Abstract
Classical protein import, mediated by the binding of a classical nuclear localization signal (NLS) to the NLS receptor, karyopherin/importin alpha, is the most well studied nuclear transport process. Classical NLSs are either monopartite sequences that contain a single cluster of basic amino acids (Lys/Arg) or bipartite sequences that contain two clusters of basic residues separated by an unconserved linker region. We have created mutations in conserved residues in each of the three NLS-binding sites/regions in Saccharomyces cerevisiae karyopherin alpha (SRP1). For each mutant we have analyzed binding to both a monopartite and a bipartite NLS cargo in vitro. We have also expressed each karyopherin alpha mutant in vivo as the only cellular copy of the NLS receptor and examined the impact on cell growth and import of both monopartite and bipartite NLS-containing cargoes. Our results reveal the functional significance of specific residues within karyopherin alpha for NLS cargo binding. A karyopherin alpha variant with a mutation in the major NLS-binding site exhibits decreased binding to both monopartite and bipartite NLS cargoes, and this protein is not functional in vivo. However, we also find that a karyopherin alpha variant with a mutation in the minor NLS-binding site, which shows decreased binding only to bipartite NLS-containing cargoes, is also not functional in vivo. This suggests that the cell is dependent on the function of at least one bipartite NLS cargo that is imported into the nucleus by karyopherin alpha. Our experiments also reveal functional importance for the linker-binding region. This study provides insight into how changes in binding to cellular NLS sequences could impact cellular function. In addition, this work has led to the creation of conditional alleles of karyopherin alpha with well characterized defects in NLS binding that will be useful for identifying and characterizing novel NLS cargoes.
Highlights
Classical protein import, mediated by the binding of a classical nuclear localization signal (NLS) to the NLS receptor, karyopherin/importin ␣, is the most well studied nuclear transport process
In the case of classical nuclear protein import, a nuclear localization signal (NLS)1 within the cargo protein is recognized by an adaptor protein, karyopherin/importin ␣, which mediates binding to the transport receptor, karyopherin/importin  [8, 9]
We have focused on four conserved residues within karyopherin ␣ that represent different binding sites/regions within the NLS-binding groove: Asp203 of the major NLS-binding site, Glu402 of the minor NLS-binding site, and Tyr283 and Arg321 of the bipartite NLS linker-binding region (Fig. 1A)
Summary
We have expressed each karyopherin ␣ mutant in vivo as the only cellular copy of the NLS receptor and examined the impact on cell growth and import of both monopartite and bipartite NLS-containing cargoes. A karyopherin ␣ variant with a mutation in the major NLS-binding site exhibits decreased binding to both monopartite and bipartite NLS cargoes, and this protein is not functional in vivo. We find that a karyopherin ␣ variant with a mutation in the minor NLS-binding site, which shows decreased binding only to bipartite NLS-containing cargoes, is not functional in vivo. In the case of classical nuclear protein import, a nuclear localization signal (NLS) within the cargo protein is recognized by an adaptor protein, karyopherin/importin ␣, which mediates binding to the transport receptor, karyopherin/importin  [8, 9]. Subsequent studies with the full-length mouse karyopherin ␣ revealed that the structure and NLS binding mode are similar for vertebrate karyopherin ␣ [14, 24]
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