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定位于特定的亚细胞间隔。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置，而且还限制在不适当的位置的功能。蛋白质的核输入通常是通过核定位信号(NLSS)与可溶性胞质受体蛋白相互作用而介导的。典型的NLSS包含*一小段碱性氨基酸(单组分)或两段间隔很近的短段碱性氨基酸(二组分)。尽管典型的NLSS长度很短，长度只有4到5个氨基酸，但它们在细胞质中与NLS受体Importin家族的成员(也称为核粘附素)产生高亲和力的相互作用。Importins作为载体将相互作用的货物引导到细胞核。*越来越明显的是，许多核蛋白不利用典型的输入途径，也不通过替代方式与典型的输入途径相互作用。事实上，在小鼠和酵母模型中的“蛋白质组范围”分析发现，40-60%*的核蛋白不包含可识别的NLS。这些蛋白质中的一些可能通过其他方式进入细胞核，而另一些可能以另一种方式使用传统的输入装置。因此，鉴定和研究非常规NLSS及其核导入机制仍然是细胞生物学领域的一个重要研究领域。我们采用并改进了一种基于转录的酵母核导入方法来鉴定和鉴定新的NLSS。这个系统有点类似于酵母1-杂交系统，因为功能性NLS与嵌合转录因子*的融合诱导了核定位，允许激活容易检测到的报告基因。这是一个健壮的、定量的、有良好特征的系统，我们和其他人已经用它来绘制和描述新的NLSS。这种酵母遗传方法的优势是，就像酵母双杂交系统一样，我们可以很容易地放大它，为那些提供核定位的序列检查大量的短肽序列。我们正在使用重组系统来筛选大量的短随机多肽文库，以寻找在酵母核进口试验中诱导核定位的新的线性*序列。*这些相互作用基序的目标也将被识别。*我们的研究将识别提供核*定位的短线性氨基酸序列。对这些信号的表征将确定用于获得进入原子核的通道的新机制。随着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标的库，它们在信号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.**