Sbg1 co-localizes and interacts with the β-glucan synthase Bgs1.

<p>(A) Sbg1 localization in cytoplasmic puncta, the division site, and cell tips. (B) Time course of arrival and ring formation of Sbg1 at the division site using Sad1 as a cell-cycle marker. (C-E) Micrograph (C), 3-D projection and line scans at the dashed line (D), and time course (E) showing co-localization of Sbg1 and Bgs1. (E) Single focal plane images of a representative cell showing Sbg1 and Bgs1 move together in cytoplasmic puncta (an example marked by white arrows). (F) Sbg1 co-IPs with Bgs1. IP using anti-GFP antibodies and cell lysates of strains expressing GFP-Bgs1, 3Flag-Sbg1, or GFP-Bgs1 3Flag-Sbg1 grown in YE5S for 48 h. The asterisk marks GFP-Bgs1. The smearing and multiple bands are likely caused by post-translational modifications.</p

Roles of the F-BAR protein Cdc15 in Bgs1 localization.

<p>(A and B) Time course and (C) micrographs of Bgs1 localization in <i>cdc15</i>Δ or <i>cdc15</i>^<i>+</i> cells. (B) Time course showing Bgs1 and Rlc1 colocalization (arrows) in ~1/3 <i>cdc15</i>Δ cells.</p

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

<div><p>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-β-glucan is the main component of primary septum and is synthesized by the conserved β-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 <i>sbg1</i> 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 β-glucan synthase Bgs1 by the novel protein Sbg1.</p></div

<i>sbg1</i> mutants are defective in contractile-ring constriction and septation.

<p>Germinated spores in (A, F, and H) were imaged 12–24 h after tetrad dissection onto YE5S plates. (A) Time course of a <i>sbg1</i>Δ cell lysed at 180 min. (B) A <i>sbg1</i>Δ cell germinated on YE5S + sorbitol and imaged after 3 d of growth. (C-E) Micrograph (C) and quantifications (D and E) of <i>sbg1</i> depletion phenotype of strain <i>81nmt1-mECitrine-sbg1</i> grown in YE5S + thiamine for 60 h. (D) Cell width at the widest part of the cell perpendicular to cell long axis. In this and other figures, asterisks indicate p < 0.01 in Student’s <i>t</i>-test and error bars are ± 1 SD. (E) Percentage of cells with ≥1 septa. (F and G) Contractile-ring formation and constriction in wt and <i>sbg1</i> mutants. <i>81nmt1-sbg1 rlc1-tdTomato</i> and control cells were grown in YE5S + thiamine liquid cultures for 36 h before imaging. Time 0 marks node appearance. (G) Timing of ring formation (node appearance to a compact ring) and constriction (a compact ring to ring constricted to a dot with highest Rlc1 pixel intensity, which includes the ring-maturation stage) in wt and <i>sbg1</i> mutants. (H and I) Ring anchoring is defective in <i>sbg1</i> mutants. Ring sliding in (H) <i>sbg1</i>Δ and (I) <i>81nmt1-sbg1</i> cells. (I) Cells were grown as in (F). Scale bars (for this and other figures except EM): 5 μm.</p

Sbg1 and Bgs1 are interdependent for localization.

<p>(A and B) Bgs1 localization depends on Sbg1. (A) Time course and (B) micrographs (left, maximum intensity projection; right, single focal plane) of <i>sbg1</i>Δ or <i>sbg1</i>^<i>+</i> cells expressing both tagged Bgs1 and Rlc1. (C) Mislocalized Sbg1 by Tom20-GBP recruits Bgs1 to mitochondria (examples marked by arrows). (D) Sbg1 depends on Bgs1 for division-site and cell-tip localization. Cells were grown at 36°C for 4 h. (E) Mislocalized Bgs1 by Tom20-GBP recruits Sbg1 to mitochondria (examples marked by arrows).</p

Sbg1 prevents the mistargeting of Bgs1 to vacuoles.

<p>(A and B) Bgs1 accumulates in vacuoles in <i>sbg1</i> mutants. FM4-64 staining of <i>sbg1</i>Δ and <i>sbg1</i>^<i>+</i> cells from spores germinated on YE5S plates for 24 h (A) or wt and <i>41nmt1-sbg1</i> cells grown in YE5S + thiamine for ~60 h (B). (C-E) Bgs1 degrades in <i>sbg1</i> mutant cells. (C) Western blotting and (D and E) quantifications (n = 6 from 2 independent experiments) of GFP-Bgs1 from cell lysates of <i>GFP-bgs1</i> and <i>81nmt1-sbg1 GFP-bgs1</i> cells grown in YE5S + thiamine for ~36 h.</p

Sbg1 plays a role in primary septum formation.

<p>EM micrographs (A-C) and quantification (D) showing septum and cell-wall defects in <i>81nmt1-sbg1</i> cells. Double headed arrows indicate septa (white) or cell wall (yellow) thickness. Red arrowheads mark gaps in the primary septum. Orange arrowheads mark asymmetric septation. Scale bars: 1 μm.</p