GTP‐blot Analysis of Small GTP‐binding Proteins

Abstract

Recombinant c-Ha-ras, ralA and rap2, but not raplA or raplB proteins retained their ability to bind [a-32P]GTP after SDSPAGE and transfer to nitrocellulose. Recombinant c-Ha-ras missing the C-terminal 23 amino acid residues failed to bind [a-32P]GTP after the blot, and the ability of recombinant ralA missing the C-terminal 28 amino acid residues to bind [a-32P]GTP was decreased manyfold. The presence of nonionic detergents of the polyoxyethylene type such as Tween 20, Triton X-100, Nonidet P40 or Lubrol PX in the incubation buffer was necessary to induce renaturation of blotted recombinant c-Ha-ras protein, whereas other types of detergents were ineffective. We propose that detergents of the polyoxyethylene type induce the refolding of some types of blotted small GTP-binding proteins and that the C-terminus is involved in the refolding process.Membranes from NIH3T3 fibroblasts overexpressing c-Ha-ras protein showed much weaker binding of [a-32P]GTP as expected from the level of ras immunoreactivity. Treatment of fibroblasts with lovastatin, an inhibitor of hydroxymethylglutaryl-coenzyme A reductase, caused the accumulation of the unfarnesylated form of c-Ha-ras in the cytosol. Examination of [a-32P]GTP-binding and immunoreactivity for cytosolic and membrane-bound c-Ha-ras revealed that binding of [CX-~~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-binding proteins in eukaryotic cells by GTP-blot analysis is hampered by the fact that these proteins differ strongly in their ability to renature after blotting to nitrocellulose.Guanine nucleotide binding regulatory proteins such as ras proteins and a-subunits of heterotrimeric GTP-binding proteins exert their function by cycling between an inactive, GDP-bound, and an active, Members of the mammalian ras protooncogene group, c-Ha-ras, c-Ki-ras and N-ras each encode small guanine nucleotide binding proteins of 21 kDa which are associated with the inner side of the plasma membrane. Mammalian ras genes acquire transformation-inducing properties by single point mutations within their coding sequences [l, 31. Cloning and sequencing of cDNAs has revealed that a large and diverse family of ras-related small GTP-binding proteins exist with calculated molecular masses between 21 -25 kDa. Groups belonging to this family include the rap, ral, rho, rac and rab proteins [4,5]. Members of the family of small GTPbinding proteins show an extraordinary conservation of sequences dedicated to guanine nucleotide binding.To crystallize the human c-Ha-ras protooncogene product, John et al. [6] expressed the protein with a truncated Cterminus of 23 amino acids. The resulting c-Ha-ras (amino acid residues 1-166) was crystallized as a complex with the slowly hydrolyzing GTP analogue guanosine 5'- [P,yimido]triphosphate (GppNHp) and analyzed by X-ray crys-

Disease-Associated Polyglutamine Stretches in Monomeric Huntingtin Adopt a Compact Structure

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Molecular cloning, bacterial expression and properties of Rab31and Rab32

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Biochemical, molecular and behavioral phenotypes of Rab3A mutations in the mouse

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