PROTEIN IMPORT INTO NUCLEUS

这个子项目是许多研究子项目中利用 资源由NIH/NCRR资助的中心拨款提供。子项目和 调查员(PI)可能从NIH的另一个来源获得了主要资金， 并因此可以在其他清晰的条目中表示。列出的机构是 该中心不一定是调查人员的机构。 许多运往核进口的货物携带核粘附素(KAP)识别的核定位信号。我们提出了量化进口速率的方法，并在体内测量单个酵母细胞中的Kap和货物浓度，为进口动力学提供了新的见解。通过系统地操纵Kap和货物的数量、类型和亲和力，我们表明，体内的进口率只受Kap和它们的货物的浓度以及它们之间的亲和力的控制。这些速率符合进口过程的简单泵漏模型。令人意外的是，我们推断，进口的主要限制因素是Kaps和Cargo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基因，如核孔复合体和Ran。很可能每一轮输入都被KAPS和核定位信号占据，当它们在细胞质中相互定位时，对其他细胞质蛋白进行采样。描述这项工作早期阶段的手稿已经出版：简单的动力学关系和非特定的竞争支配着体内的核进口速率。首页--期刊主要分类--期刊细介绍--期刊题录与文摘--期刊详细文摘内容2006 175(4)：579-93目前，这项工作正在扩大，以包括测量各种转运因子和FG-核孔素之间的解离常数。

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.