Transport of tail-anchored proteins to the inner nuclear membrane

细胞核由核膜和外核膜包围，核膜和外核膜通过核孔连接。关于蛋白质转运到内核膜（INM）的情况知之甚少。在一个模型中，跨膜蛋白可以被动地扩散穿过核孔复合物。然后它们在与核纤层的蛋白质相互作用后被隔离在INM处。另一方面，已经描述了更主动的转运，其中含有核定位信号的膜蛋白以与核输入受体的复合物的形式通过核孔易位，类似于输入到核中的可溶性蛋白。为了研究蛋白质向INM的转运，我们希望利用膜蛋白的一个子集，即尾锚定蛋白（TA-蛋白）的特性。TA-蛋白在其C-末端具有单个跨膜结构域，需要用于膜插入的翻译后机制，其不同于经典的SRP/Sec 61依赖性途径。这使我们能够在细菌中表达TA-蛋白与伴侣TRC 40的复合物，TRC 40是一种与疏水跨膜结构域结合的蛋白质。到目前为止，我们已经使用了INM蛋白emerin和LAP 2（纤层相关多肽2）作为模型底物。这两种蛋白质都可以整合到微粒体膜中，支持它们是真正的TA蛋白的观点。我们还纯化了用荧光团mCherry标记的Emerin。在毛地黄皂苷透化细胞中，mCherry-emerin以温度依赖性方式与ER膜和INM相关。我们现在正计划使用这种新的体外试验来重建emerin，LAP 2和其他TA蛋白到INM的转运。首先，我们可以分析核质转运因子的作用，其在可溶性蛋白质的核输入中的功能是公认的。在稍后的阶段，体外系统将使我们能够识别不同货物的有效运输所需的潜在胞质因子。此外，我们将在完整细胞中采用几种方法，并研究INM的其他蛋白的靶向，根据其氨基酸序列，这些蛋白也是TA蛋白途径的底物。最后，我们计划建立一个定量的显微镜系统来分析蛋白质的运输到INM在体内和体外。总之，拟议的项目将有助于更好地了解核内膜的生物发生。

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.