Probing Nuclear Localization Signal-Importin αBinding 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 (Imp alpha) 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-Imp alpha binding. Second, by fluorescence lifetime imaging microscopy, we directly monitor the occurrence of NLS-Imp alpha 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 Imp alpha 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 Imp alpha and yield the first estimate of its autoinhibition energy in living cells.