Molecular linkage underlying microtubule orientation toward cortical sites in yeast

Science

Volumn 287, Issue 5461, 2000, Pages 2257-2259

  Korinek, William S ^a   Copeland, Matthew J ^a   Chaudhuri, Amitabha ^a   Chant, John ^a  

^a HARVARD UNIVERSITY   (United States)

Author keywords

[No Author keywords available]

Indexed keywords

ALPHA TUBULIN; BINDING PROTEIN; GLUTATHIONE TRANSFERASE; HYBRID PROTEIN;

ARTICLE; COLON POLYPOSIS; MICROTUBULE; MITOSIS SPINDLE; NONHUMAN; PRIORITY JOURNAL; YEAST;

ADENOMATOUS POLYPOSIS COLI PROTEIN; CELL CYCLE PROTEINS; CELL NUCLEUS; CYTOSKELETAL PROTEINS; MICROTUBULE PROTEINS; MICROTUBULE-ASSOCIATED PROTEINS; MICROTUBULES; MITOTIC SPINDLE APPARATUS; MUTATION; NUCLEAR PROTEINS; PHENOTYPE; PROTEIN BINDING; RECOMBINANT FUSION PROTEINS; SACCHAROMYCES CEREVISIAE; SACCHAROMYCES CEREVISIAE PROTEINS; TWO-HYBRID SYSTEM TECHNIQUES;

EID: 0034708459     PISSN: 00368075     EISSN: None     Source Type: Journal    
DOI: 10.1126/science.287.5461.2257     Document Type: Article

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27. Strain PJ69-4A (19) carrying KAR9-DBD or GIC2-DBD was transformed with BIM1-AD, TUB2-AD, or the AD vector, and β-galactosidase units were measured (20). KAR9-DBD, pBK154; GIC2-DBD, pBK153; BIM1-AD, pBK155; TUB2-AD, pBK37; AD vector, pGAD-C2. β-galactosidase activity is an average of four independent runs.
28. GST and GST-fusion proteins were expressed in E. coli cells and purified with glutathione sepharose 4B beads (Pharmacia Biotech, Piscataway, NJ). All GST fusion proteins were made with the full-length protein stated except for Hof1, which was made with amino acids 1 through 286. GST, pGEX-4T-1; GST-Bim1, pBK141; GST-Hof1, pBK40; GST-Kar9, pBK140. In vitro translation used TNT Quick-Coupled Transcription/Translation Reticulocyte Lysate System (Promega). Kar9, pBK137; luciferase, luciferase T7 control DNA (Promega). Preparation of crude yeast lysates was essentially as described in Sia et al. (21). Bim1-Myc, BK514; Ynl240c-Myc, BK104. Labeled proteins and GST-fusions were incubated for 2 hours at 4°C. The beads were washed five times with buffer B [20 mM tris-HCl (pH 7.5), 0.2% NP-40, 0.25% bovine serum albumin (BSA)], including either 150 mM NaCl (Fig. 1B) or 500 mM NaCl (Fig. 1C).
29. Cultures for coprecipitations were incubated with galactose for 5 hours to induce GST-Kar9 and lysates were prepared as described above. The lysates were incubated with glutathione sepharose beads for 2 hours at 4°C and washed five times with buffer B containing 150 mM NaCl. An immunoblot was prepared and probed with 9E10 mouse monoclonal antibody to myc (Berkeley Antibody, Richmond, CA), horseradish peroxidase-conjugated goat secondary-antibody to mouse antibody (Jackson Immunologicals, Westgrove, PA), and visualized with SuperSignal chemiluminescence (Pierce, Rockford, IL). GST-KAR9, BK511; BIM1-MYC, BK514; GST-KAR9 BIM1-MYC, BK572; GST-KAR9 YNL240c-MYC, BK574.
30. bim1kar9 double mutants were isolated by meiotic tetrad analysis, and genotypes were confirmed by polymerase chain reaction (PCR). Log phase cells were fixed and stained with Hoechst 33342 (Molecular Probes, Eugene, OR). Two independent counts were performed on each strain. The average of the four counts is shown, each n > 300 cells. Wild-type, BK536 and BK537; kar9Δ, BK534 and BK538; bim1Δ, BK533 and BK539; kar9Δ bim1Δ, BK535 and BK540.
31. Standard yeast immunofluorescence techniques were used (22). Kar9-green fluorescence protein (Kar9-GFP) was induced from the MET25 promoter (pBK115) for either 2 (Fig. 3A) or 4 hours (Fig. 4) prior to fixing with 3% formaldehyde. Kar9-GFP was visualized with polyclonal rabbit antibody to GFP and CY3-conjugated secondary antibody rabbit immunoglobulin C (IgC) (Jackson Immunologicals). Microtubules were visualized with monoclonal rat antibody to YOL1/34 (Accurate Chemical,