Investigating the function and regulation of STX19

Soluble NSF attachment protein receptors (SNAREs) are the core machinery involved in membrane fusion. There are 39 SNAREs in humans that drive the fusion of different membranes and intracellular compartments, however, the machinery that regulates fusion between post-Golgi vesicles and the plasma membrane remains unclear. Syntaxin 19 (STX19) is a poorly characterised post-Golgi SNARE that localises to tubular recycling endosomes and the plasma membrane. There is limited literature describing the function of STX19 but previous studies have suggested a role in the secretion of soluble and membrane-anchored cargos. It remains unclear which pathways STX19 functions on and how STX19 function is regulated. The main aim of this study was to gain insight into the function and regulation of STX19. To do so, we used mitochondrial re-routing assays to investigate potential STX19 interactors. We found that STX19 is likely regulated by Munc18-2 through interaction at a well-conserved peptide in the STX19 N-terminus. We also validated the STX19 interaction with kinetochore-localised protein, ZWINT, and spectraplakin protein, Dystonin (DST). Additionally, we used overexpression studies to analyse the effect of STX19 expression on its SNARE binding partners and different compartmental markers. STX19 expression alters the steady-state distribution of VAMP8 and lysosomal markers, CD63 and LAMP1. We also found STX19 expression results in a striking reduction in the levels of autophagosomal markers, LC3 and P62 suggesting STX19 may function in an autophagy-dependent pathway. Finally, we have established a physiological model for studying STX19 function based on human keratinocytes. We found that STX19 is expressed upon calcium-induced differentiation and localises to distinct polarised regions of the plasma membrane in support of STX19 playing a role in the fusion of vesicles with the plasma membrane. Excitingly, we found that STX19 localises in close proximity to the activated serine/threonine kinase, AKT suggesting that STX19 could function in AKT-dependent pathways.