SPINDLE POLES AND NUCLEAR PORES--YEAST NDC1 AND MPS2

纺锤体极体(SPB)在发芽酵母细胞周期中的复制是有丝分裂纺锤体组装过程中的关键和基本事件，最终将染色体分离到后代细胞。在酵母中，SPB是唯一的微管组织中心，嵌入在核质和细胞质中的核膜(NE)成核微管中。SPB复制失败或SPB功能失败会导致类似于在许多癌细胞中观察到的亚倍体和多倍体。这个建议涉及两个基因的功能，Ndc1和mps2，这两个基因在SPB复制过程中需要将新的SPB插入NE。我们发现这些必需基因编码定位于SPB的膜蛋白。此外，Ndc1p存在于核孔复合体(NPC)中，Mps2p存在于NE和ER膜中。NPC与SPBS一样嵌入在NE中。这项建议的目的是首先通过寻找与SPB其他成分的遗传和物理相互作用来探索Ndc1p和Mps2p在SPB复制中的作用。我们还将进行基因筛查，寻找在SPB复制中发挥作用的其他基因。其次，我们将确定核包膜中Ndc1p和Mps2p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，作为我们确定它们如何发挥功能的努力的一部分。第三，我们将研究Ndc1p与NPC相关的功能。我们认为Ndc1p可能是鼻咽癌组装所必需的，就像SPB组装一样。我们提出了多种遗传学、细胞学和生物化学方法来发现Ndc1p在鼻咽癌中的作用，并确定它在这一功能中与之合作的蛋白质。最后，我们鉴定了一个新的基因NOE1，它是Ndc1-1细胞生存所必需的，其产物可能定位于SPB，提示Noe1p可能在SPB复制中发挥作用。我们将定义Noe1p与Ndc1p的作用，并确定与NOE1相互作用的基因。拟议的项目旨在了解一组不寻常的膜蛋白在SPB和NPC中的功能，并确定这些细胞器与之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.