Autophagy is an important growth regulatory process in eukaryotic cells. Dysfunctional autophagy can cause tumorigenesis and neurodegenerative diseases due to accumulation of defective cellular substructures and protein aggregates.Autophagy involves formation and elongation of an isolation membrane which sequesters cytopasmic material within double membrane-enclosed vesicles (autophagosomes). The autophagosomes eventually fuse with lysosomes where the sequestered material is degraded and is released back to the cytosol for reuse. Autophagy is induced by stresses like starvation and is mediated by core autophagy related (Atg) proteins, including the transmembrane protein mAtg9 which is required for phagophore formation. Since mAtg9 is the only transmembrane protein involved in autophagy, it has been suggested to bring the membrane to the phagophore assembly site. Autophagosome formation also requires the ubiquitin-like protein LC3. Upon induction of autophagy, cytosolic LC3 (LC3-I) becomes conjugated to phosphatidylethanolamine (PE) in the phagophore membrane (LC3-II). The conjugated LC3 on the inner membrane of the autophagosome is degraded upon fusion with a lysosome. LC3 can therefore be used as an autophagic marker. Another protein used as a marker for autophagy is the cargo receptor p62, which binds to LC3 and is constitutively degraded by autophagy. Class III PI 3-kinase and PI3P are also required for autophagy. PX domain containing proteins are known to bind to PI3P and other phosphoinositides and are found to play different roles in membrane trafficking. In order to assess whether any of these proteins are involved in autophagy a high-through-put screen was performed in the lab using a siRNA library against all human PX domain proteins where autophagosome formation was used as a read out. We found that depletion of several of the candidates either inhibited or enhanced autophagy.
One of these candidates was the PX-BAR protein SNX18. In the present study, SNX18 was studied and characterized in order to obtain a better understanding of its role in autophagy. siRNA mediated depletion of SNX18 strongly inhibited autophagy as shown by a decreased level of LC3-II. We also found that p62 degradation was reduced upon SNX18 depletion, further indicating a role for SNX18 in autophagy. Treatment with the lysosomal inhibitor bafilomycin A1 led to the suggestion that SNX18 is involved in the early stages of autophagy. As determined by immunofluorescence, endogenous SNX18 was found to colocalize with the important mAtg9 at the Golgi network under normal conditions. Interestingly, SNX18 was found to disperse from the Golgi upon starvation and induction of autophagy. To where it redistributes after starvation is yet to be determined. Moreover, SNX18 was found to interact with LC3 and the LC3 homologue GABARAP. Three potential LIRs were mapped within the unstructured region of SNX18. The significance of this interaction remains to be investigated further.
Taken together, the data suggest an important role for SNX18 in autophagy. The findings of reduced LC3-II levels and p62 degradation upon SNX18 depletion together with the observed colocalization with mAtg9 and interaction with LC3, both required for autophagosome formation, indicate that SNX18 is involved in the early stages of autophagy.