Mechanism of secretory cargo sorting at the trans-Golgi Network

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Sorting of proteins for transport to the cells surface or for secretion from cells is essential for the extracellular activities of proteins like insulin, collagens, antibodies, surface receptors, secreted proteases, and GPCRs to name a few. A major regulator of intracellular protein distribution is the trans-Golgi network (TGN) that sorts secretory proteins into specific carriers to transport them to their final destination. The sorting of lysosomal hydrolases at the TGN by the mannose-6-phosphate receptor is well understood. However, the sorting mechanism for secreted proteins remains poorly understood. We recently revealed a novel process that links the cytoplasmic actin cytoskeleton to the membrane anchored Ca2+ ATPase SPCA1 and the luminal Ca2+ binding protein Cab45 to sort of a subset of secretory proteins at the TGN. The underlying mechanism regulating the interplay between these components, however, is not clear. Thus we suggest an approach to address this issue of fundamental importance. Mass spectrometry and modern genome editing techniques in human cells, will be applied to identify other components of this sorting pathway. Super-resolution microscopy and DNA-PAINT based multiplexing will be used to map TGN organization of these components. Furthermore live cell microscopy will determine the spatio-temporal organization and its link to Ca2+ dependent sorting of the complexes. Finally we will apply in vitro reconstitution of identified components in cell-free systems and model membranes to identify the minimal machinery required for the sorting of secretory cargoes. Altogether, our findings will reveal the mechanism by which TGN sorts and targets proteins to their respective destination, a process that is essential for cell compartimentation, tissue organization and function.