Molecular Linkage Underlying Microtubule Orientation Toward Cortical Sites in Yeast
10.1126/science.287.5461.2257
Crossref journal-article
American Association for the Advancement of Science (AAAS)
Science (221)
Abstract

Selective microtubule orientation toward spatially defined cortical sites is critical to polarized cellular processes as diverse as axon outgrowth and T cell cytotoxicity. In yeast, oriented cytoplasmic microtubules align the mitotic spindle between mother and bud. The cortical marker protein Kar9 localizes to the bud tip and is required for the orientation of microtubules toward this region. Here, we show that Kar9 directs microtubule orientation by acting through Bim1, a conserved microtubule-binding protein. Bim1 homolog EB1 was originally identified through its interaction with adenomatous polyposis coli (APC) tumor suppressor, raising the possibility that an APC-EB1 linkage orients microtubules in higher cells.

Bibliography

Korinek, W. S., Copeland, M. J., Chaudhuri, A., & Chant, J. (2000). Molecular Linkage Underlying Microtubule Orientation Toward Cortical Sites in Yeast. Science, 287(5461), 2257–2259.

Authors 4
  1. William S. Korinek (first)
  2. Matthew J. Copeland (additional)
  3. Amitabha Chaudhuri (additional)
  4. John Chant (additional)
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  28. 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.
  29. 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).
  30. 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.
  31. 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; kar9 Δ BK534 and BK538; bim1 Δ BK533 and BK539; kar9 Δ bim1 Δ BK535 and BK540.
  32. 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 G (IgG) (Jackson Immunologicals). Microtubules were visualized with monoclonal rat antibody to YOL1/34 (Accurate Chemical Westbury NY) and fluorescein isothiocyanate–conjugated secondary antibody to rat IgG (Jackson Immunologicals). DNA was stained with Hoechst 33342 (Molecular Probes). Wild-type SEY6210; bim1 Δ BK529; bik1 Δ BK543.
  33. An in vitro microtubule affinity assay was performed essentially as described by Goode and Feinstein (23) with the following notes. Bovine brain tubulin protein (Cytoskeleton Denver CO) was polymerized for 20 min at 35°C and stabilized with 20 μM taxol (Cytoskeleton). Polymerized tubulin (50 to 100 μg) was incubated with test proteins for 20 min. Reactions were then loaded into 800-μl Ultra Clear centrifuge tubes (Beckman) over 550 μl of a glycerol cushion buffer (30% glycerol 1 mM guanosine triphosphate 80 mM Na-Pipes (pH 6.9) 1 mM MgCl 2 1 mM EGTA) (Cytoskeleton) and were supplemented with 20 μM taxol. In addition to about 10 ng of in vitro–translated Kar9 100 ng of purified GST-Bim1 and/or 5 μg microtubules reactions in Fig. 3C also contained 1 μg of BSA (New England Biolabs Beverly MA) and 100 ng of His 6 -tagged Kar9 protein (pBK161).
  34. Yeast strains plasmids and DNA primers used in this study are listed in table 1 in the supplementary data which is available at www.sciencemag.org/feature/data/1047752.shl. Gene deletions were made in an isogenic background and confirmed by PCR (24). Standard media genetic methods and DNA manipulations were used (25 26). Yeast cultures were grown at 30°C and were in exponential phase at time of experiments.
  35. We are grateful to members of the J. Chant laboratory and to K. Kaplan for helpful discussions and insight. We thank R. Losick for critically reading the manuscript T. Chen for performing some supporting experiments Y. Ho for the tagged YNL240c control and J. Kahana and P. Silver for the antibodies to GFP. Supported by NIH grant GM49782 (J.C.) and grants GM07620-19 through GM07620-21 from an Institutional National Research Service Award Training Program in Genetics (W.S.K.).
Dates
Type When
Created 23 years, 1 month ago (July 27, 2002, 5:48 a.m.)
Deposited 1 year, 7 months ago (Jan. 13, 2024, 5:47 a.m.)
Indexed 1 month, 3 weeks ago (July 8, 2025, 7:30 a.m.)
Issued 25 years, 5 months ago (March 24, 2000)
Published 25 years, 5 months ago (March 24, 2000)
Published Print 25 years, 5 months ago (March 24, 2000)
Funders 0

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@article{Korinek_2000, title={Molecular Linkage Underlying Microtubule Orientation Toward Cortical Sites in Yeast}, volume={287}, ISSN={1095-9203}, url={http://dx.doi.org/10.1126/science.287.5461.2257}, DOI={10.1126/science.287.5461.2257}, number={5461}, journal={Science}, publisher={American Association for the Advancement of Science (AAAS)}, author={Korinek, William S. and Copeland, Matthew J. and Chaudhuri, Amitabha and Chant, John}, year={2000}, month=mar, pages={2257–2259} }