Bim1p/Yeb1p mediates the Kar9p-dependent cortical attachment of cytoplasmic microtubules

Abstract

In Saccharomyces cerevisiae, positioning of the mitotic spindle depends on the interaction of cytoplasmic microtubules with the cell cortex. In this process, cortical Kar9p in the bud acts as a link between the actin and microtubule cytoskeletons. To identify Kar9p-interacting proteins, a two-hybrid screen was conducted with the use of full-length Kar9p as bait, and three genes were identified: BIM1, STU2, and KAR9 itself. STU2 encodes a component of the spindle pole body. Bim1p is the yeast homologue of the human microtubule-binding protein EB1, which is a binding partner to the adenomatous polyposis coli protein involved in colon cancer. Eighty-nine amino acids within the third quarter of Bim1p was sufficient to confer interaction with Kar9p. The two-hybrid interactions were confirmed with the use of coimmunoprecipitation experiments. Genetic analysis placed Bim1p in the Kar9p pathway for nuclear migration. Bim1p was not required for Kar9p's cortical or spindle pole body localization. However, deletion of BIM1 eliminated Kar9p localization along cytoplasmic microtubules. Furthermore, in the bim1 mutants, the cytoplasmic microtubules no longer intersected the cortical dot of Green Fluorescent Protein-Kar9p. These experiments demonstrate that the interaction of cytoplasmic microtubules with the Kar9p cortical attachment site requires the microtubule-binding protein Bim1p.

Figure 1

Kar9p interacts with Bim1p, Stu2p, and Kar9p by two-hybrid analysis. (A) The wild-type two-hybrid strain (PJ69-4A) containing pGDB-KAR9 (pMR4150) and the indicated pGAD-BIM1 library-positive plasmids were serially diluted 10-fold and spotted onto plates lacking uracil and leucine (−ura leu) or adenine (−ade). Plates were photographed after 2 d at 30°C. (B) A two-hybrid reporter strain deleted for BIM1 (MY7309) containing either GDB-KAR9 (pMR4150) or empty GDB vector (pMR3763) was transformed with the indicated STU2 plasmids and assayed for activation of the HIS3 reporter gene. The microtubule-binding domain of Stu2p (MT) has been mapped to amino acids 558–658 (Wang and Huffaker, 1997). Three independent STU2 transformants are shown. Plates were photographed after 3 d at 30°C. (C) The wild-type two-hybrid strain (PJ69-4A) containing pGDB-KAR9 (pMR4150) and the indicated KAR9 library plasmids were serially diluted 10-fold and spotted onto plates lacking uracil and leucine (−ura leu) or adenine (−ade). Plates were photographed after 2 d at 30°C. (D and E) The Genetics Computer Group (Madison, WI) programs Plotsimilarity and Pretty were used to compare the EB1 homologues from human (accession number I52726), mouse (accession number AAA96320), catfish (accession number AAD31035), D. melanogaster (accession number AAD27859), urochordate (accession number CAA67697), C. elegans (accession number CAA20332), S. pombe (accession number Q10113), and S. cerevisiae (accession number NP 010932). (E) The BLOSUM62 amino acid substitution matrix was used for calculations. Additional parameters were set as GapLengthWeight, 2; GapWeight, 8. A consensus was noted if six or more residues were similar. The location of the Kar9p-binding domain is indicated by closed circles.

