Ciliate Molecular Biology Conference 2025 (CMB 2025)

In the ciliate Tetrahymena thermophila, a subset of secretory proteins can be exocytosed via specialized vesicles called mucocysts. Mucocysts belong to the very broad family of lysosome-related organelles (LROs) including secretory LROs in animal cells, whose formation involves contributions from both secretory and endocytic trafficking. One factor in the formation of animal cell LROs is control of luminal ion concentrations, for which key determinants are proton-translocating Vacuolar-type ATPases (V-ATPase). In addition, mucocyst formation in Tetrahymena relies of proteolytic maturation of cargo proteins similar to secretory granules, including insulin granules. However, the key proteases involved are unrelated. In this study, we began with expression profiling to identify a V-ATPase a-subunit in T. thermophila that is targeted to mucocysts. Cells lacking the V-ATPase-a1 gene show defects in the targeting of mucocyst core proteins as well as a key cathepsin required for their proteolytic maturation, and are thus defective in both mucocyst biogenesis and exocytosis. Further, the regulation of the proprotein maturase in ciliates depends like insulin granule prohormone convertases on a targeted V-ATPase-a1p, but with interesting mechanistic differences.  First, mucocyst maturation does not depend on stable acidification, consistent with prior observations in the ciliate Paramecium. Secondly, in the absence of the mucocyst-targeting subunit of the V-ATPase complex, we detect clear defects in mucocyst-associated membrane trafficking, suggesting that the V-ATPase plays direct or indirect roles at this level.