Mechanisms in COPII-Dependent Transport

项目摘要 精确的蛋白质在分泌途径中的运输对细胞的功能和生长至关重要。我们的研究 该计划的重点是依赖于外壳的分选机制，这种机制推动蛋白质在 内质网和高尔基复合体。新生的分泌蛋白在内质网和 然后，完全折叠的蛋白质被选择性地包装到COPII包被的囊泡中，以便顺行运输到 高尔基情结。这条向前的通道被来自高尔基山脉的逆行运输所平衡，高尔基山脉 选择性地在COPI包被的囊泡中将蛋白质返回到内质网。确保只递送折叠的内分泌物 蛋白质，这一被称为内质网质量控制的过程在很大程度上保留了内质网中的新生蛋白质，直到正确 折叠或以末端错误折叠的蛋白质为目标进行降解。维护的协调机制 细胞器识别、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被很好地理解。 我们确定了一组跨膜货物受体，它们在毛层依赖的分类和质量中起作用。 在早期分泌途径中的控制。这项研究计划将解决有关货物受体如何 识别它们的客户，并催化蛋白质的净定向运输。在过去的资助期内，我们 将Erv41-Erv46复合体表征为逆行受体，通过 Erv46中一个保守的硫氧还蛋白样结构域的活性。我们最近的发现表明Erv41-Erv46 受体还可以回收错误折叠的分泌蛋白，这些蛋白已经离开了内质网。这样做的具体目的是 提案将测试Erv46亚基识别以下显示的疏水特征的分子模型 在高尔基车厢的低pH环境中逃逸的货物。在返回ER后，氧化还原活动在 Erv46硫氧还蛋白样结构域在中性pH下从受体释放结合的货物。通过这些 在早期分泌途径中，机制、pH和氧化还原梯度驱动有效的蛋白质分选。这 模型将在以下实验目标中进行测试。目的1：确定Erv41的分子机制- Erv46货物在Golgi隔间的pH值降低时结合。目标2：确定 在ER中，货物从Erv41-Erv46高效释放。目标3：测试Erv41-Erv46识别的模型 并直接结合到错误折叠的货物上，以便主动回收到急诊室。我们将通过以下方式严格测试我们的模型 利用迭代的遗传、细胞和生化方法监测体内、细胞内的蛋白质功能 自由分析和纯化因子的重建实验中。定义了分子机制 潜在的保守蛋白质分选过程将提供对细胞组织和 有助于治疗与分泌途径功能有关的人类疾病。

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.