Autophagosome–lysosome fusion is independent of V-ATPase-mediated acidification

TLDR

V‑ATPase maintains lysosomal acidity, which is essential for hydrolase activity and has been presumed necessary for fusion of lysosomes with autophagosomes and endosomes. In Drosophila fat bodies, V‑ATPase loss blocks autophagic flux yet lysosomes still fuse with autophagosomes and endosomes to form giant autolysosomes, whereas BafilomycinA1 blocks fusion independently of pH, a defect mimicked by SERCA depletion and reversed by SERCA activation, demonstrating that lysosomal acidification is not required for fusion.

Abstract

The ATP-dependent proton pump V-ATPase ensures low intralysosomal pH, which is essential for lysosomal hydrolase activity. Based on studies with the V-ATPase inhibitor BafilomycinA1, lysosomal acidification is also thought to be required for fusion with incoming vesicles from the autophagic and endocytic pathways. Here we show that loss of V-ATPase subunits in the Drosophila fat body causes an accumulation of non-functional lysosomes, leading to a block in autophagic flux. However, V-ATPase-deficient lysosomes remain competent to fuse with autophagosomes and endosomes, resulting in a time-dependent formation of giant autolysosomes. In contrast, BafilomycinA1 prevents autophagosome-lysosome fusion in these cells, and this defect is phenocopied by depletion of the Ca(2+) pump SERCA, a secondary target of this drug. Moreover, activation of SERCA promotes fusion in a BafilomycinA1-sensitive manner. Collectively, our results indicate that lysosomal acidification is not a prerequisite for fusion, and that BafilomycinA1 inhibits fusion independent of its effect on lysosomal pH.