SPINDLE POLES AND NUCLEAR PORES--YEAST NDC1 AND MPS2

在芽殖酵母细胞周期中，纺锤体极体（SPB）复制是有丝分裂纺锤体组装的关键和必要事件，纺锤体最终将染色体分离到后代细胞中。 在酵母中，SPB是唯一的微管组织中心，并且嵌入核被膜（NE）中，使核质和细胞质中的微管成核。SPB复制或SPB功能的失败导致非整倍性和多倍性，类似于在许多癌细胞中观察到的。该建议解决了两个基因的功能，NDC 1和MPS 2，这是需要在SPB复制插入新的SPB到NE。 我们已经发现这些必需基因编码定位于SPB的膜蛋白。 此外，Ndc 1 p被发现在核孔复合物（NPC）和Mps 2 p被发现在NE和ER膜。 像SPB一样，NPC嵌入在NE中。 本提案的目的是首先，探索Ndc 1 p和Mps 2 p在SPB复制中的功能，通过寻找与其他SPB组件的遗传和物理相互作用。 我们还将进行遗传筛选，以寻找在SPB复制中起作用的其他基因。 其次，我们将确定Ndc 1 p和Mps 2 p在核膜中的拓扑结构。此外，我们将确定这些蛋白质是否可以进入核质和/或细胞质，作为我们努力确定它们如何发挥作用的一部分。 第三，我们将研究Ndc 1 p的NPC相关功能。 我们认为Ndc 1 p可能需要NPC组装，如SPB组装。 我们提出了各种遗传学，细胞学和生物化学的方法来发现Ndc 1 p在NPC中的作用，并确定与它合作的蛋白质。 最后，我们已经确定了一个新的基因，NOE 1，这是所需的ndc 1 -1细胞的活力，其产品可能是本地化的SPB，这表明一个可能的作用NOE 1 p SPB复制。 我们将定义Noe 1 p相对于Ndc 1 p的作用，并确定与NOE 1相互作用的基因。 该项目旨在了解SPB和NPC中一组不寻常的膜蛋白的功能，并确定与这些细胞器相互作用的其他蛋白质。

Spindle pole body (SPB) duplication during the budding yeast cell cycle is a critical and essential event in assembly of the mitotic spindle that ultimately segregates the chromosomes to the progeny cells. In yeast, the SPB is the sole microtubule organizing center and is embedded in the nuclear envelope (NE) nucleating microtubules in the nucleoplasm and in the cytoplasm. Failure in SPB duplication or in SPB function leads to anueploidy and polyploidy similar to that observed in many cancer cells. This proposal addresses the function of two genes, NDC1 and MPS2, that are required during SPB duplication to insert the new SPB into the NE. We have found that these essential genes encode membrane proteins that localize to the SPB. In addition, Ndc1p is found in nuclear pore complexes (NPC) and Mps2p is found in the NE and the ER membranes. NPCs, like SPBs, are embedded in the NE. The aims of this proposal are to first, explore the function of Ndc1p and Mps2p in SPB duplication by looking for genetic and physical interactions with other SPB components. We will also carry out genetic screens to look for additional genes that function in SPB duplication. Second, we will determine the topology of Ndc1p and Mps2p in the nuclear envelope. Furthermore, we will determine whether these proteins have access to the nucleoplasm and/or the cytoplasm as part of our efforts to determine how they function. Third, we will study the NPC-related function of Ndc1p. We believe that Ndc1p may be required for NPC assembly, like SPB assembly. We propose a variety of genetic, cytological and biochemical approaches to discover the role of Ndc1p at NPCs and to identify proteins with which it collaborates in this function. Finally, we have identified a novel gene, NOE1, that is required for the viability of ndc1-1 cells and whose product may be localized to the SPB, suggesting a possible role for Noe1p in SPB duplication. We will define the role of Noe1p with respect to Ndc1p, and identify genes that interact with NOE1. The proposed project is aimed at understanding the function of a set of unusual membrane proteins at the SPB and the NPC, and at identifying other proteins with which the interact at these organelles.