Purification of protein A-tagged yeast ran reveals association with a novel karyopherin beta family member, Pdr6p.
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.
Lau
D
D
Biochemie-Zentrum Heidelberg, Im Neuenheimer Feld 328, D-69120 Heidelberg, Germany.
Künzler
M
M
Braunwarth
A
A
Hellmuth
K
K
Podtelejnikov
A
A
Mann
M
M
Hurt
E
E
D002451:Cell Compartmentation; D049452:Green Fluorescent Proteins; D008164:Luminescent Proteins / Q000235:genetics; D020559:Monomeric GTP-Binding Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D009685:Nuclear Envelope / Q000737:chemistry*; D009687:Nuclear Proteins / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D011485:Protein Binding; D029701:Saccharomyces cerevisiae Proteins; D028961:beta Karyopherins; D020931:ran GTP-Binding Protein / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism
C080324:GSP1 protein, S cerevisiae; D008164:Luminescent Proteins; D009687:Nuclear Proteins; D029701:Saccharomyces cerevisiae Proteins; D028961:beta Karyopherins; D049452:Green Fluorescent Proteins; C080325:GSP2 protein, S cerevisiae; D020559:Monomeric GTP-Binding Proteins; D020931:ran GTP-Binding Protein