Sbg1 Is a Novel Regulator for the Localization of the ß-Glucan Synthase Bgs1 in Fission Yeast.

Glucan synthases synthesize glucans, complex polysaccharides that are the major components in fungal cell walls and division septa. Studying regulation of glucan synthases is important as they are essential for fungal cell survival and thus popular targets for anti-fungal drugs. Linear 1,3-ß-glucan is the main component of primary septum and is synthesized by the conserved ß-glucan synthase Bgs1 in fission yeast cytokinesis. It is known that Rho1 GTPase regulates Bgs1 catalytic activity and the F-BAR protein Cdc15 plays a role in Bgs1 delivery to the plasma membrane. Here we characterize a novel protein Sbg1 that is present in a complex with Bgs1 and regulates its protein levels and localization. Sbg1 is essential for contractile-ring constriction and septum formation during cytokinesis. Sbg1 and Bgs1 physically interact and are interdependent for localization to the plasma membrane. Bgs1 is less stable and/or mis-targeted to vacuoles in sbg1 mutants. Moreover, Sbg1 plays an earlier and more important role in Bgs1 trafficking and localization than Cdc15. Together, our data reveal a new mode of regulation for the essential ß-glucan synthase Bgs1 by the novel protein Sbg1.

Real-Time Visualization and Quantification of Contractile Ring Proteins in Single Living Cells.

Single-cell microscopy provides a powerful tool to visualize cellular and subcellular processes in wild-type and mutant cells by observing fluorescently tagged proteins. Here, we describe three simple methods to visualize fission yeast cells: gelatin slides, coverslip-bottom dishes, and tetrad fluorescence microscopy. These imaging methods and data analysis using free software make it possible to quantify protein localization, dynamics, and concentration with high spatial and temporal resolution. In fission yeast, the actomyosin contractile ring is essential for cytokinesis. We use the visualization and quantification of contractile ring proteins as an example to demonstrate how to use these methods.

Regulation of Rho-GEF Rgf3 by the arrestin Art1 in fission yeast cytokinesis.

Rho GTPases, activated by guanine nucleotide exchange factors (GEFs), are essential regulators of polarized cell growth, cytokinesis, and many other cellular processes. However, the regulation of Rho-GEFs themselves is not well understood. Rgf3 is an essential GEF for Rho1 GTPase in fission yeast. We show that Rgf3 protein levels and localization are regulated by arrestin-related protein Art1. art1∆ cells lyse during cell separation with a thinner and defective septum. As does Rgf3, Art1 concentrates to the contractile ring starting at early anaphase and spreads to the septum during and after ring constriction. Art1 localization depends on its C-terminus, and Art1 is important for maintaining Rgf3 protein levels. Biochemical experiments reveal that the Rgf3 C-terminus binds to Art1. Using an Rgf3 conditional mutant and mislocalization experiments, we found that Art1 and Rgf3 are interdependent for localization to the division site. As expected, active Rho1 levels at the division site are reduced in art1∆ and rgf3 mutant cells. Taken together, these data reveal that the arrestin family protein Art1 regulates the protein levels and localization of the Rho-GEF Rgf3, which in turn modulates active Rho1 levels during fission yeast cytokinesis.