Mechanisms in COPII-Dependent Transport

项目摘要 分泌途径中蛋白质的准确转运对细胞功能和生长至关重要。我们的研究 该计划的重点是涂层依赖的分选机制，驱动蛋白质之间的运输 内质网（ER）和高尔基复合体。新生分泌蛋白在ER翻译， 然后将完全折叠的蛋白质选择性地包装到COPII包被的囊泡中，用于顺行运输， 高尔基复合体。这一前向途径通过高尔基体的逆向转运来平衡， 选择性地将蛋白质返回到COPI包被的囊泡中的ER。为了确保仅递送折叠的分泌物， 蛋白质，一个被称为ER质量控制的过程在很大程度上保留了ER中的新生蛋白质， 折叠的或靶向末端错误折叠的蛋白质进行降解。协调机制， 细胞器身份、推进折叠的生物合成货物和保留错误折叠的蛋白质还没有很好地理解。 我们鉴定了一组跨膜货物受体，其在外套依赖性分选和质量中起作用。 控制早期分泌途径。这项研究计划将解决货物受体如何 识别它们的客户并催化蛋白质的净定向流量。在过去的一段时间里，我们 Erv 41-Erv 46复合物是一种逆行受体，通过 Erv 46中保守的硫氧还蛋白样结构域的活性。我们最近的发现表明Erv 41-Erv 46 受体还回收已经离开ER的错误折叠的分泌蛋白。具体目标是 该提案将测试Erv 46亚基识别由Erv 46亚基显示的疏水特征的分子模型。 逃逸货物在低pH环境中的高尔基室。返回ER后， Erv 46硫氧还蛋白样结构域在中性pH下从受体释放结合的货物。 在早期分泌途径中，pH和氧化还原梯度驱动有效的蛋白质分选机制。这 模型将在以下实验目标中进行测试。目的1：明确Erv 41的分子机制。 Erv 46在Golgi隔室pH降低时的货物结合。目标2：确定 在ER中，货物从Erv 41-Erv 46有效释放。目标3：测试Erv 41-Erv 46识别的模型 并直接与错误折叠的货物结合，以主动回收到ER。我们将严格测试我们的模型， 利用迭代的遗传、细胞和生物化学方法来监测体内、细胞中的蛋白质功能， 游离测定和用纯化因子的重构实验。定义分子机制， 保守的蛋白质分选过程的基础将提供对细胞组织的基本见解， 有助于治疗与分泌途径功能相关的人类疾病。

Project Summary Accurate protein transport in the secretory pathway is vital for cell function and growth. Our research program is focused on coat-dependent sorting mechanisms that drive protein trafficking between the endoplasmic reticulum (ER) and Golgi complex. Nascent secretory proteins are translated at the ER and then fully folded proteins are selectively packaged into COPII coated vesicles for anterograde transport to the Golgi complex. This forward pathway is balanced by retrograde transport from the Golgi, which selectively returns proteins to the ER in COPI coated vesicles. To ensure delivery of only folded secretory proteins, a process known as ER quality control largely retains nascent proteins in the ER until correctly folded or targets terminally misfolded proteins for degradation. The coordinated mechanisms that maintain organelle identity, advance folded biosynthetic cargo and retain misfolded proteins are not well understood. We identified a set of transmembrane cargo receptors that function in coat-dependent sorting and quality control in the early secretory pathway. This research plan will address key questions on how cargo receptors recognize their clients and catalyze net directional traffic of proteins. In the past funding period, we characterized the Erv41-Erv46 complex as a retrograde receptor that retrieves ER-resident proteins through the activity of a conserved thioredoxin-like domain in Erv46. Our recent findings indicate the Erv41-Erv46 receptor also retrieves misfolded secretory proteins that have exited the ER. The specific aims of this proposal will test the molecular model that the Erv46 subunit recognizes hydrophobic features displayed by escaped cargo in the low pH environment of Golgi compartments. After return to the ER, redox activity on the Erv46 thioredoxin-like domain releases bound cargo from the receptor at neutral pH. Through these mechanisms, pH and redox gradients drive efficient protein sorting in the early secretory pathway. This model will be tested in the following experimental aims. Aim 1: Define the molecular mechanisms of Erv41- Erv46 cargo binding at reduced pH of the Golgi compartment. Aim 2: Determine the mechanism by which cargo is efficiently released from Erv41-Erv46 in the ER. Aim 3: Test the model that Erv41-Erv46 recognizes and binds directly to misfolded cargo for active retrieval to the ER. We will rigorously test our models by exploiting iterative genetic, cellular and biochemical approaches to monitor protein function in vivo, in cell free assays and in reconstitution experiments with purified factors. Defining the molecular mechanisms that underlie conserved protein sorting processes will provide fundamental insights on cellular organization and contribute to treatments for human diseases connected to secretory pathway function.