How lysosomes SNARE autophagosomes

Credit: ComStock

The fusion of lysosomes with other cellular compartments must be tightly regulated to control the degradative functions of lysosomal hydrolases. Specific membrane fusion between lysosomes and late endosomes — involving SNARE complexes — has been well studied, but little is known about the mechanisms of lysosome–autophagosome fusion. This paper identifies syntaxin 17 (STX17) as an autophagosomal SNARE. STX17 has a unique structure characterized by two transmembrane domains that mediates STX17 recruitment only to fully formed autophagosomes.

In mouse embryonic fibroblasts (MEFs), STX17 colocalized with the autophagosome marker LC3 under starvation conditions (which are known to induce autophagy) but not in resting cells. A truncation mutant of STX17 lacking the amino-terminal SNARE domain also colocalized with LC3 in starved cells, which shows that the autophagosomal localization of STX17 is not a result of transfer from another organelle during SNARE-mediated membrane fusion.

Knockdown of STX17 resulted in the accumulation of LC3⁺ autophagosomes in human HeLa cells even under normal growth conditions. These LC3⁺ autophagosomes did not colocalize with late endosomal or lysosomal markers and did not show any features of cytoplasmic degradation, which suggests that fusion between autophagosomes and late endosomes and/or lysosomes is blocked in the absence of STX17.

In other systems, membrane fusion requires a complex of Qa-, Qb-, Qc- and R-SNARE proteins. STX17 is a Qa-SNARE and the authors observed that it functions with the lysosomal R-SNARE vesicle-associated membrane protein 8 (VAMP8) and the Qbc-SNARE synaptosomal-associated protein 29 (SNAP29) to mediate membrane fusion. STX17 co-immunoprecipitated with VAMP8 and SNAP29, and endogenous interaction between STX17 and VAMP8 or SNAP29 was enhanced under starvation conditions. Moreover, knockdown of VAMP8 or SNAP29 induced the accumulation of autophagosomes, presumably as a result of decreased lysosome-mediated degradation.

“fusion between autophagososmes and late endosomes and/or lysosomes is blocked in the absence of STX17”

Importantly, lysosome–autophagosome membrane fusion must not occur before complete closure of the autophagosome membrane if the cytoplasmic contents are to be efficiently degraded. STX17 did not colocalize with unclosed autophagosomes but was recruited specifically to the outer membrane of completed autophagosomes. This specific recruitment was shown to be mediated by the unusual structure of STX17. Whereas most SNARE proteins have a single transmembrane domain with their carboxyl terminus in the organelle lumen, STX17 has two poorly hydrophobic transmembrane domains that form a hairpin structure, and both the C terminus and the N terminus of STX17 were shown to be cytosolic. A mutant of STX17 in which the two transmembrane domains had been replaced with the transmembrane domain of an ER-resident protein could not localize to autophagosomes, which shows that these domains are involved in specific targeting of this SNARE. Both transmembrane domains contain Gly zipper motifs, which when disrupted by Gly to Leu substitution resulted in significantly decreased colocalization with LC3⁺ autophagosomes.

The authors showed that a large amount of STX17 is present in the cytosol — which they suggest is owing to its low hydrophobicity compared with other SNARE proteins — from where it might be recruited directly to autophagosomes after membrane closure. This calls into question the requirement for autophagosomes to fuse with endosomes to acquire SNARE proteins before lysosomal fusion. However, the mechanism by which the two transmembrane domains of STX17 might facilitate specific recruitment from the cytosol requires further investigation.