Lysosome-related organelles (LROs) are a heterogeneous family of organelles found in many cell types, whose similarities to lysosomes include acidification by vacuolar-type proton ATPases (V-ATPases). However, some organelles with hallmarks of LROs are nonetheless non-acidic. Here, we investigate this phenomenon using the ciliate Tetrahymena thermophila, which has secretory LROs called mucocysts. Using three approaches, we show that mature mucocysts, poised for exocytosis, are non-acidic. However, mucocysts forming in the cytoplasm are acidic, and a specific V-ATPase a-subunit is present and indispensable for mucocyst biogenesis. In the absence of this subunit, cells show defects in at least two features of mucocyst formation, namely heterotypic vesicle fusion of mucocyst precursors and proprotein processing. The stage specificity of acidification can be explained by our finding that several other canonical V-ATPase subunits are present in the forming mucocysts but not in mature mucocysts. Based on our data, we argue that a specific V-ATPase complex is targeted to newly forming, immature mucocysts and subsequently disassembles at a later stage in the maturation pathway.
Keywords: LRO; Mucocyst; V-ATPase; V-ATPase-a1p; pH; pHluorin.
© 2025. Published by The Company of Biologists.