Pradhan, A.; Tadasare, N.; Sarkar, D.; Bansal, L. K.; Turkewitz, A. P.; Kumar, S.

In the ciliate Tetrahymena thermophila, a subset of proteins are exocytosed via lysosome-related organelles (LROs) called mucocysts. Formation of many LROs depends on modulation of pH by vacuolar-type ATPases (V-ATPases). Here, we used expression profiling to identify a V-ATPase a-subunit that is targeted to mucocysts. Cells lacking the V-ATPase-a1 gene are defective in mucocyst biogenesis and exocytosis. However, the requirement for this subunit may not be limited to the canonical role of V-ATPases in proton pumping, since we find no evidence that mature mucocysts are acidified. Consistent with this, we find that while the a1-subunit is present in both immature and mature mucocysts, several other V-ATPase subunits are absent from the latter. These data suggest that the V-ATPase complex disassembles during mucocyst maturation, and that the a1-subunit performs roles independent of the holo-complex. Cells lacking the a1-subunit are defective in the targeting of both structural and enzymatic mucocyst proteins, and moreover appear deficient in fusion between vesicles from the secretory and endocytic pathways that is a feature of normal mucocyst maturation.