S. pombe Sec8p is homologous to proteins from S. cerevisiae and human. Amino acid sequences were aligned using ClustalX and Boxshader programs. Identical amino acids are shaded in black, and conservative substitutions are shaded in gray. Hs, Homo sapiens; Sc, S. cerevisiae; Sp, S. pombe.

Alignment of S. pombe Sec6p, Sec10p, Sec15p, and Exo70p with related proteins from S. cerevisiae, rat, and human. Amino acid sequences are aligned using ClustalX and Boxshader programs. Identical amino acids are shaded in black, and conservative substitutions are shaded in gray. Rn, Rattus norvegicus; Hs, Homo sapiens; Sp, S. pombe; Sc, S. cerevisiae.

Localization of exocyst components in S. pombe. (A) Exocyst components localize to the division site and cell tips. sec8-GFP sec6-Myc cells were stained with antibodies against GFP and Myc. In the merged micrograph, Sec8-GFP is in red, Sec6-Myc in green, and DNA in blue. Arrowheads: tip localization; 1, 4: a single ring structure; 2, 3: double ring structures. (B) Sec8-GFP appeared as two rings in late mitotic cells using confocal microscopy and 3D viewing. Top panel, cell with 0° rotation; lower panel, cell with 139° rotation. (C and D) Merged micrographs of Sec8-GFP with Sec10-Myc (C) and Exo70-Myc (D). Sec8-GFP is in red, Sec10-Myc or Exo70-Myc in green, and DNA in blue. (E) The localization of Sec10p in relation to Myo2p, an actomyosin ring marker. cdc25-22 cells expressing Sec10p-GFP were synchronized in G2 by incubation at 36°C for 4 h and allowed to enter mitosis by shifting to 24°C. Cells were stained with antibodies against GFP and Myo2p. (F) The localization of Sec10p is dependent on intact F-actin structures. cdc25-22 cells expressing Sec10p-GFP weretreated with either LatA in DMSO (bottom panels) or DMSO alone (top panels) upon release into mitosis. Samples were stained with antibodies against GFP and α-tubulin to visualize Sec10-GFP and microtubules. (G and H) Localization of Sec10p to the division site is lost in cdc12-112 (G) and cdc15-140 (H) mutants. Cells were grown at 24°C, shifted to 36°C for 4 h, and stained with antibodies against GFP and α-tubulin to visualize Sec10-GFP and microtubules. (I) The localization of Sec10p is insensitive to brefeldin A treatment. cdc25-22 cells expressing Sec10-GFP or Gma12-GFP (as a marker for the Golgi) were treated with either brefeldin A in ethanol (bottom panels) or ethanol alone (top panels) upon release into mitosis. Samples were stained with DAPI and with antibodies against GFP to visualize Sec10-GFP or Gma12-GFP and DNA. Bars: (A–D) 10 μm; (E–I) 5 μm.

sec8 mutants are defective in exocytosis. (A-D) sec8-1 mutant and sec8 shut-off cells accumulate large numbers of secretory vesicles. (A and B) Wild-type (A1, A2) and sec8-1 (B1–B3) mutant cells were grown at 24°C and shifted to 36°C for 4 h, fixed, and processed for electron microscopic analysis. Higher magnifications of the framed regions in A1 and B1 are also shown (A1′ and B1′). Arrows point at presumed secretory vesicles; arrowheads point at division septa. (C and D) sec8 shut-off cells were grown in medium lacking (C) or containing (D) thiamine at 30°C, fixed, and processed for electron microscopic analysis. Note that in the mutant cell forming a septum (D), vesicles were accumulated in the vicinity of the septum. The average number of vesicles in wild-type (5 cells were quantified) and sec8-1 (6 cells were quantified) were 1 and 53, respectively. Bars: 0.5 μm in the two magnified boxes; 1 μm in all other cells. (E) sec8-1 secretes less activity of acid phosphatase. Wild-type (squares) and sec8-1 (triangles) cells were assayed for secreted acid phosphatase activity at 24°C (open) and 36°C (filled).

Phenotype of mut2-1 (sec8-1) cells. (A) Growth curves of wild-type and mut2-1 cells. Cell numbers of wild-type and mut2-1 cells were quantified by counting cells using a hemacytometer after temperature shift from 24 to 36°C. ●, wild-type; ○, mut2-1. (B) mut2-1 is defective in cell separation. mut2-1 cells were grown at 24°C to log phase and shifted to the restrictive temperature of 36°C. Samples were collected just before the temperature shift (0 h) and 4 h later and stained with 4′,6-diamidino-2-phenylindole (DAPI) and rhodamine-conjugated phalloidin to visualize DNA and F-actin (top and middle panels) or with Calcofluor to visualize septa (bottom panels). A constricting actomyosin ring is shown with an arrow. Bar: 10 μm.

sec8-1 cells are not defective in polarized cell growth. Wild-type cells (A) and sec8-1 cells (B) were synchronized in G1 by growth in nitrogen-free medium for 18 h at 24°C, and shifted to 36°C for 1 h to inactivate the Sec8-1p protein. Cells were then resuspended in rich medium to allow cell cycle progression at 36°C. Samples were collected just before resuspending in rich medium (0 h) and at intervals thereafter and stained with Calcofluor to visualize septa. Bar: 10 μm.

Sec6p, Sec8p, Sec10p, and Exo70p associate in vivo. (A) Protein extracts were prepared from cells expressing Sec8-GFP (lanes 1), Sec8-GFP and Sec6-Myc (lanes 2), or Sec6-Myc alone (lanes 3). Total lysates (right panel) and immunoprecipitates prepared using anti-GFP antibodies (left panel) were blotted using anti-Myc (upper panels) and anti-GFP antibodies (lower panels). (B and C) Similar experiments were conducted using cells expressing Sec8-GFP, Sec10-Myc and Sec8-GFP, or Sec10-Myc (B) or cells expressing Sec8-GFP, Exo70-Myc and Sec8-GFP, or Exo70-Myc (C).

(A, D, and E) Null mutants of sec8 (A), sec6 (D), and sec10 (E) are defective in cleavage of the septum. Mutant spores were germinated in appropriate selective media and stained with DAPI, phalloidin, and Calcofluor to visualize DNA, F-actin, and septa, respectively. (B) Germinated sec8-null mutant cells do not contain detectable maternal Sec8p (see text for details). (C) sec8 shut-off cells are defective in cell separation. 81nmt1-sec8 cells were grown in thiamine-containing medium for 14 h at 30°C and stained with DAPI, phalloidin, and Calcofluor to visualize DNA, F-actin, and septa, respectively. Bar: 10 μm.