Abstract
The small GTPase Ran (encoded by GSP1 and GSP2 in yeast) plays a central role in nucleocytoplasmic transport. GSP1 and GSP2 were tagged with protein A and functionally expressed in a gsp1 null mutant. After affinity purification of protein A-tagged Gsp1p or Gsp2p by IgG-Sepharose chromatography, known karyopherin beta transport receptors (e.g. Kap121p and Kap123p) and a novel member of this protein family, Pdr6p, were found to be associated with yeast Ran. Subsequent tagging of Pdr6p with green fluorescent protein revealed association with the nuclear pore complexes in vivo. Thus, functional tagging of yeast Ran allowed the study of its in vivo distribution and interaction with known and novel Ran-binding proteins.
Highlights
Ran belongs to the Ras superfamily of small guanine nucleotide-binding proteins and is highly conserved within the eukaryotic kingdom
The main accessory proteins, which assist Ran during transport, are NTF2, a RanGDP-binding protein at the nuclear envelope required for nuclear import of Ran [9], RanGAP1 in (Rna1p) in S. cerevisiae, a cytoplasmically located Ran-GTPase activating protein, and RanGEF (Prp20p in S. cerevisiae), a nuclear guanine nucleotide exchange protein, as catalysts of the Ran GTPase cycle
Yeast Ran (Gsp1p and Gsp2p) Tagged with ProtA Is Functional—The protein A (ProtA) tagging strategy was successfully applied in the past to affinity purify in a single step a protein of interest from yeast and identify its interacting partners [26]
Summary
Ran belongs to the Ras superfamily of small guanine nucleotide-binding proteins and is highly conserved within the eukaryotic kingdom (for review, see Refs. 1, 2). To study the dynamic intracellular localization of Ran and its interaction with partner proteins in the living cell, Gsp1p and Gsp2p were tagged with GFP1 and ProtA, respectively. These tools allowed us to study the in vivo localization of Gsp1p and to identify a novel member of the karyopherin  transport receptor family called Pdr6p.
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