Recognition Mechanisms of Nuclear Localization and Export Signals

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2009-06-19

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Abstract

The transport of proteins between the nucleus and cytoplasm of cells is mediated by the Karyopherin beta family of proteins. Karyopherin betas recognize their substrates through either a nuclear localization or export signal depending on the direction of transport. Even though there are ten yeast import Karyopherin betas, for the past thirteen years there was only one well characterized nuclear localization signal, the classical nuclear localization signal. However, a second signal, the proline-tyrosine nuclear localization signal recognized by Karyopherin beta2, was recently identified through X-ray crystallography and biochemical studies of Karyopherin beta2 bound to one of its substrates. These studies identified rules for the recognition of the proline-tyrosine nuclear localization signal by Karyopherin beta2. The signal must have overall basic charge, structural disorder and a weak consensus sequence of an amino-terminal basic or hydrophobic-enriched region followed by a carboxyl-terminal arginine residue separated from a proline and tyrosine residue by two to five residues. The proline-tyrosine nuclear localization signal is also recognized by the Saccharomyces cerevisiae homolog of Karyopherin beta2, Karyopherin 104, demonstrating the generality of this import mechanism across eukaryotes. Thermodynamic analyses of the two known substrates of Karyopherin 104, Hrp1p and Nab2p, revealed physical properties governing its binding. The proline-tyrosine nuclear localization signal is an extended signal with significant sequence diversity. The signal is comprised of three binding epitopes, each of which can have varying energetic strengths in different substrates. The multivalent nature of the signal increases the diversity of the signal as well as the difficulty of identifying new substrates. A bioinformatics search identified putative proline-tyrosine nuclear localization signals which were validated through biochemical studies. Additionally, one of the proteins identified, Tfg2p, was verified as a bona fide Karyopherin 104 substrate. Analysis of Tfg2p's cellular localization revealed that its nuclear localization was not solely determined by the presence of a nuclear localization signal, but was also dependent on its retention in the nucleus. Furthermore, crystallographic studies of substrate Snurportin1 bound to the export karyopherin CRM1 revealed that its nuclear export signal has two binding epitopes implying that the multivalent nature of targeting signals may not be limited to the proline-tyrosine nuclear localization signal.

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Karyopherins, Transcription Factors, Saccharomyces cerevisiae Proteins

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