SRP-like ribonucleoprotei n complex in hyperthermophilic archaea. The gene encoding the Ffh homologue in the hyperthermophilic archaeote Acidianus ambivalens has been cloned an d sequenced. Recombinant Ffh protein was expressed in E. coli and subjected to biochemical and functional studies. A. ambivalens Ffh encodes a 50.4-kDa protein that is structured by three di stinct regions: the N-terminal hydrophilic N-region (N), the GTP/GDP-bindin g domain (G) and a C-terminal located C-domain (C). The A. ambivalens Ffh s equence shares 44-46% sequence similarity with Ffh of methanogenic archaea, 34-36% similarity with eukaryal SRP54 and 30-34% similarity with bacterial Ffh. A polyclonal antiserum raised against the first two domains of A. amb ivalens Ffh reacts specifically with a single protein (apparent molecular m ass: 46 kDa, termed p46) present in cytosolic and in plasmamembrane cell fr actions of A. ambivalens. Recombinant Ffh has a melting point of t(m) = 89 degrees C. Its intrinsic GTPase activity obviously depends on neutral pH an d low ionic strength with a preference for chloride and acetate salts. High est rates of GTP hydrolysis have been achieved at 81 degrees C in presence of 0.1-1 mM Mg2+. GTP hydrolysis is significantly inhibited by high glycero l concentrations, and the GTP hydrolysis rate also markedly decreases by ad dition of detergents. The K-m for GTP is 13.7 mu M at 70 degrees C and GTP hydrolysis is strongly inhibited by GDP (K-i = 8 mu M). A. ambivalens Ffh, which includes an RNA-binding motif in the C-terminal domain, is shown to b ind specifically to 7S RNA of the related crenarchaeote Sulfolobus solfatar icus. Comparative sequence analysis reveals the presence of typical signal sequen ces in plasma membrane as well as extracellular proteins of hyperthermophil ic crenarchaea which strongly supposes recognition events by an Ffh contain ing SRP-like particle in these organisms.