Journal: The Journal of Cell Biology

Article Title: A Novel Function of Saccharomyces cerevisiae CDC5 in Cytokinesis

doi:

Figure Lengend Snippet: Direct interactions between cdc5ΔN and septins may lead to aberrant septin ring structures. Strain 1783 expressing a GST-cdc5ΔN under control of GAL1 promoter (KLY1053) were additionally integrated with TUB1-GFP (KLY1069), YFP-CDC3 (KLY1075), CYK2-GFP (KLY1071), or MYO1-GFP (KLY1073). These cells were cultured under the induction conditions for 12 h and subjected to microscopic examination. (A) Overexpression of GST-cdc5ΔN induces an apparent G1 arrest. Internal cell bodies possess disassembled spindles, whereas peripheral cell bodies possess elongated spindles, suggesting that only the cells at the edge continue to divide (see text). Bar: 5 μm. (B) Induction of various aberrant septin structures by overexpression of GST-cdc5ΔN . It appeared that a large fraction of septin double rings (visualized as YFP-Cdc3) were distantly placed from each other (open arrowheads) or that septin rings were disassembled with remnants of the YFP-Cdc3 signal present at the mother-bud neck (arrowheads). In addition, tiny septin ring structures (arrows) or abnormally large rings (barbed arrows) without an apparent bud formation were often present (see text for detail). Bar: 5 μm. (C) Two-hybrid interactions between Cdc5 and septins. Diagram shows structures of various Cdc5 constructs used in these analyses. To enhance the protein stability, a destruction-box–deficient form of Cdc5 was used in place of the wild-type Cdc5. Grey boxes indicate the kinase domain in the NH 2 terminus of Cdc5. Closed boxes in the COOH-terminal domain indicate the polo-box, whereas the gray indicates the polo-box with FAA mutations. The numbers in the table indicate the Miller units of β-galactosidase activity averaged from two independent experiments. Cl, Cla I; RV, Eco RV; Sn, Sna BI; Cdc5, Cdc5 lacking the NH 2 -terminal residues 6–71 ; cdc5 Δ C, COOH-terminal domain deletion; cdc5ΔN , NH 2 -terminal domain deletion; cdc5ΔN /FAA, NH 2 -terminal domain deletion with FAA mutations in the polo-box; a and b, control plasmids: pEG202-NLS (DNA binding domain fusion vector) and pJG4-5 (activation domain fusion vector). (D) Coimmunoprecipitation of cdc5ΔN with Cdc11. To examine in vivo interactions between cdc5ΔN and septins, cellular lysates (supernatant of 15,000 g ; S15) were prepared from strains bearing CDC11-TEV-9myc at the CDC11 locus and expressing either GAL1-EGFP-cdc5ΔN (SKY1732) or GAL1-EGFP-cdc5ΔN/FAA (SKY1734). Cdc11 was immunoprecipitated with an anti–Myc antibody cross linked to sepharose beads. To elute Cdc11 and its associated proteins, immunoprecipitates were digested with TEV protease. The resulting eluates were mixed with Laemmli sample buffer and subjected to SDS-PAGE and Western blot analyses with either anti–GFP antibody or anti–Cdc11 antibody. To determine the efficiency of coprecipitation, a fraction of S15 before immunoprecipitation was also loaded. Due to the TEV cleavage, Cdc11 protein eluted from immunoprecipitates possesses a molecular size smaller than Cdc11-TEV-9Myc. cdc5ΔN , S15 lysates from SKY1732; cdc5ΔN /FAA, S15 lysates from SKY1734, S15, a fraction of S15 lysates; bead, anti–Flag antibody crossed linked to sepharose beads; anti–Myc, anti–Myc antibody crossed linked to sepharose.

Article Snippet: An anti–Myc antibody cross linked to sepharose beads (BabCO) was used to precipitate Cdc11-TEV-9Myc.

Techniques: Expressing, Control, Cell Culture, Over Expression, Construct, Activity Assay, Binding Assay, Plasmid Preparation, Activation Assay, In Vivo, Immunoprecipitation, SDS Page, Western Blot