PROTEIN IMPORT INTO NUCLEUS

这个子项目是许多研究子项目中的一个 由NIH/NCRR资助的中心赠款提供的资源。子项目和 研究者（PI）可能从另一个NIH来源获得了主要资金， 因此可以在其他CRISP条目中表示。所列机构为 研究中心，而研究中心不一定是研究者所在的机构。 许多用于核进口的货物携带核定位信号，这些信号被核转运蛋白（karyopherins，Kaps）识别。我们目前的方法来定量进口率和测量Kap和货物浓度在体内的单个酵母细胞，提供新的见解进口动力学。通过系统地操纵的数量，类型和亲和力的Kaps和货物，我们表明，在体内的进口率只是由Kaps和他们的货物和它们之间的亲和力的浓度。这些速率适合于进口过程的简单泵泄漏模型。出乎意料的是，我们推断输入的主要限制因素是Kaps和货物在压倒性的非特异性竞争背景下在细胞质中找到彼此的能力较差，而不是其他更明显的候选者，例如核孔复合体和Ran。很可能每个输入轮的大部分被Kaps和核定位信号占据，当它们在细胞质中彼此定位时，核定位信号采样其他细胞质蛋白。描述这项工作的早期阶段的手稿已经发表：简单的动力学关系和非特异性竞争在体内控制核输入率。Timney BL，Tetenbaum-Novatt J，Agate DS，威廉姆斯R，Zhang W，Chait BT，Rout MP.J Cell Biol. 2006 175（4）：579-93.目前这项工作正在扩大到包括各种运输因子和FG-核孔蛋白之间的解离常数的测量。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Many cargoes destined for nuclear import carry nuclear localization signals that are recognized by karyopherins (Kaps). We present methods to quantitate import rates and measure Kap and cargo concentrations in single yeast cells in vivo, providing new insights into import kinetics. By systematically manipulating the amounts, types, and affinities of Kaps and cargos, we show that import rates in vivo are simply governed by the concentrations of Kaps and their cargo and the affinity between them. These rates fit to a straightforward pump-leak model for the import process. Unexpectedly, we deduced that the main limiting factor for import is the poor ability of Kaps and cargos to find each other in the cytoplasm in a background of overwhelming nonspecific competition, rather than other more obvious candidates such as the nuclear pore complex and Ran. It is likely that most of every import round is taken up by Kaps and nuclear localization signals sampling other cytoplasmic proteins as they locate each other in the cytoplasm. a manuscript describing the early stages of this work has been published: Simple kinetic relationships and nonspecific competition govern nuclear import rates in vivo. Timney BL, Tetenbaum-Novatt J, Agate DS, Williams R, Zhang W, Chait BT, Rout MP.J Cell Biol. 2006 175(4):579-93. Currently this work is being expanded to include measurements of the dissociation constants between a variety of transport factors and FG-nucleoporins.