Transport of tail-anchored proteins to the inner nuclear membrane

细胞核被内核膜和外核膜包围，它们通过核孔连接。关于蛋白质向核膜（INM）的转运，我们所知甚少。在一种模型中，跨膜蛋白可以被动地通过核孔复合物扩散。在与核层蛋白相互作用后，它们被隔离在INM上。另一方面，已经描述了一种更主动的运输，其中含有核定位信号的膜蛋白通过核孔与核输入受体的复合物进行转运，类似于输入细胞核的可溶性蛋白。到目前为止，还没有确定膜蛋白运输到INM所需的特定因子。为了研究蛋白质到INM的运输，我们想利用膜蛋白子集的特性，尾部锚定蛋白（ta蛋白）。ta蛋白在其c端有一个单一的跨膜结构域，这需要一个与经典的SRP/ sec61依赖途径不同的翻译后膜插入机制。这使我们能够在细菌中与伴侣蛋白TRC40复合物中表达ta蛋白，TRC40是一种结合到疏水跨膜结构域的蛋白质。到目前为止，我们已经使用了inm蛋白emerin和LAP2 （lamina associated polypeptide 2）作为模型底物。这两种蛋白质都可以在翻译后整合到微粒体膜上，这支持了它们是真正的ta蛋白的概念。我们还纯化了用荧光团mCherry标记的emerin。在洋地黄苷渗透细胞中，樱桃-emerin以温度依赖的方式与er膜和INM相关。我们现在计划使用这种新的体外实验来重建emerin， LAP2和其他ta蛋白到INM的运输。首先，我们可以分析核细胞质转运因子的作用，它在可溶性蛋白的核输入中的作用是确定的。在后期阶段，体外系统将使我们能够确定有效运输不同货物所需的潜在细胞质因子。此外，我们将在完整细胞中采用几种方法，并研究INM的其他蛋白质的靶向性，根据它们的氨基酸序列，它们也是ta蛋白途径的底物。最后，我们计划建立一个定量显微镜系统来分析蛋白质在体内和体外向INM的转运。总之，拟议的项目将有助于更好地了解核膜的生物发生。

The nucleus is surrounded by the inner and the outer nuclear membrane, which are connected via the nuclear pores. Rather little is known about transport of proteins to the inner nuclear membrane (INM). In one model, transmembrane proteins can passively diffuse across the nuclear pore complex. They are then sequestered at the INM upon interaction with proteins of the nuclear lamina. On the other hand, a more active transport has been described, where membrane proteins containing nuclear localization signals translocate through the nuclear pore in a complex with nuclear import receptors, similar to soluble proteins that are imported into the nucleus. Factors that are specifically required for transport of membrane proteins to the INM have not been identified so far.To investigate transport of proteins to the INM, we want to take advantage of the properties of a subset of membrane proteins, the tail-anchored proteins (TA-proteins). TA-proteins have a single transmembrane domain at their very C-terminus, requiring a post-translational mechanism for membrane insertion that is distinct from the classic, SRP/Sec61-dependent pathway. This enables us to express TA-proteins in bacteria in a complex with the chaperone TRC40, a protein that binds to the hydrophobic transmembrane domain. So far, we have used the INM-proteins emerin and LAP2 (lamina associated polypeptide 2) as model substrates. Both proteins could be post-translationally integrated into microsomal membranes, supporting the notion that they are bona fide TA-proteins. We also purified emerin tagged with the fluorophore mCherry. In digitonin-permeabilized cells, mCherry-emerin associated with the ER-membrane and the INM in a temperature-dependent manner. We are now planning to use this novel in vitro assay to reconstitute transport of emerin, LAP2 and other TA-proteins to the INM. First, we can analyze the role of nucleocytoplasmic transport factors, whose function in nuclear import of soluble proteins is well-established. At a later stage, the in vitro system will allow us to identify potential cytosolic factors that are required for efficient transport of the different cargoes. Furthermore, we will follow several approaches in intact cells and investigate the targeting of other proteins of the INM that, according to their amino acid sequence, are also substrates of the TA-protein pathway. Finally, we plan to establish a quantitative microscopy system to analyze transport of proteins to the INM in vivo and in vitro. Together, the proposed project will contribute to a better understanding of the biogenesis of the inner nuclear membrane.