Quantification of nuclear protein transport using induced heterodimerization.
Trafficking of proteins between the cytoplasm and nucleus occurs exclusively across the nuclear pore complex of eucaryotic cells. Fundamental aspects of this process affect temporal and spatial parameters, the latter carried out by specific import [nuclear localization sequence (NLS)] and export [nuclear export sequence (NES)] sequences. In this study, we focused on the adaptation of a protein heterodimerization assay to kinetically measure Crm1-mediated nuclear export in living cells using the rapalog AP21967, a heterodimerizing agent and NLS- and NES-containing fusion proteins equipped with distinct AP21967-specific binding motifs. In HeLa cells, we observed rapid nuclear export of the NLS-containing fusion protein in the presence of AP21967, with the extent of this process being a function of the number of AP21967-binding motifs. AP21967-induced nuclear export was specifically inhibited by the Crm1-binding molecule leptomycin B. Half maximal export was achieved after approximately 10 min. We further applied protein heterodimerization in HeLa cells to study induced NLS-mediated nuclear import. Only in the presence of heterodimerizer AP21967 nuclear import of a cytoplasmically localizing fusion protein was observed. Induced protein heterodimerization is thus a valuable tool to quantitatively study nucleocytoplasmic protein trafficking in cultured cells, in a non-invasive, time-saving manner.
Busch
Albert
A
University of Würzburg, Institute of Anatomy and Cell Biology, Würzburg, Germany.
Kiel
Tilman
T
Hübner
Stefan
S
D021581:Active Transport, Cell Nucleus / Q000187:drug effects / Q000502:physiology; D002467:Cell Nucleus / Q000187:drug effects / Q000378:metabolism*; D003593:Cytoplasm / Q000187:drug effects / Q000378:metabolism*; D005231:Fatty Acids, Unsaturated / Q000494:pharmacology; D006367:HeLa Cells; D006801:Humans; D028884:Karyopherins / Q000737:chemistry / Q000378:metabolism*; D049790:Nuclear Export Signals; D019913:Nuclear Localization Signals / Q000737:chemistry / Q000378:metabolism; D055503:Protein Multimerization; D018160:Receptors, Cytoplasmic and Nuclear / Q000737:chemistry / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000378:metabolism; D020123:Sirolimus / Q000031:analogs & derivatives / Q000494:pharmacology; D000097606:Exportin 1 Protein
D005231:Fatty Acids, Unsaturated; D028884:Karyopherins; D049790:Nuclear Export Signals; D019913:Nuclear Localization Signals; D018160:Receptors, Cytoplasmic and Nuclear; D011993:Recombinant Fusion Proteins; D020123:Sirolimus; D000097606:Exportin 1 Protein; C539629:AP 21967; C038753:leptomycin B