A V-ATPase complex, required for biogenesis of a lysosome-related organelle (LRO) in Tetrahymena thermophila, disassembles during LRO maturation

Lysosome-related organelles (LROs) are a heterogeneous family of organelles found in many cell types, whose significant similarities to lysosomes include their acidification by vacuolar-type ATPases (V-ATPases). However, some organelles with hallmarks of LROs are nonetheless non-acidic. Here we investigate this poorly understood phenomenon using the ciliate Tetrahymena thermophila, which have prominent secretory LROs called mucocysts. Using three different approaches, we show that mature mucocysts, poised for exocytosis, are non-acidic. Nonetheless, a specific V-ATPase a-subunit is targeted to mucocysts and is indispensable for mucocyst biogenesis and secretion. Consistent with this, immature mucocysts in the cytoplasm appear to be transiently acidified. The stage specificity of acidification can be explained by our finding that several canonical V-ATPase subunits are present in the immature, but not mature, mucocysts. Our data argue that a mucocyst-specific V-ATPase complex is targeted to newly-forming mucocysts and subsequently disassembles at a late stage in Tetrahymena LRO maturation, and that this depends on the AP-3 trafficking adaptor.