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Characterization of a novel sorting pathway at the Trans Golgi Network (TGN) (SORTMECH)
Start date: Apr 1, 2013, End date: Mar 31, 2017 PROJECT  FINISHED 

"The Trans Golgi Network (TGN) is the central sorting station for newly synthesised proteins in the cell. Similar to a postal distributioncenter, incoming cargo is selected, sorted, and packaged to be carried to its final cellular destination by the postman in charge. Due to theinvolvement of numerous different cargoes and pleiomorhic carriers for the different destinations this process of protein sorting at the TGNis highly sophisticated. While the sorting event of lysosomal hydrolases at the TGN via mannose-6-phosphate receptor and clathrin-coatedvesicles to the lysosome is well characterised, the relevant mechanism for other luminal proteins at the TGN remains elusive.We recently discovered a new role for the actin severing protein twinstar in Drosophila and its orthologs in yeast (cof1) and mammaliancells (ADF and cofilin1) in regulation of luminal protein sorting at the TGN. This process is a novel calcium dependent sorting mechanismthat directly links the actin cytoskeleton and ADF/cofilin to the TGN localized P-Type calcium ATPase SPCA1.In the project described herein we will characterize the new sorting mechanism in detail. New biochemical tools will identify components ofthe sorting machinery on both cytosolic and luminal faces of the TGN. Cutting edge Mass Spectrometry will dissect the composition of TGNassociated or luminal protein complexes. FLIM experiments will elucidate the temporal and spatial dynamics of the complexes. Highthroughput screening in yeast will yield new components on a genome wide level. Functional assays utilising siRNA and reconstitution willdefine their precise role in cargo sorting in vitro and in vivo. The complementary investigation of a Drosophila model will elucidate thephysiological relevance of the process in the intact organism and its impact on developmental biology. Altogether, this work represents anessential experimental set up to answer a yet completely unresolved question in cell biology."
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