Figure 1

Kar9p interacts with Bim1p, Stu2p, and Kar9p by two-hybrid analysis. (A) The wild-type two-hybrid strain (PJ69-4A) containing pGDB-KAR9 (pMR4150) and the indicated pGAD-BIM1 library-positive plasmids were serially diluted 10-fold and spotted onto plates lacking uracil and leucine (−ura leu) or adenine (−ade). Plates were photographed after 2 d at 30°C. (B) A two-hybrid reporter strain deleted for BIM1 (MY7309) containing either GDB-KAR9 (pMR4150) or empty GDB vector (pMR3763) was transformed with the indicated STU2 plasmids and assayed for activation of the HIS3 reporter gene. The microtubule-binding domain of Stu2p (MT) has been mapped to amino acids 558–658 (Wang and Huffaker, 1997). Three independent STU2 transformants are shown. Plates were photographed after 3 d at 30°C. (C) The wild-type two-hybrid strain (PJ69-4A) containing pGDB-KAR9 (pMR4150) and the indicated KAR9 library plasmids were serially diluted 10-fold and spotted onto plates lacking uracil and leucine (−ura leu) or adenine (−ade). Plates were photographed after 2 d at 30°C. (D and E) The Genetics Computer Group (Madison, WI) programs Plotsimilarity and Pretty were used to compare the EB1 homologues from human (accession number I52726), mouse (accession number AAA96320), catfish (accession number AAD31035), D. melanogaster (accession number AAD27859), urochordate (accession number CAA67697), C. elegans (accession number CAA20332), S. pombe (accession number Q10113), and S. cerevisiae (accession number NP 010932). (E) The BLOSUM62 amino acid substitution matrix was used for calculations. Additional parameters were set as GapLengthWeight, 2; GapWeight, 8. A consensus was noted if six or more residues were similar. The location of the Kar9p-binding domain is indicated by closed circles.

Figure 2

Kar9p coimmunoprecipitates with Bim1p, Stu2p, and Kar9p. (A) A kar9Δ stain (MS4263) containing HA epitope–tagged KAR9 (pMR4723), nontagged KAR9 (pMR4722), or galactose-inducible BIM1::V5 (pMR4620) plasmids were grown to mid exponential phase, and extracts were prepared for immunoprecipitation as described in MATERIALS AND METHODS. V5 antibody conjugated to Sepharose beads was used to immunoprecipitate Bim1p::V5. It was also used to probe Western blots. The HA antibody HA.11(16B12) conjugated to agarose beads was used to immunoprecipitate Kar9p::HA and probe the resulting Western blot. The HA antibody 12CA5 was used as a probe in the V5 immunoprecipitation experiment. Identical results were obtained when the two HA antibodies were used in any combination for either the immunoprecipitation step or subsequent Western blots. (B) Stu2p::HA was immunoprecipitated from extracts prepared from wild-type (MS1556) or kar9Δ (MS4306) strains containing HA epitope–tagged STU2 (pWP70) or empty vector (pRS415; Sikorski and Hieter, 1989), as described in MATERIALS AND METHODS. Proteins from the precipitates were analyzed by Western blotting with the use of rabbit anti-Kar9p and the HA antibody 12CA5. (C) GFP–Kar9p was immunoprecipitated with the use of anti-GFP (a kind gift from Pamela Silver) from wild-type strains transformed with Kar9p:HA (pMR4723), Kar9p with no tag (pMR4722), and P_GAL1-GFP-Kar9p (pMR3464). Proteins from the precipitates were analyzed by Western blotting with the use of rabbit anti-Kar9p and the HA antibody 12CA5.

Figure 3

Bim1p is required for localization of GFP–Kar9p along microtubules but not for Kar9p localization at the SPB or cortex. (A) Wild-type (MS1556) and bim1Δ (MS7310) strains containing GFP–Kar9p (pMR3465) were grown to early exponential phase and induced for GFP-Kar9p expression. The percentage of cells exhibiting GFP-Kar9p expression in medium to large-budded cells with a single nucleus is shown under each representative cell (n = 720 for wild-type cells and 660 for bim1Δ cells). The average of three independent experiments is shown. Five percent of wild-type and 6% of bim1Δ cells exhibited GFP–Kar9p localization patterns that are not depicted. (B) A bim1Δ strain (MS7310) containing GFP–Kar9p (pMR3465) was grown and prepared as described in A.

Figure 4

Cytoplasmic microtubules do not intersect the Kar9p dot in bim1Δ cells. Wild-type and bim1Δ cells containing GFP–Kar9p were fixed for double-label indirect immunofluorescence with the use of anti-GFP, anti-tubulin, and DAPI, as described in MATERIALS AND METHODS (n = 96 for wild-type cells and 105 for bim1Δ cells).