Insertion of the nuclear pore complex and the yeast spindle pole body into the nuclear envelope.

在这里，我们将研究如何大的蛋白质结构成为插入到双膜的核膜（NE）。这仍然是细胞生物学中一个悬而未决的大问题。我们将使用核孔复合体（NPC）和纺锤体极体（SPB），酵母中心体，从芽殖和分裂酵母作为模型来解决这个问题。新出现的图像是NPC在细胞核内的间期组装。核孔蛋白在内核膜（INM）下方积聚，使该膜变形，最终INM和外核膜（ONM）融合。使用芽殖酵母，我们最近确定了旁系同源物Brr 6/Brl 1，NE的两个完整的膜蛋白，作为NPC生物发生位点的组成部分，其在NPC生物发生的后期发挥作用，可能在INM/ONM融合中。从这两种蛋白质开始，我们的目标是了解NPC如何在完整的NE中组装。我们将通过遗传学和生物化学的方法来检测芽殖酵母Brr 6/Brl 1的功能。特别是，我们将插入纯化的Brr 6/Brl 1到蛋白质脂质体，并测量其性能。使用脂质传感器分析局部脂质变化将指示在NPC生物发生期间Brr 6/Brl 1是否调节脂质。有趣的是，我们的数据表明，芽殖酵母SPB的NE插入需要NPC的帮助，NPC停靠在生长的子SPB（dSPB）上，该子SPB首先位于ONM的细胞质面上。这些NPC可以提供插入dSPB的ONM/INM融合位点或将NPC/SPB的共享因子（Ndc 1）递送至dSPB。我们将通过电子断层扫描分析SPB复制，以了解dSPB和NPC之间的关系。将使用超分辨率活细胞成像来阐明靠近dSPB的NPC的命运。接下来，将芽殖酵母NPC/SPB生物发生与裂殖酵母进行比较。令人惊讶的是，分裂酵母同源物SpBrr 6（分裂酵母缺乏BRL 1）是将SPB插入NE所需的，而我们的数据反对Brr 6/Brll在芽殖酵母中的这种功能。SpBrr 6是否在NPC生物发生中起作用尚不清楚。为了了解Brr 6样蛋白的一般功能，重要的是要知道SpBrr 6是否也在NPC生物发生中发挥作用。免疫电子显微镜将指示分裂酵母SpBrr 6与NPC和SPB的定位。条件致死Spbrr 6（ts）细胞的电子显微镜将有助于理解SpBrr 6在NE插入SPB和NPC生物发生过程中的功能。两者合计，这一建议将阐明NE是如何调制，以允许SPB插入和NPC生物发生。

Here, we will investigate how large proteinaceous structures become inserted into the double membrane of the nuclear envelope (NE). This is still one of the big open questions in cell biology. We will address this using the nuclear pore complex (NPC) and the spindle pole body (SPB), the yeast centrosome, from budding and fission yeast as models. The emerging picture is that NPCs assemble in interphase from within the nucleus. Nucleoporins accumulate underneath the inner nuclear membrane (INM), deform this membrane and eventually the INM and outer nuclear membrane (ONM) fuse. Using budding yeast, we recently identified the paralogues Brr6/Brl1, two integral membrane proteins of the NE, as components of NPC biogenesis sites that function late in NPC biogenesis, perhaps in INM/ONM fusion. Starting with these two proteins, we aim to understand how NPCs assemble in the intact NE. We will test the functions of budding yeast Brr6/Brl1 by genetic and biochemical approaches. In particular, we will insert purified Brr6/Brl1 into proteo-liposomes and measure their properties. Analysis of local lipid changes using lipid sensors will indicate whether Brr6/Brl1 modulate lipids during NPC biogenesis. Interestingly, our data suggest that the NE insertion of the budding yeast SPB requires the assistance of NPCs that dock onto the growing daughter SPB (dSPB) that first sits on the cytoplasmic face at the ONM. These NPCs may provide the ONM/INM fusion site into which the dSPB is inserted or deliver shared factors of NPCs/SPBs (Ndc1) to the dSPB. We will analyze SPB duplication by electron tomography in order to see the relationship between dSPB and NPCs. Super resolution live cell imaging will be used to clarify the fate of the NPCs close to the dSPB. Next, budding yeast NPC/SPB biogenesis will be compared with fission yeast. Surprisingly, the fission yeast homologue SpBrr6 (fission yeast lacks BRL1) is needed for the insertion of the SPBs into the NE while our data argue against such a function of Brr6/Brl1 in budding yeast. Whether SpBrr6 plays a role in NPC biogenesis is unclear. To understand the general functions of Brr6-like proteins, it is important to know whether SpBrr6 also has a role in NPC biogenesis. Immunoelectron microscopy will indicate the localization of fission yeast SpBrr6 with NPCs and SPBs. Electron microscopy of conditional lethal Spbrr6(ts) cells will help to understand the functions of SpBrr6 during NE insertion of the SPB and NPC biogenesis. Taken together, this proposal will clarify how the NE is modulated in order to allow SPB insertion and NPC biogenesis.