Regulation, organization and physiological implications of SPPL3-mediated Golgi glycosylation enzyme proteolysis and secretion

• Applicant: Dr. Matthias Voss
• Subject Area: Biochemistry
• Term: since 2025
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 568973770

The Golgi apparatus is a central organelle of the eukaryotic secretory pathway. In humans, it is equipped with close to 200 different membrane-anchored enzymes that ensure proper glycan decoration of secreted and membrane proteins as well as lipids. Importantly, aberrations in Golgi glycosylation can lead to severe disease. The conserved and constitutively expressed Golgi-localized intramembrane protease SPPL3 cleaves Golgi glycosylation enzymes off their membrane anchors, enabling their subsequent secretion. Accordingly, loss of SPPL3 activity affects numerous glycosylation-dependent processes in vitro, often with profound translational implications. It is, however, unknown which cellular mechanisms ensure Golgi localization of SPPL3, how spatial access of SPPL3 to its substrates is regulated and which factors determine whether and to what extent a given Golgi membrane protein is cleaved by SPPL3. In fact, in light of the widespread susceptibility of Golgi glycosylation enzymes to SPPL3-mediated intramembrane proteolysis, it is conceivable that SPPL3 contributes to Golgi proteostasis and the correct intra-Golgi steady-state localization of glycosylation enzymes. Finally, SPPL3 cleavage products can be found in body fluids such as blood and Golgi enzymes thus may have a currently not recognized function in the extracellular space once they have been liberated by SPPL3. The proposed project aims on the one hand to dissect on a cell-intrinsic level how SPPL3 intra-Golgi localization and its substrate access are governed. To this end, interaction partners of SPPL3 will be identified through proximity biotinalytion of steady-state Golgi-localized and dynamically trafficking SPPL3 and a parallel candidate-based approach. The identified molecular interactions of SPPL3 will be subject to in-depth functional characterization to establish their capacity to regulate Golgi localization of SPPL3 and the underlying molecular mechanism. In addition, we will comprehensively examined alterations in SPPL3-dependent Golgi enzyme secretion following targeting of Golgi proteins known to regulate trafficking and localization of Golgi glycosyltransferases to molecularly understand how substrate-protease encounters in the Golgi apparatus are regulated and how this could be exploited to fine-tune SPPL3 activity. In order to get first insights into the physiological function of Golgi glycosylation enzymes secreted in a SPPL3-dependent manner, mouse models enabling an inducible, tissue-specific deletion of Sppl3 expression in vivo will be used to investigate the tissue source of blood-borne Golgi enzymes and to explore whether such SPPL3 cleavage products in peripheral blood contribute to glycan re-modelling in the extracellular space. Collectively, this will provide insights into the organization and implications of Golgi enzyme secretion and will help to predict how changes in SPPL3 expression precisely affect glycosylation-dependent physiological processes.

• DFG Programme: Research Grants