Identification of short linear peptides that function as protein interaction motifs and confer nuclear import

我们的目标是更多地了解调节蛋白质定位的过程。许多蛋白质通过特定的定位信号精确地靶向特定的亚细胞区室。正确的定位是必要的，以确保适当的活动不仅在细胞内的适当位点，而且还限制在不适当的位点的功能。蛋白质的核输入通常通过经由核定位信号（NLS）与可溶性胞质受体蛋白的相互作用来介导。标准NLS包含 或者是一段短的碱性氨基酸（单分裂的）或者是两段间隔很近的短的碱性氨基酸（双分裂的）。尽管 典型的NLS长度较短，长度仅为4或5个氨基酸，它们在细胞质中与 NLS受体的输入蛋白家族成员（也称为核转运蛋白）。输入蛋白作为载体将相互作用的货物导向细胞核。 越来越明显的是，许多核蛋白不利用经典输入途径或通过替代方式与经典输入途径相互作用。事实上，在小鼠和酵母模型中的“蛋白质组范围”分析发现， 的核蛋白不包含可识别的NLS。 这些蛋白质中的一些可以通过其他方式进入细胞核，而其他蛋白质可以以替代方式使用常规的输入装置。因此，非常规核武器系统及其核扩散机制的识别和研究， 输入仍然是细胞生物学领域中的重要研究领域。 我们采用并改进了一种基于转录的酵母细胞核输入试验来鉴定和表征新的NLS。 该系统有点类似于酵母单杂交系统，在功能性NLS与嵌合转录因子融合方面 诱导核定位，允许容易检测的报告基因的激活。这是一个强大的，定量的 我们和其他人已经用来映射和表征新的NLS。这种酵母遗传学方法的优势在于 就像酵母双杂交 系统，我们可以很容易地扩大规模，以检查大量的短肽序列，为那些赋予 核定位我们正在使用重组系统来筛选一个大型的短随机肽库，以寻找新的线性肽。 在酵母核输入测定中诱导核定位的序列。 这些相互作用基序的目标也将被确定。 我们的研究将确定短的线性氨基酸序列，赋予核 本地化这些信号的表征将确定用于获得细胞核通道的新机制。 由于这些信号作为蛋白质相互作用基序发挥作用，我们还将扩大已知的线性基序及其靶点，这些基序在信号传导和调控网络中发挥关键作用。

Our goal is to learn more about processes regulating protein localization. Many proteins are precisely targeted to particular sub-cellular compartments by specific localization signals. Correct localization is necessary to ensure that the appropriate activity is not only at the appropriate site within the cell, but also to restrict function at inappropriate sites. Nuclear import of proteins is typically mediated by interaction with soluble cytosolic receptor proteins via nuclear localization signals (NLSs). Canonical NLSs contain either a short stretch of basic amino acids (monopartite) or two closely spaced short stretches of basic amino acids (bipartite). Despite the short length of canonical NLSs, which are as little as 4 or 5 amino acids in length, they make high affinity interactions in the cytosol with members of the importin family of NLS receptors (also known as karyopherins). Importins function as carriers to direct the interacting cargo to the nucleus. It has become increasingly apparent that the many nuclear proteins do not utilize the canonical import pathway or interact with the canonical import pathway via alternative means. Indeed, "proteome wide" analyses in murine and yeast models found that 40-60% of nuclear proteins do not contain a recognizable NLS. Some of these proteins may access the nucleus by other means, while other may use the conventional import apparatus in an alternative manner. As such, identification and studies of non-conventional NLSs and their mechanisms of nuclear import remains an important area of investigation in the field of cell biology. We adopted and improved a transcription based nuclear import assay in yeast to identify and characterize novel NLSs. This system is somewhat akin to a yeast 1-hybrid system, in that fusion of a functional NLS to a chimeric transcription factor induces nuclear localization, allowing activation of easily detected reporter genes. This is a robust, quantitative and well characterized system that we and others have used to map and characterize novel NLSs. The advantage of this yeast genetic approach is that, like the yeast 2-hybrid system, we can readily scale it up to examine massive numbers of short peptide sequences for those that confer nuclear localization. We are doing this using a recombination system to screen a large library of short random peptides for novel linear sequences that induce nuclear localization in the yeast nuclear import assay. The targets of these interaction motifs will also be identified. Our studies will identify short linear amino acid sequences that confer nuclear localization. Characterization of these signals will identify novel mechanisms utilized to gain passage to the nucleus. As these signals function as protein interaction motifs, we will also be expanding the known repertoire of linear motifs and their targets, which play key roles in signalling and regulatory networks.