LAMP-2 is required for incorporating syntaxin-17 into autophagosomes and for their fusion with lysosomes

Abstract

Autophagy is an evolutionarily conserved process used for removing surplus and damaged proteins and organelles from the cytoplasm. The unwanted material is incorporated into autophagosomes that eventually fuse with lysosomes, leading to the degradation of their cargo. The fusion event is mediated by the interaction between the Qa-SNARE syntaxin-17 (STX17) on autophagosomes and the R-SNARE VAMP8 on lysosomes. Cells deficient in lysosome membrane-associated protein-2 (LAMP-2) have increased numbers of autophagosomes but the underlying mechanism is poorly understood. By transfecting LAMP-2-deficient and LAMP-1/2­-double-deficient mouse embryonic fibroblasts (MEFs) with a tandem fluorescent-tagged LC3 we observed a failure of fusion between the autophagosomes and the lysosomes that could be rescued by complementation with LAMP-2A. Although we observed no change in expression and localization of VAMP8, its interacting partner STX17 was absent from autophagosomes of LAMP-2-deficient cells. Thus, LAMP-2 is essential for STX17 expression by the autophagosomes and this absence is sufficient to explain their failure to fuse with lysosomes. The results have clear implications for situations associated with a reduction of LAMP-2 expression.

Role and mechanisms of autophagy in acetaminophen‐induced liver injury

“…LAMP‐2, a lysosomal membrane protein, is also required for the fusion of autophagosomes with lysosomes. Lack of LAMP‐2 prevents the localization of STX17 and SNAP‐29 on the autophagosomes resulting in defective fusion of autophagosomes with lysosomes . After the sequestered cargo is delivered inside the lysosome, it is broken down by resident hydrolases of the lysosome and the resulting macromolecules are released back into the cytosol as new energy sources and building blocks for cell survival (Figure ).…”

“…Lack of LAMP-2 prevents the localization of STX17 and SNAP-29 on the autophagosomes resulting in defective fusion of autophagosomes with lysosomes. 53 After the sequestered cargo is delivered inside the lysosome, it is broken down by resident hydrolases of the lysosome and the resulting macromolecules are released back into the cytosol as new energy sources and building blocks for cell survival ( Figure 1). In addition to providing new building blocks and energy sources, autophagy also helps to remove damaged organelles such as mitochondria as another important mechanism for cell survival.…”

Genome-wide identification of loci associated with growth in rainbow trout

“…Five SNPs in LAMP2 have been identified in windows explaining the highest genetic variability (~6%) in this category. LAMP2 is essential during autophagy for the fusion of autophagosomes with lysosomes [67]. ATP6V1H is a vacuolar (H+)-ATPase, which is required to acidify the phagosome/lysosome for proper processing [68].…”

The coordination of membrane fission and fusion at the end of autophagosome maturation

“…Atg8 proteins function at both steps, accelerating membrane fission [13, 14] and coordinating the fusion apparatus [14]. After fission, cues on the autophagosome and possibly the lysosome promote SNARE insertion on the closed autophagosome [13, 15]. Interestingly, different macroautophagic cargoes are associated with different SNARE families [14, 16] and thus future work will have to establish whether tight fission/fusion coordination is conserved for all autophagosome SNARE-mediated events or is unique to classic starvation and basal autophagy pathways.…”

“…Working in mouse embryonic fibroblasts, Hubert et al show that STX17 recruitment requires the lysosomal protein LAMP-2 [15]. When LAMP-2 is knocked out, induction of macroautophagy still drives the formation of LC3-positive autophagosomes, but STX17 recruitment fails and fusion between autophagosomes and lysosomes is lost [15]. Lysosome-related SNARE machinery including Rab7 and VAMP8 are unaffected, suggesting STX17 recruitment is not tied to the formation of a SNARE-fusion assembly site.…”