The functions of the yeast TRAPP complexes and the longin domain-containing protein Tca17 in membrane traffic

蛋白质和脂质在细胞区室之间的运动对于建立和维持细胞区室的独特性至关重要。许多疾病都与不当的蛋白质靶向有关，因此了解其工作原理将有助于了解某些疾病状态。在膜系统中，运动是由从供体隔室中产生的小泡介导的，它们必须融合并将其内容物运送到适当的受体隔室。在这一过程中最上游的事件之一是囊泡识别，涉及被称为系绳因子的大蛋白质复合物。一个这样的束缚因子是一个叫做TRAPP的相关复合物家族。在酵母中，有三种这样的复合物，称为TRAPP I， II和III。每种复合物都参与不同的转运事件，其中TRAPP I参与内质网（ER）和高尔基体之间的转运，TRAPP II参与高尔基体晚期的转运，TRAPP III参与自噬。迄今为止，只有两种TRAPP复合物在人类中被鉴定出来，代表了酵母TRAPP II和III复合物的等价物。利用随机诱变技术，我们在酵母TRAPP复合物中发现了一系列编码Trs23亚基的基因突变。我们专注于一个单一的突变，它删除了一个在高等真核生物中没有发现的酵母特异性结构域，我们称之为SMS结构域。该结构域对蛋白在酵母中的功能不是必需的，但确实影响TRAPP I在重组系统和体内的组装。因此，我们假设酵母TRAPP I在体内不存在，仅在细胞裂解时可见。如果这是真的，它将对TRAPP在膜转运事件中的作用产生重要影响，这表明TRAPP复合物可能参与多种事件。这将进一步暗示TRAPP复合物的活性存在翻译后调控。本建议试图用多方面的办法来解决这些问题。我们将首先建立新的、简单的和定量的方法来测量内质网和高尔基体之间以及酵母中高尔基体和质膜之间的运输。这将使我们能够测试所有三种TRAPP复合物的功能，以支持这些车厢之间的交通。我们将评估从饥饿细胞（正在进行自噬）中提取的细胞质是否会改变复合物的活性。我们还将关注一个名为Tca17的亚基，来自高等真核生物的证据表明它可能在TRAPP功能中发挥调节作用。事实上，我们已经发现了TCA17基因和蛋白磷酸酶之间的遗传相互作用，这表明磷酸化在TRAPP功能中起作用。这些研究的结果将导致关于捆绑因子如何起作用以及它们是否确实针对某一特定运输事件的范式转变。

The movement of proteins and lipids between cellular compartments is crucial for the establishing and maintaining the distinct nature of the compartments. Many diseases are linked to improper protein targeting, thus understanding how this works will provide insight into certain disease states. In the endomembrane system, the movement is mediated by small vesicles that emerge from a donor compartment and must fuse and deliver their contents to the appropriate acceptor compartment. One of the most upstream events in this process is vesicle recognition that involves large protein complexes referred to as tethering factors. One such tethering factor is a family of related complexes called TRAPP. In yeast, there are three such complexes referred to as TRAPP I, II and III. Each complex has been implicated in different transport events, with TRAPP I functioning in transport between the endoplasmic reticulm (ER) and Golgi, TRAPP II functioning in transport at the late Golgi and TRAPP III functioning in autophagy. To date, only two TRAPP complexes have been identified in humans, representing the equivalents of the yeast TRAPP II and III complexes. Using random mutagenesis, we identified a series of yeast mutations in the gene encoding the Trs23 subunit, found in all three yeast TRAPP complexes. We focused on a single mutation that deleted a yeast-specific domain not found in higher eukaryotes that we called the SMS domain. This domain was not essential for the function of the protein in yeast but did affect the assembly of TRAPP I in a recombinant system and in vivo. Thus we hypothesize that yeast TRAPP I does not exist in vivo and is only seen upon cell lysis. If this is true it will have important implications for the role of TRAPP in membrane trafficking events, suggesting that TRAPP complexes can be involved in multiple events. This would further imply that there is post-translational regulation of the activity of TRAPP complexes. The present proposal seeks to address these questions using a multifaceted approach. We will first establish new, simple and quantitative assays to measure transport between the ER and Golgi and between the Golgi and plasma membrane in yeast. This will allow us to test the function of all three TRAPP complexes for their ability to support traffic between these compartments. We will assess whether cytosol taken from starved cells (undergoing autophagy) alters the activity of the complexes. We will also focus on a subunit called Tca17 for which evidence from higher eukaryotes suggests it may play a regulatory role in TRAPP function. Indeed, we have found a genetic interaction between the TCA17 gene and a protein phosphatase, suggesting a role for phosphorylation in TRAPP function. The results of these studies will result in a paradigm shift as to how tethering factors function and whether they are indeed specific for one particular transport event.