Probing nuclear localization signal-importin alpha binding equilibria in living cells.
The regulated process of protein import into the nucleus of a eukaryotic cell is mediated by specific nuclear localization signals (NLSs) that are recognized by protein-import receptors. In this study, we present fluorescence-based methods to quantitatively address the physicochemical details of NLS recognition by the receptor protein importin alpha (Impalpha) in living cells. First, by combining fluorescence recovery after photobleaching measurements and protein-concentration calibration, we quantitatively define nuclear import saturability and afford an affinity value for NLS-Impalpha binding. Second, by fluorescence lifetime imaging microscopy, we directly monitor the occurrence of NLS-Impalpha interaction and measure its effective dissociation constant (K(D)) in the actual cellular environment. Our kinetic and thermodynamic analyses independently indicate that the subsaturation of Impalpha with the expressed NLS cargo regulates nuclear import rates in living cells, in contrast to what can be predicted on the basis of available in vitro data. Finally, our experiments also provide evidence for the regulation of nuclear import mediated by the intrasteric importin beta-binding domain of Impalpha and yield the first estimate of its autoinhibition energy in living cells.
National Enterprise for nanoScience and nanoTechnology, Scuola Normale Superiore, and Consiglio Nazionale delle Ricerche-Istituto Nazionale per la Fisica della Materia, Piazza S. Silvestro 12, 56126 Pisa, Italy. Electronic address: f.cardarelli@sns.it.
Francesco
F
Cardarelli
National Enterprise for nanoScience and nanoTechnology, Scuola Normale Superiore, and Consiglio Nazionale delle Ricerche-Istituto Nazionale per la Fisica della Materia, Piazza S. Silvestro 12, 56126 Pisa, Italy; IIT@NEST, Center for Nanotechnology Innovation, Piazza S. Silvestro 12, 56126 Pisa, Italy.
Ranieri
R
Bizzarri
National Enterprise for nanoScience and nanoTechnology, Scuola Normale Superiore, and Consiglio Nazionale delle Ricerche-Istituto Nazionale per la Fisica della Materia, Piazza S. Silvestro 12, 56126 Pisa, Italy; IIT@NEST, Center for Nanotechnology Innovation, Piazza S. Silvestro 12, 56126 Pisa, Italy.
Michela
M
Serresi
National Enterprise for nanoScience and nanoTechnology, Scuola Normale Superiore, and Consiglio Nazionale delle Ricerche-Istituto Nazionale per la Fisica della Materia, Piazza S. Silvestro 12, 56126 Pisa, Italy; IIT@NEST, Center for Nanotechnology Innovation, Piazza S. Silvestro 12, 56126 Pisa, Italy.
Lorenzo
L
Albertazzi
National Enterprise for nanoScience and nanoTechnology, Scuola Normale Superiore, and Consiglio Nazionale delle Ricerche-Istituto Nazionale per la Fisica della Materia, Piazza S. Silvestro 12, 56126 Pisa, Italy; IIT@NEST, Center for Nanotechnology Innovation, Piazza S. Silvestro 12, 56126 Pisa, Italy.
Fabio
F
Beltram
D000818:Animals; D016466:CHO Cells; D006224:Cricetinae; D003412:Cricetulus; D006367:HeLa Cells; D006801:Humans; D019913:Nuclear Localization Signals / Q000235:genetics / Q000378:metabolism*; D010449:Peptide Mapping / Q000379:methods; D028901:alpha Karyopherins / Q000235:genetics / Q000378:metabolism*; D028961:beta Karyopherins / Q000235:genetics / Q000378:metabolism
D019913:Nuclear Localization Signals; D028901:alpha Karyopherins; D028961:beta Karyopherins