Purification of Protein A-tagged Yeast Ran Reveals Association with a Novel Karyopherin β Family Member, Pdr6p*

The small GTPase Ran (encoded by GSP1and GSP2 in yeast) plays a central role in nucleocytoplasmic transport. GSP1 and GSP2 were tagged with protein A and functionally expressed in a gsp1null mutant. After affinity purification of protein A-tagged Gsp1p or Gsp2p by IgG-Sepharose chromatography, known karyopherin β 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.

[1]  T. Nishimoto,et al.  Self-organization of microtubule asters induced in Xenopus egg extracts by GTP-bound Ran. , 1999, Science.

[2]  Yixian Zheng,et al.  Stimulation of microtubule aster formation and spindle assembly by the small GTPase Ran. , 1999, Science.

[3]  A. Dickmanns,et al.  A Role for RanBP1 in the Release of CRM1 from the Nuclear Pore Complex in a Terminal Step of Nuclear Export , 1999, The Journal of cell biology.

[4]  D. Tollervey,et al.  A Novel In Vivo Assay Reveals Inhibition of Ribosomal Nuclear Export in Ran-Cycle and Nucleoporin Mutants , 1999, The Journal of cell biology.

[5]  L H Lee,et al.  Crm1p mediates regulated nuclear export of a yeast AP‐1‐like transcription factor , 1998, The EMBO journal.

[6]  G. Lipowsky,et al.  NTF2 mediates nuclear import of Ran , 1998, The EMBO journal.

[7]  Takashi Miyata,et al.  When Overexpressed, a Novel Centrosomal Protein, RanBPM, Causes Ectopic Microtubule Nucleation Similar to γ-Tubulin , 1998, The Journal of cell biology.

[8]  M. Künzler,et al.  Yeast Los1p Has Properties of an Exportin-Like Nucleocytoplasmic Transport Factor for tRNA , 1998, Molecular and Cellular Biology.

[9]  Pamela A. Silver,et al.  Regulated nucleo/cytoplasmic exchange of HOG1 MAPK requires the importin β homologs NMD5 and XPO1 , 1998, The EMBO journal.

[10]  U. Scherf,et al.  The hCSE1/CAS protein is phosphorylated by HeLa extracts and MEK-1: MEK-1 phosphorylation may modulate the intracellular localization of CAS. , 1998, Biochemical and biophysical research communications.

[11]  M. Moore.  Ran and Nuclear Transport* , 1998, The Journal of Biological Chemistry.

[12]  E. O’Shea,et al.  Phosphorylation regulates association of the transcription factor Pho4 with its import receptor Pse1/Kap121. , 1998, Genes & development.

[13]  G. Blobel,et al.  Transport routes through the nuclear pore complex. , 1998, Current opinion in cell biology.

[14]  K. Weis,et al.  Importins and exportins: how to get in and out of the nucleus. , 1998, Trends in biochemical sciences.

[15]  Matthias Mann,et al.  Mtr10p functions as a nuclear import receptor for the mRNA‐binding protein Npl3p , 1998, The EMBO journal.

[16]  I. Mattaj,et al.  Nucleocytoplasmic transport: the soluble phase. , 1998, Annual review of biochemistry.

[17]  Dirk Görlich,et al.  RanBP1 is crucial for the release of RanGTP from importin β‐related nuclear transport factors , 1997, FEBS letters.

[18]  P. Bork,et al.  A Novel Class of RanGTP Binding Proteins , 1997, The Journal of cell biology.

[19]  N Jones,et al.  Regulation of yAP‐1 nuclear localization in response to oxidative stress , 1997, The EMBO journal.

[20]  A. Podtelejnikov,et al.  Linking genome and proteome by mass spectrometry: large-scale identification of yeast proteins from two dimensional gels. , 1996, Proceedings of the National Academy of Sciences of the United States of America.

[21]  P. Silver,et al.  A GTPase Controlling Nuclear Trafficking: Running the Right Way or Walking RANdomly? , 1996, Cell.

[22]  D. Tollervey,et al.  Nuclear pore proteins are involved in the biogenesis of functional tRNA. , 1996, The EMBO journal.

[23]  S. Sazer.  The search for the primary function of the Ran GTPase continues. , 1996, Trends in cell biology.

[24]  P. D’Eustachio,et al.  The small nuclear GTPase Ran: How much does it run? , 1996, BioEssays : news and reviews in molecular, cellular and developmental biology.

[25]  P. Silver,et al.  The GTP-bound form of the yeast Ran/TC4 homologue blocks nuclear protein import and appearance of poly(A)+ RNA in the cytoplasm. , 1995, Proceedings of the National Academy of Sciences of the United States of America.

[26]  F. Melchior,et al.  Mechanisms of nuclear protein import. , 1995, Current opinion in cell biology.

[27]  P. Grandi,et al.  Purification of NSP1 reveals complex formation with ‘GLFG’ nucleoporins and a novel nuclear pore protein NIC96. , 1993, The EMBO journal.

[28]  D. Goldfarb,et al.  Regulation of RNA processing and transport by a nuclear guanine nucleotide release protein and members of the Ras superfamily. , 1993, The EMBO journal.

[29]  A. Lee,et al.  GSP1 and GSP2, genetic suppressors of the prp20-1 mutant in Saccharomyces cerevisiae: GTP-binding proteins involved in the maintenance of nuclear organization , 1993, Molecular and cellular biology.

[30]  A. Goffeau,et al.  Multiple or pleiotropic drug resistance in yeast. , 1991, Biochimica et biophysica acta.

[31]  J. Sambrook,et al.  Molecular Cloning: A Laboratory Manual , 2001 .