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区室的独特性质至关重要。许多疾病都与蛋白质靶向不当有关，因此了解其工作原理将有助于深入了解某些疾病状态。在内膜系统中，这种运动是由小泡介导的，这些小泡从供体隔室中出现，必须融合并将其内容物输送到适当的受体隔室。在这个过程中的最上游事件之一是囊泡识别，涉及大的蛋白质复合物称为拴系因子。一个这样的拴系因子是一个家族的相关复合物称为TRAPP。在酵母中，有三种这样的复合物，称为TRAPP I、II和III。每个复合物都涉及不同的运输事件，TRAPP I在内质网（ER）和高尔基体之间的运输中起作用，TRAPP II在晚期高尔基体的运输中起作用，TRAPP III在自噬中起作用。迄今为止，在人类中仅鉴定出两种TRAPP复合物，代表酵母TRAPP II和III复合物的等价物。使用随机诱变，我们确定了一系列的酵母突变的基因编码的Trs23亚基，发现在所有三个酵母TRAPP复合物。我们专注于一个单一的突变，删除了酵母特异性结构域没有发现在高等真核生物，我们称之为SMS域。该结构域对于蛋白质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不存在于体内，并且仅在细胞裂解时可见。如果这是真的，它将有重要的意义，TRAPP的膜运输事件中的作用，表明TRAPP复合物可以参与多种事件。这将进一步暗示TRAPP复合物的活性存在翻译后调节。本提案力求采用多层面办法解决这些问题。我们将首先建立新的，简单的和定量的检测方法来测量ER和高尔基体之间的运输和高尔基体和质膜在酵母。这将使我们能够测试所有三个TRAPP复合体的功能，以确定它们支持这些隔间之间交通的能力。我们将评估从饥饿细胞（经历自噬）中提取的胞质溶胶是否改变复合物的活性。我们还将关注一个称为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，表明磷酸化在TRAPP功能中的作用。这些研究的结果将导致一个范式的转变，如何拴系因素的功能，以及他们是否确实是特定的一个特定的交通事件。

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.