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首页> 外文期刊>Journal of Molecular Biology >Classical NLS proteins from Saccharomyces cerevisiae.
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Classical NLS proteins from Saccharomyces cerevisiae.

机译：来自酿酒酵母的经典NLS蛋白。

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Proteins can enter the nucleus through various receptor-mediated import pathways. One class of import cargos carries a classical nuclear localization signal (cNLS) containing a short cluster of basic residues. This pathway involves importin alpha (Impalpha), which possesses the cNLS binding site, and importin beta (Impbeta), which translocates the import complex through the nuclear pore complex. The defining criteria for a cNLS protein from Saccharomyces cerevisiae are an in vivo import defect in Impalpha and Impbeta mutants, direct binding to purified Impalpha, and stimulation of this binding by Impbeta. We show for the first time that endogenous S. cerevisiae proteins Prp20, Cdc6, Swi5, Cdc45, and Clb2 fulfill all of these criteria identifying them as authentic yeast cNLS cargos. Furthermore, we found that the targeting signal of Prp20 is a bipartite cNLS and that of Cdc6 is a monopartite cNLS. Basic residues present within these motifs are of different significance for the interaction with Impalpha.We determined the binding constants for import complexes containing the five cNLS proteins by surface plasmon resonance spectrometry. The dissociation constants for cNLS/alpha/beta complexes differ considerably, ranging from 1 nM for Cdc6 to 112 nM for Swi5, suggesting that the nuclear import kinetics is determined by the strength of cNLS/Impalpha binding. Impbeta enhances the affinity of Impalpha for cNLSs approximately 100-fold. This stimulation of cNLS binding to Impalpha results from a faster association in the presence of Impbeta, whereas the dissociation rate is unaffected by Impbeta. This implies that, after entry into the nucleus, the release of Impbeta by the Ran guanosine triphosphatase (Ran GTPase) from the import complex is not sufficient to dissociate the cNLS/Impalpha subcomplex. Our observation that the nucleoporin Nup2, which had been previously shown to release the cNLS from Impalpha in vitro, is required for efficient import of all the genuine cNLS cargos supports a general role of Nup2 in import termination.
机译：蛋白质可以通过各种受体介导的导入途径进入细胞核。一类进口货物带有经典的核定位信号（cNLS），其中包含一小批基本残留物。该途径涉及具有cNLS结合位点的importin alpha（Impalpha）和importin beta（Impbeta），后者通过核孔复合体使导入复合物移位。来自酿酒酵母（Saccharomyces cerevisiae）的cNLS蛋白的定义标准是Impalpha和Impbeta突变体的体内输入缺陷，直接结合至纯化的Impalpha以及通过Impbeta刺激这种结合。我们首次显示内源啤酒酵母蛋白Prp20，Cdc6，Swi5，Cdc45和Clb2满足所有这些标准，将其鉴定为真实的酵母cNLS货物。此外，我们发现Prp20的靶向信号是二分cNLS，而Cdc6的靶向信号是二分cNLS。这些基序中存在的碱性残基对于与Impalpha的相互作用具有不同的意义。我们通过表面等离子体共振光谱法确定了包含5种cNLS蛋白的输入复合物的结合常数。 cNLS /α/β复合物的解离常数相差很大，范围从Cdc6的1 nM到Swi5的112 nM，这表明核的进口动力学取决于cNLS / Impalpha结合的强度。 Impbeta将Impalpha对cNLS的亲和力提高了约100倍。 cNLS与Impalpha结合的这种刺激源自在存在Impbeta时更快的关联，而解离速率不受Impbeta的影响。这意味着进入核中后，Ran鸟苷三磷酸酶（Ran GTPase）从导入复合物中释放的Impbeta不足以解离cNLS / Impalpha亚复合物。我们的观察表明，核仁蛋白Nup2（以前已显示可在体外从Impalpha释放cNLS）是有效进口所有真正的cNLS货物所必需的，这支持了Nup2在进口终止中的一般作用。

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