Karyopherin binding interactions and nuclear import mechanism of nuclear pore complex protein Tpr.
BACKGROUND
Tpr is a large protein with an extended coiled-coil domain that is localized within the nuclear basket of the nuclear pore complex. Previous studies 1 involving antibody microinjection into mammalian cells suggested a role for Tpr in nuclear export of proteins via the CRM1 export receptor. In addition, Tpr was found to co-immunoprecipitate with importins alpha and beta from Xenopus laevis egg extracts 2, although the function of this is unresolved. Yeast Mlp1p and Mlp2p, which are homologous to vertebrate Tpr, have been implicated in mRNA surveillance to retain unspliced mRNAs in the nucleus34. To augment an understanding of the role of Tpr in nucleocytoplasmic trafficking, we explored the interactions of recombinant Tpr with the karyopherins CRM1, importin beta and importin alpha by solid phase binding assays. We also investigated the conditions required for nuclear import of Tpr using an in vitro assay.


RESULTS
We found that Tpr binds strongly and specifically to importin alpha, importin beta, and a CRM1 containing trimeric export complex, and that the binding sites for importins alpha and beta are distinct. We also determined that the nuclear import of Tpr is dependent on cytosolic factors and energy and is efficiently mediated by the importin alpha/beta import pathway.


CONCLUSIONS
Based on the binding and nuclear import assays, we propose that Tpr is imported into the nucleus by the importin alpha/beta heterodimer. In addition, we suggest that Tpr can serve as a nucleoporin binding site for importin beta during import of importin beta cargo complexes and/or importin beta recycling. Our finding that Tpr bound preferentially to CRM1 in an export complex strengthens the notion that Tpr is involved in protein export.
Ben-Efraim
Iris
I
Department of Cell Biology, The Scripps Research Institute, La Jolla, CA 92037, USA. ibenefra@gmail.com
Frosst
Phyllis D
PD
Gerace
Larry
L
D001665:Binding Sites; D028884:Karyopherins / Q000378:metabolism; D028861:Nuclear Pore Complex Proteins / Q000378:metabolism*; D018160:Receptors, Cytoplasmic and Nuclear / Q000378:metabolism; D011994:Recombinant Proteins / Q000378:metabolism; D028901:alpha Karyopherins / Q000378:metabolism* / Q000502:physiology; D028961:beta Karyopherins / Q000378:metabolism* / Q000502:physiology; D000097606:Exportin 1 Protein
D028884:Karyopherins; D028861:Nuclear Pore Complex Proteins; D018160:Receptors, Cytoplasmic and Nuclear; D011994:Recombinant Proteins; D028901:alpha Karyopherins; D028961:beta Karyopherins; D000097606:Exportin 1 Protein