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Characterising the factors involved in ER-to-Golgi anterograde trafficking 10.17863/CAM.93885 Yakunin, Igor Munro, Sean The Golgi apparatus is a major hub of protein transport, modification and sorting. Secreted and membrane proteins are synthesised in the Endoplasmic Reticulum (ER) and are packaged into carrier vesicles at ER exit sites. The carriers are transported to the cis-Golgi apparatus, where they fuse with the Golgi membrane and release cargo into the Golgi apparatus for further processing. Vesicle recognition at the Golgi apparatus occurs in two steps: long-range recognition via tethering and short-range fusion. The focus of my PhD revolved around the organisation of ER-to-Golgi traffic with a focus on the mechanisms underlying tethering of ER-to-Golgi carriers and the role of tethering in trafficking. Previous work had shown that two long coiled-coil proteins, GMAP210 and GM130, are able to capture vesicles arriving from the ER. The mechanism by which these two proteins recognise other membranes is unclear. To study the role of GM130, I generated a stable GM130 knockout cell line using CRISPRCas9 gene editing, and observed a striking disruption in ER exit site morphology. The Golgi-apposed ER exit sites (which I term central ER exit sites), were completely dispersed, with only peripheral ER exit sites remaining. This phenotype was also recreated when p115, a GM130 binding partner of unclear function, was knocked down using siRNA. The ER exit site dispersal phenotype was unique to GM130, as the loss of GMAP210 and giantin did not produce a similar effect. Therefore, GM130 possesses a unique role among the cis-Golgi coiled-coil proteins of organising ER exit sites. I next determined which regions of GM130 are necessary for ER exit site localisation. I have discovered a novel Rab2 binding site at the C terminus of GM130, that can direct GM130 to the Golgi apparatus in addition to its canonical GRASP65-mediated localisation mechanism. I have also identified the 154-447 region of GM130 as contributing to ER exit site localisation, possibly through the recruitment of the Ste20 kinases. Having shown that loss of p115 produces the same ER exit site phenotype as loss of GM130, I looked for novel binding partners of p115. This revealed an interaction between p115 and Sec16A, an ER exit site protein. I then mapped the parts of Sec16A and p115 required for their interaction. This allowed me to generate mutants of both Sec16A and p115 that no longer bound to each other, and I proved that the interaction is direct using an in-vitro direct binding assay. I next investigated the role of Sec16A, p115 and GM130 in trafficking. Using a lentiviral CRISPR method, I was able to generate transient knockouts of these 3 proteins. Loss of all three proteins resulted in a marked disruption to ER exit site organisation. I then used the Retention Using Streptavidin Hooks (RUSH) assay to look at the effect of the loss of these proteins on cargo trafficking. While the effect of GM130 was moderate, the loss of p115 and Sec16A produced a complete block to anterograde trafficking. I was then able to investigate the importance of different protein-protein interactions for anterograde trafficking. Using different mutants of p115, I showed that the loss of binding between p115 and GM130 or Sec16A has the same effect, with the double mutant having no additional effect. In summary, I have identified and characterised a novel direct protein-protein connection between ER exit sites and the Golgi apparatus, and shown that this connection has a role in anterograde trafficking. University of Cambridge 2022-09-01 Thesis Doctoral Doctor of Philosophy (PhD) eng https://www.repository.cam.ac.uk/handle/1810/346464 https://apollo8-f-pro.lib.cam.ac.uk/bitstreams/31ae3d15-c38b-46a0-a756-53a4bc74a325/download 72ab9ab666d9e47447571738ef02a102 https://www.rioxx.net/licenses/all-rights-reserved/ Golgi apparatus Endoplasmic Reticulum Protein trafficking GM130 p115/USO1 Sec16A