Functional analysis of a family of TANGO1-like proteins. What is the influence on transport of large cargos and are so-called megacarriers generated?

高等真核生物产生各种各样的蛋白质和分子，其中很大一部分在细胞外到达目的地。为了到达外部，这些蛋白质被翻译成内质网，被运送到高尔基体，并从那里到达细胞表面。从内质网到高尔基体的运输是由COPII囊泡促进的，其大小约为60-90nm。有趣的是，一些被运输的蛋白质非常大。一个例子是胶原蛋白VII，它以400纳米长的刚性三螺旋的形式出现。据推测，这些大型货物是用比通常的囊泡更大的巨型载体运输的。到目前为止，我们既不知道这些囊泡是否真的存在，也不知道它们是如何形成的。然而，已知内质网出口定位蛋白TANGO1在VII型胶原的运输中起作用。当TANGO1被耗尽时，细胞不能再将胶原VII从内质网运送到高尔基体。有趣的是，这些细胞的大量蛋白质分泌不受影响。通过序列比较，我发现了一个TANGO1样（Tali）蛋白家族，它像TANGO1一样定位于内质网出口位点。我想探究这些蛋白是否与TANGO1一起在巨载体的产生中发挥作用，或者它们是否在内质网的囊泡生物发生的其他步骤中被需要。该提案的另一个目的是研究大蛋白质是如何运输的。为了阐明巨载体在大货物运输中的作用，并深入了解其机制，我将在体外重建内质网水平的囊泡生物发生。

Higher eukaryotes produce a wide range of proteins and molecules, a large portion of these have their destination outside of the cell. In order to reach the outside, these proteins are translated into the endoplasmatic reticulum, are transported to the Golgi and from there to the cell surface. The transport from the endoplasmatic reticulum to the Golgi is facilitated by COPII vesicles, that have a defined size of about 60-90nm. Interestingly, some of the transported proteins are quit large. An example is Collagen VII that comes in the form of a 400nm long, rigid triple helix. It has been speculated that these large cargos are transported in megacarriers, larger than usual vesicles. So far it is neither known whether these vesicles truly exist, nor how they form.It is known however, that the ER exit site localized protein TANGO1 plays a role in the transport of Collagen VII. When TANGO1 is depleted, cells can no longer shuttle Collagen VII from the endoplasmatic reticulum to the Golgi. Interestingly, bulk protein secretion is not affected in these cells. Trough sequence comparison I discovered a family of TANGO1-like (Tali) proteins that like TANGO1 localize to ER exit sites. I want to explore if these proteins, together with TANGO1, play a role in the generation of megacarriers or whether they are needed in other steps of vesicle biogenesis at the endoplasmatic reticulum.An additional aim of the proposal is to investigate how large proteins are transported anyway. To clarify the involvement of megacarriers in the transport of large cargo and to gain insight into the mechanism, I will in Vitro reconstitute vesicle biogenesis at the level of the ER.