GTP-BLOT ANALYSIS OF SMALL GTP-BINDING PROTEINS - THE C-TERMINUS IS INVOLVED IN RENATURATION OF BLOTTED PROTEINS

Recombinant c-Ha-ras, ralA and rap2, but not raplA or raplB proteins r etained their ability to bind [alpha-P-32]GTP after SDS/PAGE and trans fer to nitrocellulose. Recombinant c-Ha-ras missing the C-terminal 23 amino acid residues failed to bind [alpha-P-32]GTP after the blot, and the ability of recombinant ralA missing the C-terminal 28 amino acid residues to bind [alpha-P-32]GTP was decreased manyfold. The presence of nonionic detergents of the polyoxyethylene type such as Tween 20, T riton X-100, Nonidet P40 or Lubrol PX in the incubation buffer was nec essary to induce renaturation of blotted recombinant c-Ha-ras protein, whereas other types of detergents were ineffective. We propose that d etergents of the polyoxyethylene type induce the refolding of some typ es of blotted small GTP-binding proteins and that the C-terminus is in volved in the refolding process. Membranes from NIH3T3 fibroblasts ove rexpressing c-Ha-ras protein showed much weaker binding of [alpha-P-32 ]GTP as expected from the level of ras immunoreactivity. Treatment of fibroblasts with lovastatin, an inhibitor of hydroxymethylglutaryl-coe nzyme A reductase, caused the accumulation of the unfarnesylated form of c-Ha-ras in the cytosol. Examination of [alpha-P-32]GTP-binding and immunoreactivity for cytosolic and membrane-bound c-Ha-ras revealed t hat binding of [alpha(-32)P]GTP to unprocessed c-Ha-ras was increased about threefold compared to the same amount of processed c-Ha-ras. Our results demonstrate that detection and quantification of small GTP-bi nding proteins in eukaryotic cells by GTP-blot analysis is hampered by the fact that these proteins differ strongly in their ability to rena ture after blotting to nitrocellulose.