Nuclear import of TFIIB is mediated by Kap114p, a karyopherin with multiple cargo-binding domains.
Nuclear import and export is mediated by an evolutionarily conserved family of soluble transport factors, the karyopherins (referred to as importins and exportins). The yeast karyopherin Kap114p has previously been shown to import histones H2A and H2B, Nap1p, and a component of the preinitiation complex (PIC), TBP. Using a proteomic approach, we have identified several potentially new cargoes for Kap114p. These cargoes include another PIC component, the general transcription factor IIB or Sua7p, which interacted directly with Kap114p. Consistent with its role as a Sua7p import factor, deletion of KAP114 led to specific mislocalization of Sua7p to the cytoplasm. An interaction between Sua7p and TBP was also detected in cytosol, raising the possibility that both Sua7p and TBP can be coimported by Kap114p. We have also shown that Kap114p possesses multiple overlapping binding sites for its partners, Sua7p, Nap1p, and H2A and H2B, as well as RanGTP and nucleoporins. In addition, we have assembled an in vitro complex containing Sua7p, Nap1p, and histones H2A and H2B, suggesting that this Kap may import several proteins simultaneously. The import of more than one cargo at a time would increase the efficiency of each import cycle and may allow the regulation of coimported cargoes.
Hodges
Jennifer L
JL
Center for Cell Signaling, Department of Microbiology, University of Virginia Health Sciences Center, Charlottesville, USA.
Leslie
Jennifer H
JH
Mosammaparast
Nima
N
Guo
Yurong
Y
Shabanowitz
Jeffrey
J
Hunt
Donald F
DF
Pemberton
Lucy F
LF
D021581:Active Transport, Cell Nucleus; D001665:Binding Sites; D001692:Biological Transport; D015153:Blotting, Western; D018797:Cell Cycle Proteins / Q000378:metabolism; D002851:Chromatography, High Pressure Liquid; D003593:Cytoplasm / Q000378:metabolism; D003600:Cytosol / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D005656:Fungal Proteins / Q000737:chemistry; D017353:Gene Deletion; D017930:Genes, Reporter; D005982:Glutathione Transferase / Q000378:metabolism; D049452:Green Fluorescent Proteins / Q000378:metabolism; D006657:Histones / Q000737:chemistry; D028884:Karyopherins / Q000378:metabolism*; D008853:Microscopy; D008954:Models, Biological; D009687:Nuclear Proteins / Q000737:chemistry / Q000378:metabolism*; D056490:Nucleosome Assembly Protein 1; D010957:Plasmids / Q000378:metabolism; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D021381:Protein Transport; D040901:Proteomics / Q000379:methods; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry / Q000378:metabolism*; D021241:Spectrometry, Mass, Electrospray Ionization; D035581:Transcription Factor TFIIB / Q000737:chemistry*; D028961:beta Karyopherins; D020931:ran GTP-Binding Protein / Q000737:chemistry
D018797:Cell Cycle Proteins; D005656:Fungal Proteins; D006657:Histones; C400425:KAP114 protein, S cerevisiae; D028884:Karyopherins; C533556:NAP1 protein, S cerevisiae; C533557:NAP1L1 protein, human; D009687:Nuclear Proteins; D056490:Nucleosome Assembly Protein 1; D029701:Saccharomyces cerevisiae Proteins; C108121:TNPO1 protein, human; D035581:Transcription Factor TFIIB; D028961:beta Karyopherins; D049452:Green Fluorescent Proteins; D005982:Glutathione Transferase; D020931:ran GTP-Binding Protein