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

<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 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

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 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

Meiosis and FSM formation defects in <i>pom2Δ</i> cells.

<p>(A) Representative DIC and fluorescence micrographs showing defects in meiosis and FSM formation in <i>pom2Δ</i> cells. <i>pom2⁺</i> (JW2619 and JW2620) or <i>pom2Δ</i> strains (JW2617 and JW2618) were crossed on SPA5S plates to induce meiosis and sporulation for 11 h before imaging. In merged images, nuclear membrane (Cut11-mRFP), FSM (GFP-Psy1), and nuclear DNA are in red, green, and blue, respectively. Representative images are shown. Images a-g are examples of asci with other than 4 nuclei, h-k are asci with 4 nuclei. (B) Quantification of meiosis and FSM formation defects in <i>pom2⁺</i> or <i>pom2Δ</i> cells as shown in (A). Asci with 4 well-separated DNA masses and a normal sphere-shaped FSM enclosing each DNA mass were classified as no defect; asci with other than 4 DNA masses but normal FSM morphology with each FSM enclosing one DNA mass, or 4 DNA masses with abnormal nuclei segregation pattern but normal FSM morphology were classified as meiosis defect only; spore membrane formation defect only includes asci with 4 DNA masses but FSM defects: either one or more DNA masses were not enclosed by FSM or two DNA masses were enclosed within one FSM; other asci were classified as defects in both meiosis and FSM formation. Bar, 3 µm.</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

List of <i>S. pombe</i> strains used in this study.

<p>List of <i>S. pombe</i> strains used in this study.</p

Pom2 localizes to mitochondria throughout the cell cycle and to the division site during cytokinesis.

<p>(A) Pom2 localizes to mitochondria. Cells expressing Pom2-tdTomato (strain JW1607) stained with mitochondrial dye DiOC₆(3) (top) or unstained (middle) and DiOC₆(3) stained wild-type cells (bottom, strain JW81) were imaged using exactly the same settings. (B) Pom2 co-localizes with Rlc1 in the contractile ring. Cells expressing Pom2-3GFP (bottom, strain JW1605), Rlc1-tdTomato (middle, JW1341), or both (top, JW1714) were imaged using exactly the same settings. Pom2 rings are marked by arrowheads. (C and D) Timing of Pom2 localization to the division site during cytokinesis. Temperature-sensitive <i>cdc25-22 pom2-3YFP sad1-mEGFP</i> cells (JW2646) were arrested at late G2 by growing at 36°C for 4 h before release to 25°C at time zero. Samples were taken every ten minutes, imaged with seven Z-sections spaced at 0.8 µm and quantified. (C) Maximum intensity projection of whole cells with Pom2-3YFP ring signals marked by arrowheads. (D) Graph of the time course of the fraction of cells with Pom2-3YFP ring signals (left Y-axis) and the mean spindle pole bodies (SPB) separation (right Y-axis). (E) Mildly overexpressed Pom2 localizes to the contractile ring. Wild type 972 and <i>41nmt1-GFP-pom2</i> (JW40) strains were grown in YE5S (repressing condition) at 25°C for 33 h before imaging. Images shown are maximum-intensity projections of 28 Z-sections with 0.2-µm spacing. Arrowheads indicate the contractile rings. Bars, 5 µm.</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