Contractile vacuoles (CVs) are key players of osmoregulation in many protists. To investigate the mechanism of CV function in Chlamydomonas, we isolated novel osmoregulatory mutants. 4 isolated mutant cell lines carried the same 33,641 b deletion rendering the cell lines unable to grow under strong hypotonic conditions. One mutant cell line (Osmo75) was analyzed in detail. Mutant cells contained a variable CV morphology with most cells displaying multiple small CVs. In addition enlarged 1 or 2 CVs or no light microscopically visible CVs at all were observed. These findings suggest that the mutant is impaired in homotypic vacuolar and exocytotic membrane fusion. Furthermore the mutants displayed a long flagella phenotype. One of the affected genes is the only SEC6 homologue in Chlamydomonas (CreSEC6). The SEC6 protein is a component of the exocyst complex required for efficient exocytosis. Transformation of the Osmo75 mutant with CreSEC6GFP construct rescued the mutant completely (osmoregulation and flagellar length). Rescued strains overexpressed CreSEC6 (as GFP-tagged protein) and displayed a modified CV activity. CVs were significantly larger, whereas the CV contraction interval remained unchanged leading to increased water efflux rates. Electron microspical analysis of Osmo75 showed that the mutant is able to form the close contact zones between the PM (plasma membrane) and the CV membrane observed during late diastole and systole. These results indicate that the CreSEC6 is essential for CV function and required for homotypic vesicle fusion during diastole and water expulsion during systole. In addition CreSEC6 is not only necessary for CV function, but possibly influencing the CV cycle in an indirect way and flagellar length control in Chlamydomonas.
KK-B carried out all experiments except the rescue of Osmo75 and analyzed the data. LS performed the rescue of Osmo75 using CreSEC6-GFP. BB conceived and designed the study, analyzed the data and wrote the draft of the manuscript. All authors read and approved the final manuscript.