A novel membrane complex is required for docking and regulated exocytosis of lysosome-related organelles in Tetrahymena thermophila

Author summaryAll cells, whether single-celled protists or multicellular organisms, interact dynamically with their environments. One important mode of interaction is the release of molecules, a phenomenon called secretion, which can then modify the environment to promote the organism's well-being. Moreover, many cells have the capacity to rapidly adjust the pathways that underlie secretion, allowing them to tailor their secretory behavior in response to changes in their surroundings. A dramatic example of this is the capacity to synthesize and then store reservoirs of secretory molecules, whose eventual release is triggered when the cell senses specific environmental conditions. This phenomenon is called 'regulated exocytosis' and has been long studied in animals, because it serves as the basis for communication between different cells and tissues. Many single-celled organisms can also secrete via regulated exocytosis, and understanding the mechanisms involved could have practical consequences for developing therapies against several devastating human parasites. In this paper, we took a genetic approach to identifying factors involved in exocytosis in a single-celled protist, the ciliate Tetrahymena thermophila. We find that a novel gene, which appears to be present only in Tetrahymena and its relatively close evolutionary relatives, plays an important role in the pathway. Our results add another layer to recent findings that cells like Tetrahymena evolved unique mechanisms for regulated exocytosis, expanding our appreciation of cellular biodiversity. In the ciliate Tetrahymena thermophila, lysosome-related organelles called mucocysts accumulate at the cell periphery where they secrete their contents in response to extracellular events, a phenomenon called regulated exocytosis. The molecular bases underlying regulated exocytosis have been extensively described in animals but it is not clear whether similar mechanisms exist in ciliates or their sister lineage, the Apicomplexan parasites, which together belong to the ecologically and medically important superphylum Alveolata. Beginning with a T. thermophila mutant in mucocyst exocytosis, we used a forward genetic approach to uncover MDL1 (Mucocyst Discharge with a LamG domain), a novel gene that is essential for regulated exocytosis of mucocysts. Mdl1p is a 40 kDa membrane glycoprotein that localizes to mucocysts, and specifically to a tip domain that contacts the plasma membrane when the mucocyst is docked. This sub-localization of Mdl1p, which occurs prior to docking, underscores a functional asymmetry in mucocysts that is strikingly similar to that of highly polarized secretory organelles in other Alveolates. A mis-sense mutation in the LamG domain results in mucocysts that dock but only undergo inefficient exocytosis. In contrast, complete knockout of MDL1 largely prevents mucocyst docking itself. Mdl1p is physically associated with 9 other proteins, all of them novel and largely restricted to Alveolates, and sedimentation analysis supports the idea that they form a large complex. Analysis of three other members of this putative complex, called MDD (for Mucocyst Docking and Discharge), shows that they also localize to mucocysts. Negative staining of purified MDD complexes revealed distinct particles with a central channel. Our results uncover a novel macromolecular complex whose subunits are conserved within alveolates but not in other lineages, that is essential for regulated exocytosis in T. thermophila.