Dissecting cargo recruitment processes at ER export sites

• Applicant: Dr. Christoph Kaether
• Subject Area: Cell Biology
• Term: from 2020 to 2024
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 431549950

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.

• DFG Programme: Research Grants