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。规范NLSS包含 一小段碱性氨基酸(单组分)或两段排列紧密的碱性氨基酸(二组分)。尽管 短长度的典型的NLSS，长度只有4或5个氨基酸，它们在细胞质中与 NLS受体(也称为核粘附素)Importin家族的成员。Importins作为载体，将相互作用的货物引导到原子核。 越来越明显的是，许多核蛋白没有利用典型的输入途径，也没有通过替代手段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“蛋白质组广泛”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，非常规NLSS及其核机制的识别和研究 进口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进口实验，以鉴定和表征新的NLSS。该系统在功能NLS与嵌合转录因子融合方面有点类似于酵母1-杂交系统 诱导核定位，允许激活容易检测到的报告基因。这是一个稳健的、量化的 和我们和其他人已经用来定位和表征新的NLSS的良好特征的系统。这种酵母遗传方法的优势 就像酵母2-杂交种一样 系统，我们可以很容易地放大它来检查大量的短肽序列 核本地化。我们正在使用重组系统来筛选一个大的短随机多肽文库，以寻找新的线性 在酵母核进口试验中诱导核定位的序列。 这些互动主题的目标也将被确定。 我们的研究将确定短的线性氨基酸序列，赋予核 本地化。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。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的库，这些基序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.