Abstract
Many important regulatory proteins, including cell cycle regulators and transcription factors, contain a phosphorylation site within or adjacent to a classic nuclear localization signal (NLS) sequence. Previous studies show that the nuclear localization of these cargoes can be regulated by phosphorylation at these sites. It was hypothesized that this phosphorylation regulates the nuclear import of NLS cargo proteins by modulating the interaction of the cargo with the classic nuclear transport receptor, importin alpha. In this study, we utilize in vitro solution binding assays and in vivo analyses to directly test this model. We demonstrate that mimicking phosphorylation at a site adjacent to an NLS decreases the binding affinity of the NLS for importin alpha. This decrease in cargo affinity for importin alpha correlates with a decrease in nuclear accumulation in vivo. Through these analyses, we show that the cell cycle-dependent nuclear import of the Saccharomyces cerevisiae transcription factor Swi6p correlates with a phosphorylation-dependent change in affinity for importin alpha. Furthermore, we present data using the SV40 NLS to suggest that this form of regulation can be utilized to artificially modulate the nuclear import of a cargo, which is usually constitutively targeted to the nucleus. This work defines one molecular mechanism for regulating nuclear import by the classic NLS-mediated transport pathway.
Highlights
The nuclear envelope establishes an essential regulatory barrier, which eukaryotic cells can use to control cellular processes such as gene expression and cell cycle progression
It was hypothesized that this phosphorylation regulates the nuclear import of nuclear localization signal (NLS) cargo proteins by modulating the interaction of the cargo with the classic nuclear transport receptor, importin ␣
We demonstrate that mimicking phosphorylation at a site adjacent to an NLS decreases the binding affinity of the NLS for importin ␣
Summary
The other two forms of regulation, methods 1 and 2 above, could be due to phosphorylation at any site within the protein. The proximal position of the phosphorylation site to the NLS sequence suggests that the nuclear import of these cargoes may be modulated by directly regulating the binding affinity of the NLS for the NLS receptor. A complete understanding of phosphorylation-mediated regulation of nuclear import by modulation of the interaction between an NLS and importin ␣ requires a quantitative model for the import of a cargo that correlates the in vitro interaction energies with the in vivo localization of a protein. We have utilized site-directed mutagenesis, in vitro binding assays, and in vivo analyses to investigate this mechanism of regulating nuclear transport Through these analyses we show that mimicking phosphorylation of residues adjacent to an NLS decreases the affinity of that NLS for importin ␣. We propose that this mode of regulation could be exploited to artificially manipulate the steady state localization of proteins
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