Dissecting cargo recruitment processes at ER export sites

三分之一的蛋白质与分泌途径有关。分泌运输始于内质网（ER）和内质网出口位点（ERES）。在这里，COPII外壳与Sar1和几个辅助蛋白一起介导货物从内质网转运。COPII的上游是额外的未表征的募集/过滤机制，被分泌抑制剂fl -06抑制。我们对FLI-06的抗性进行了全基因组CRISPR/Cas功能丧失筛选。小ER膜蛋白YIPF5或GOT1B的缺失介导了对fl -06的抗性。在具有激活转录的互补全基因组功能获得筛选中，我们鉴定出GOT1A，这是GOT1B的一个非常接近的同源物。在VSVG-EYFP转运试验和细胞增殖试验中，我们证实了YIPF5或GOT1B的缺失或GOT1A的过表达介导了对fl -06的抗性。YIPF5与YIF1A和YOS1（也称为IER3IP1）处于一个复合体中，后者在某种形式的小头畸形中发生突变。YIPF5和GOT1A/B直接相互作用，两者都与COPII组分相互作用。这些蛋白在内质网输出中的分子功能尚不清楚。我们的假设是：YIPF5和GOT1B是过滤或招募机制的一部分，控制对ERES的访问，从而控制er输出。GOT1A的作用与GOT1B相似，但对fl -06不敏感。为了验证这一假设，我们将分析GOT1A和B的交换突变体，以确定功能域，并找出它们之间的区别。我们将使用交换/删除突变体的共免疫沉淀实验来确定YIPF5与GOT1A/B相互作用的重要区域。在已建立的运输试验（VSVG和RUSH）中，我们将调查YIPF5和GOT1A/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，我们将使用活体显微复制以及共免疫沉淀和去糖基化检测。为了测试FLI-06是否直接结合YIPF5/GOT1B，我们将进行共免疫沉淀实验，并使用易出错PCR进行变异组学筛选，以鉴定FLI-06耐药点突变。作为过滤/招募机制的一部分，YIPF5和GOT1A/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，我们将使用bira标记的YIPF5或GOT1B，并在不同的条件下进行BioID标记：基础、饥饿（分泌减少）、被FLI-06阻断和被VSVG等过表达蛋白激活。与GOT1B/YIPF5相互作用的生物素化蛋白将通过MassSpec进行鉴定。hit将通过共免疫沉淀和敲低实验进行验证。为了验证YIPF5/GOT1A/B确实是过滤机制的一部分的假设，我们将分析在没有GOT1B/YIPF5的情况下，错误折叠的突变体ldl -受体和通常保留ER的突变体CFTR将在多大程度上从ER中输出。综上所述，我们的研究探讨了COPII上游的货物选择/招募机制以及YIPF5和GOT1A/B在其中所起的作用。

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.