Activity of the SPCA1 calcium ATPase couples sphingomyelin synthesis to sorting of secretory proteins in the trans-Golgi network

Newly synthesized lipids and secretory proteins are sorted in the trans-Golgi Network (TGN) into secretory vesicles for their transport to the plasma membrane or secretion. Sorting of transmembrane proteins as well as soluble lysosomal hydrolases at the TGN has been studied extensively in the past. However, the molecular mechanisms underlying how lipid metabolism and sorting of secretory proteins are coupled to establish distinct trafficking routes for cargo and lipid transport, are still unknown. A previously described sorting machinery at the TGN is required for packaging of secretory cargos in a Ca2+-dependent manner. The Ca2+-ATPase SPCA1 promotes Ca2+-import in an ADF/Cofilin- and F-actin-dependent manner. The TGN-luminal protein Cab45 together with active Ca2+-import are then required for efficient sorting of secretory cargos. Biochemical oligomerization assays showed that Cab45 selectively binds to its cargo Lysozyme C (LyzC) and reversibly oligomerizes upon Ca2+-addition thereby promoting the sorting of secretory cargo in an oligomerization-driven sorting mechanism. In my thesis I used a comparative proximity biotinylation proteomics approach of isolated sphingomyelin (SM)-rich secretory vesicles to identify Cab45 in these types of carriers. By using live-cell microscopy, Cab45 and its cargo LyzC was shown to exit the Golgi and to be secreted in SM-rich vesicles. Development of a retention using selective hooks (RUSH)-based cargo sorting assay allowed to monitor cargo sorting kinetics of LyzC and the lysosomal hydrolase cathepsin D. Depletion of Cab45 and SM synthesis at the Golgi lead to a significant kinetic sorting delay of LyzC vesicle formation while cathepsin D sorting was unaffected. To assess the effects of local SM synthesis at the TGN on Ca2+-import activity by SPCA1, a FRET-based Ca2+-import assay revealed that disruption of SM synthesis in the TGN significantly decreased Ca2+-flux activity by SPCA1. Furthermore, by combing a UV-crosslink competent metabolic precursor of SM and immunoprecipitation experiments SPCA1 was shown to closely associate with SM in TGN membranes. These results demonstrate that local SM synthesis promotes SPCA1 Ca2+-import activity and thus is required for sorting of secretory proteins into SM-rich vesicles in a Cab45-dependent oligomerization-driven sorting mechanism. These new insights propel our understanding of the Golgi apparatus as the major trafficking hub and cargo sorting station within cells