The SEC6 protein is required for function of the contractile vacuole inChlamydomonas reinhardtii

Abstract

SummaryContractile vacuoles (CVs) are essential for osmoregulation in many protists. To investigate the mechanism of CV function in Chlamydomonas, we isolated novel osmoregulatory mutants. Four of the isolated mutant cell lines carried the same 33,641 base deletion, rendering the cell lines unable to grow under strong hypotonic conditions. One mutant cell line (Osmo75) was analyzed in detail. The CV morphology was variable in mutant cells, and most cells had multiple small CVs. In addition, one or two enlarged CVs or no visible CVs at all, were observed by light microscopy. These findings suggest that the mutant is impaired in homotypic vacuolar and exocytotic membrane fusion. Furthermore the mutants had long flagella. One of the affected genes is the only SEC6 homologue in Chlamydomonas (CreSEC6). The SEC6 protein is a component of the exocyst complex that is required for efficient exocytosis. Transformation of the Osmo75 mutant with a CreSEC6-GFP construct rescued the mutant completely (osmoregulation and flagellar length). Rescued strains overexpressed CreSEC6 (as a GFP-tagged protein) and displayed a modified CV activity. CVs were larger, whereas the CV contraction interval remained unchanged, leading to increased water efflux rates. Electron microscopy analysis of Osmo75 cells showed that the mutant is able to form the close contact zones between the plasma membrane and the CV membrane observed during late diastole and systole. These results indicate that 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 influences the CV cycle in an indirect manner and flagellar length in Chlamydomonas.

An organelle K+ channel is required for osmoregulation in Chlamydomonas reinhardtii

“…As KCN11 was localized to the contractile vacuole membrane, we examined the contractile vacuole functional properties of the cells. In the growth medium, the average contractile vacuole periods between wild-type (WT) cells and kcn11 mutants were similar, with 20.72±0.53 s (mean±s.e.m., n=45) for WT cells and 20.43±0.51 s (n=45) for the mutants, which are consistent with previous report (Komsic-Buchmann et al, 2012) ( Fig. 4A,B).…”

“…During diastole of the contractile vacuole cycle, liquid-filled vesicles continue to fuse with the contractile vacuole Komsic-Buchmann et al, 2012;Luykx et al, 1997;Parkinson et al, 2014). Vesicle fusion requires the function of the exocyst because contractile vacuole formation is compromised in a sec6 mutant in C. reinhardtii (Komsic-Buchmann et al, 2012).…”

“…3A). Instead of localization to the plasma membrane, KCN11 was exclusively found at two vesicle-like structures located on each side of the base of the flagella, which are reminiscent of the two contractile vacuoles (Komsic-Buchmann et al, 2012;Luykx et al, 1997). Three dimensional reconstructions of the images show that KCN11-HA staining highlights two bladder-like structures, indicating these structures are contractile vacuoles (Fig.…”

“…reinhardtii is a fresh water unicellular green alga. Two contractile vacuoles, which pulsate alternately, are present in the cell anterior, on opposite sides of the plane that include the flagellar basal bodies (Komsic-Buchmann et al, 2012;Luykx et al, 1997). In this work, we have identified a voltage-gated K + channel that is localized to the contractile vacuole membrane of C. reinhardtii, and crucially required for the contractile vacuole cycling in water and functions in osmoregulation.…”

Critical Function of a Chlamydomonas reinhardtii Putative Polyphosphate Polymerase Subunit during Nutrient Deprivation

“…cerevisiae Vtc1p is present in a complex that is involved in acidocalcisome membrane fusion events and polyphosphate synthesis (Ogawa et al, 2000;Uttenweiler et al, 2007;Hothorn et al, 2009), while S. cerevisiae Vtc4p is a polyphosphate polymerase (Hothorn et al, 2009). Other proteins associated with various vacuole types have been analyzed through mutational studies and shown to be involved in contractile vacuole function, autophagy, cytokinesis, and lysosomal inactivation of Parainfluenza virus (Komsic-Buchmann et al, 2012;Ding et al, 2014;Tenenboim et al, 2014). The Vtc proteins of S. cerevisiae appear to assemble into a complex whose formation requires all of the polypeptide subunits of the complex.…”