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 Ca2+ 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.
Highlights
The ATP-dependent proton pump V-ATPase ensures low intralysosomal pH, which is essential for lysosomal hydrolase activity
We show that (1) genetic depletion of V-ATPase subunits disrupts lysosomal acidity, blocks autophagic flux and leads to an accumulation of large non-functional autolysosomes; (2) defects in lysosomal acidification do not prevent autophagosome–lysosome fusion; (3) in contrast, BafA1 inhibits autophagosome–lysosome fusion in Drosophila as it does in mammalian cells; and (4) BafA1 potentially targets the Ca2 þ sarco/endoplasmic reticulum Ca2 þ -ATPase (SERCA) pump to inhibit vesicle fusion
We first examined the role of V-ATPase subunits in autophagy by characterizing the number and size of structures labelled with mCherry-tagged Atg8a, a specific marker of autophagosomes and autolysosomes[2,14]
Summary
The ATP-dependent proton pump V-ATPase ensures low intralysosomal pH, which is essential for lysosomal hydrolase activity. We show that (1) genetic depletion of V-ATPase subunits disrupts lysosomal acidity, blocks autophagic flux and leads to an accumulation of large non-functional autolysosomes; (2) defects in lysosomal acidification do not prevent autophagosome–lysosome fusion; (3) in contrast, BafA1 inhibits autophagosome–lysosome fusion in Drosophila as it does in mammalian cells; and (4) BafA1 potentially targets the Ca2 þ sarco/endoplasmic reticulum Ca2 þ -ATPase (SERCA) pump to inhibit vesicle fusion. These findings show that lysosomal acidification and fusion are two separable, independent events. Better understanding of the mode of action of autophagy inhibitors such as BafA1 is essential for further development of such potential therapeutic compounds
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