Dissecting cargo recruitment processes at ER export sites

剖析 ER 出口站点的货物招募流程

• 批准号: 431549950
• 负责人: Dr. Christoph Kaether
• 金额: --
• 依托单位: Leibniz-Institut für Alternsforschung - Fritz-Lipmann-Institut e.V. (FLI)
• 依托单位国家: 德国
• 项目类别: Research Grants
• 资助国家: 德国
• 项目状态: 未结题
• 来源: https://gepris.dfg.de/gepris/projekt/431549950?language=en
• 关键词: Dissecting; cargo; recruitment; processes; ER

A third of all proteins associate with the secretory pathway. Secretory transport starts at the endoplasmic reticulum (ER) at ER exit sites (ERES). Here the COPII coat together with Sar1 and several auxiliary proteins mediates the transport of cargo out of the ER. Upstream of COPII are additional uncharacterized recruitment/filter mechanisms that are inhibited by the secretion inhibitor FLI-06. We performed a genome-wide CRISPR/Cas loss-of-function screen for resistance to FLI-06. Loss of YIPF5 or GOT1B, small ER membrane proteins, mediated resistance to FLI-06. In a complementary genome-wide gain-of-function screen with activated transcription we identified GOT1A, a very close homologue of GOT1B. In a VSVG-EYFP transport assay and in cell proliferation assays we confirmed that loss of YIPF5 or GOT1B or overexpression of GOT1A mediated resistance to FLI-06. YIPF5 is in a complex with YIF1A and YOS1 (also called IER3IP1), the latter being mutated in a certain form of microcephaly. YIPF5 and GOT1A/B interact directly, and both interact with COPII components. The molecular function of these proteins in ER export is not clear.Our hypothesis: YIPF5 and GOT1B are part of a filter or recruitment mechanism that controls the access to ERES and thus ER-export. GOT1A has a similar role as GOT1B but is insensitive to FLI-06.To test the hypothesis we will analyze swapping mutants of GOT1A and B to determine functional domains and to find out what the difference is between them. We will define regions important for interaction of YIPF5 with GOT1A/B using co-immunoprecipitation experiments of swapping/deletion mutants. In established transport assays (VSVG and RUSH) we will investigate to what extend YIPF5 and GOT1A/B interact with cargo and/or COPII and if and how they enter ERES. For this, we will use live-micros¬copy as well as co-immunoprecipitation and deglycosylation assays. To test whether FLI-06 directly binds YIPF5/GOT1B, we will perform co-immunoprecipitation experiments and will conduct a variomics screen using error-prone PCR to identify FLI-06 resistant point mutations. Being part of a filter/recruitment mechanism, YIPF5 and GOT1A/B will interact dynamically with other proteins. To analyze their interactome, we will use BirA-tagged YIPF5 or GOT1B and perform BioID labeling in different conditions: basal, starvation (reduced secretion), blocked by FLI-06 and activated by overexpressed proteins like VSVG. Biotinylated proteins interacting with GOT1B/YIPF5 will be identified by MassSpec. Hits will be validated by co-immunoprecipitation and knockdown experiments. To test the hypothesis that YIPF5/GOT1A/B are indeed part of a filter mechanisms, we will analyze to what extend misfolded mutant LDL-receptor and mutant CFTR, normally ER-retained, will be exported out of the ER in the absence of GOT1B/YIPF5.Taken together, our study investigates mechanisms of cargo selection/recruitment upstream of COPII and the role that YIPF5 and GOT1A/B play therein.

三分之一的蛋白质与分泌途径有关。分泌转运起始于内质网（ER）的内质网出口位点（ERES）。在这里，COPII外壳与Sar 1和几种辅助蛋白一起介导货物从ER中转运出来。COPII的上游是额外的未表征的募集/过滤机制，这些机制被分泌抑制剂FLI-06抑制。我们进行了全基因组CRISPR/Cas功能丧失筛选，以获得对FLI-06的抗性。YIPF 5或GOT 1B（小ER膜蛋白）的缺失介导了对FLI-06的抗性。在具有激活转录的互补全基因组功能获得性筛选中，我们鉴定了GOT 1A，它是GOT 1B的非常接近的同源物。在VSVG-EYFP转运测定和细胞增殖测定中，我们证实YIPF 5或GOT 1B的缺失或GOT 1A的过表达介导了对FLI-06的抗性。YIPF 5与YIF 1A和YOS 1（也称为IER 3 IP 1）形成复合体，后者以某种形式的小头畸形发生突变。YIPF 5和GOT 1A/B直接相互作用，并且都与COPII组件相互作用。我们的假设是：YIPF 5和GOT 1B是一种过滤或募集机制的一部分，它控制着ERES的进入，从而控制着ER的输出。GOT 1A与GOT 1 B具有相似的功能，但对FLI-06不敏感。为了验证这一假设，我们将分析GOT 1A和B的交换突变体，以确定功能结构域，并找出它们之间的差异。我们将使用交换/缺失突变体的免疫共沉淀实验来确定YIPF 5与GOT 1A/B相互作用的重要区域。在已建立的转运试验（VSVG和RUSH）中，我们将研究YIPF 5和GOT 1A/B与货物和/或COPII相互作用的程度，以及它们是否以及如何进入ERES。为此，我们将使用活显微镜以及免疫共沉淀和去糖基化测定。为了测试FLI-06是否直接结合YIPF 5/GOT 1B，我们将进行免疫共沉淀实验，并将使用易错PCR进行变异组学筛选以鉴定FLI-06抗性点突变。作为过滤/募集机制的一部分，YIPF 5和GOT 1A/B将与其他蛋白质动态相互作用。为了分析它们的相互作用组，我们将使用BirA标记的YIPF 5或GOT 1B，并在不同条件下进行BioID标记：基础，饥饿（分泌减少），被FLI-06阻断和被过表达的蛋白质如VSVG激活。将通过MassSpec鉴定与GOT 1B/YIPF 5相互作用的生物素化蛋白质。将通过免疫共沉淀和敲低实验验证命中。为了验证YIPF 5/GOT 1A/B确实是过滤机制的一部分这一假设，我们将分析在GOT 1B/YIPF 5缺失的情况下，错误折叠的突变型LDL受体和突变型CFTR（通常保留在ER上）将在多大程度上输出到ER外。