Investigating the role of Scyl1 in the secretory pathway and how its loss causes disease in humans

Abstract

The secretory pathway is responsible for the synthesis, modification and transport of a range of proteins and lipids that are found within the endomembrane system and also secreted to the extracellular space. Scyl1 is a protein that localises predominantly to the ER-Golgi intermediate complex (ERGIC) and cis-Golgi, with a pool also found at the trans-Golgi network. It is known to interact with coat protein complex I (COPI), Arf GTPases and GORAB, and based on these interactions is believed to play a role in retrograde trafficking within the Golgi, by acting as a COPI scaffold protein. Loss of functional Scyl1 protein in humans leads to the multi-systemic CALFAN (low γ-glutamyl-transferase cholestasis, acute liver failure and neurodegeneration) syndrome, which is characterised by ataxia, peripheral neuropathy, acute liver failure, cholestasis and skeletal abnormalities. Both the cellular function of Scyl1 and the mechanism by which its mutation leads to CALFAN syndrome are poorly defined. In this study the roles of Scyl1 within the secretory pathway were investigated. We report that Scyl1 localises to ER exit sites (ERES) where it binds to the coat protein complex II (COPII) outer coat protein Sec31A. In addition, we found COPI at these sites in close association with both Scyl1 and Sec31A. Utilising a mitochondrial relocation assay we mapped the binding sites within Scyl1 and Sec31A that are responsible for these interactions. Using patient-derived CALFAN fibroblasts and mouse embryonic fibroblasts (MEFs), in which Scyl1 was knocked out using CRISPR/Cas9, we were unable to find a role for Scyl1 in the trafficking of type I collagen, despite its retention within the ER under certain conditions. We also confirm an interaction between Scyl1 and the COG complex, a multi-subunit tethering complex that plays a key role in the recycling of glycosylation enzymes within the Golgi. Alongside this, we observed aberrant glycosylation of LAMP1 and LAMP2 in both patient-derived CALFAN fibroblasts and Scyl1 KO MEFs, and we found N-propeptide processing of type-I procollagen was altered compared to that in wild-type fibroblasts, suggesting a role for Scyl1 in efficient trafficking of enzymes within the Golgi. Taken together, we show that Scyl1 can interact with a number of proteins within the secretory pathway. Our data is consistent with potential roles for Scyl1 in both ER to Golgi trafficking and in retrograde trafficking of Golgi-resident enzymes between Golgi cisternae. On this basis three models for the function of Scyl1 in anterograde trafficking from the ER are proposed, alongside a model for the role of Scyl1 in retrograde trafficking, based on its interaction with COPI and the COG complex.

Date of Award	1 Aug 2022
Original language	English
Awarding Institution	The University of Manchester
Supervisor	Philip Woodman (Supervisor) & Martin Lowe (Supervisor)