In vivo analysis of importin alpha proteins reveals cellular proliferation inhibition and substrate specificity.
The "classical" nuclear import pathway depends on importins alpha and beta. Humans have only one importin beta, while six alpha importins have been described. Whether or not distinct alpha importins are essential for specific import pathways in living human cells is unclear. We used RNA interference technology to specifically down-regulate the expression of ubiquitously expressed human alpha importins in HeLa cells. Down-regulation of importins alpha3, alpha5, alpha7, and beta strongly inhibited HeLa cell proliferation, while down-regulation of importins alpha1 and alpha4 had only a minor effect or no effect. Nucleoplasmin import was not prevented by down-regulation of any alpha importin, indicating that the importin alpha/beta pathway was generally not affected. In contrast, importin alpha3 or alpha5 down-regulation specifically inhibited the nuclear import of the Ran guanine nucleotide exchange factor, RCC1. Coinjection of recombinant alpha importins and RCC1 into down-regulated cells demonstrated that these transport defects were specifically caused by the limited availability of importin alpha3 in both cases. Thus, importin alpha3 is the only alpha importin responsible for the classical nuclear import of RCC1 in living cells.
Quensel
Christina
C
Osteeklinik Damp GmbH, Sente-Deern-Ring 30, 24351 Osteebad Damp, Germany.
Friedrich
Beate
B
Sommer
Thomas
T
Hartmann
Enno
E
Kohler
Matthias
M
D017209:Apoptosis; D015153:Blotting, Western; D018797:Cell Cycle Proteins / Q000378:metabolism; D002467:Cell Nucleus / Q000378:metabolism; D049109:Cell Proliferation; D002469:Cell Separation; D003513:Cycloheximide / Q000494:pharmacology; D003600:Cytosol / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D015536:Down-Regulation; D005434:Flow Cytometry; D020662:Guanine Nucleotide Exchange Factors / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D008856:Microscopy, Fluorescence; D009687:Nuclear Proteins / Q000378:metabolism; D011500:Protein Synthesis Inhibitors / Q000494:pharmacology; D034622:RNA Interference; D034741:RNA, Small Interfering / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry; D013379:Substrate Specificity; D013997:Time Factors; D014162:Transfection; D028901:alpha Karyopherins / Q000235:genetics* / Q000378:metabolism; D028961:beta Karyopherins / Q000378:metabolism
D018797:Cell Cycle Proteins; D020662:Guanine Nucleotide Exchange Factors; C423142:KPNA1 protein, human; C106259:KPNA4 protein, human; C494630:KPNA6 protein, human; D009687:Nuclear Proteins; D011500:Protein Synthesis Inhibitors; C062521:RCC1 protein, human; D034741:RNA, Small Interfering; D011994:Recombinant Proteins; D028901:alpha Karyopherins; D028961:beta Karyopherins; D003513:Cycloheximide