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Vol. 17, Issue 1, 178-191, January 2006

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The CLIP-170 Homologue Bik1p Promotes the Phosphorylation and Asymmetric Localization of Kar9p

Department of Biology, University of Rochester, Rochester, NY 14627

Submitted June 27, 2005; Revised October 5, 2005; Accepted October 12, 2005
Monitoring Editor: Tim Stearns

	ABSTRACT

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION ACKNOWLEDGMENTS REFERENCES

 
Accurate positioning of the mitotic spindle in Saccharomyces cerevisiae is coordinated with the asymmetry of the two poles and requires the microtubule-to-actin linker Kar9p. The asymmetric localization of Kar9p to one spindle pole body (SPB) and microtubule (MT) plus ends requires Cdc28p. Here, we show that the CLIP-170 homologue Bik1p binds directly to Kar9p. In the absence of Bik1p, Kar9p localization is not restricted to the daughter-bound SPB, but it is instead found on both SPBs. Kar9p is hypophosphorylated in bik1 mutants, and Bik1p binds to both phosphorylated and unphosphorylated isoforms of Kar9p. Furthermore, the two-hybrid interaction between full-length KAR9 and the cyclin CLB5 requires BIK1. The binding site of Clb5p on Kar9p maps to a short region within the basic domain of Kar9p that contains a conserved phosphorylation site, serine 496. Consistent with this, Kar9p is found on both SPBs in clb5 mutants at a frequency comparable with that seen in kar9-S496A strains. Together, these data suggest that Bik1p promotes the phosphorylation of Kar9p on serine 496, which affects its asymmetric localization to one SPB and associated cytoplasmic MTs. These findings provide further insight into a mechanism for directing centrosomal inheritance.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION ACKNOWLEDGMENTS REFERENCES

 
Positioning of the mitotic spindle is critical for cell division in all eukaryotes. In the budding yeast Saccharomyces cerevisiae, the mitotic spindle is positioned across the plane of cytokinesis located at the mother-bud neck, yielding an asymmetric cell division. This requires both actin cables and cytoplasmic microtubules (cMTs) (Theesfeld et al., 1999; Sullivan and Huffaker, 1992). The minus ends of cMTs emanate from a centrosome-like structure called the spindle pole body (SPB) embedded in the nuclear envelope (Byers and Goetsch, 1975).

Spindle positioning in yeast is governed by two sequential processes, termed the Kar9p pathway and the dynein pathway (Miller and Rose, 1998). Proteins in the Kar9p pathway function to move the nucleus up to the bud neck and align the mitotic spindle along the mother-bud axis before anaphase (DeZwaan et al., 1997; Miller and Rose, 1998). In this process, cMTs are oriented toward both the bud neck and growing bud (Segal et al. 2000a; Kusch et al., 2002). This involves a molecular bridge between actin and cMTs consisting of the type V myosin Myo2p, Kar9p, and the MT binding protein Bim1p (Beach et al., 2000; Korinek et al., 2000; Lee et al., 2000; Miller et al., 2000; Yin et al., 2000; Hwang et al., 2003). The kinesin Kip3p and the XMAP215/ TOG1 homologue Stu2p also contribute to the Kar9p pathway (DeZwaan et al., 1997; Miller et al., 1998; Kosco et al., 2001). Together with the dynactin complex, dynein (DYN1/DHC1) and Num1p at the cortex exert pulling and sliding forces on cMTs (Muhua et al., 1994; Adames and Cooper, 2000; Heil-Chapdelaine et al., 2000; Yeh et al., 2000; Farkasovsky and Kuntzel, 2001; Lee et al., 2003). This moves the spindle across the plane of cytokinesis at (Yeh et al., 1995) or just after (Kahana et al., 1995) the onset of anaphase. The CLIP-170 homologue Bik1p and the kinesin Kip2p also function in the dynein pathway (Miller et al., 1998; Miller and Rose, 1998).

The two pathways were initially defined by genetic analysis and are partially redundant (Miller and Rose, 1998). Double mutations within the same pathway are no more deleterious than either single mutation alone (Muhua et al., 1994). However, simultaneous mutations between components of each pathway result in cell death, or synthetic lethality (Miller and Rose, 1998; Heil-Chapdelaine et al., 2000). Although the kinesin Kip2p is genetically required for the dynein pathway, recent work has revealed a function for Kip2p in the Kar9p mechanism (Miller et al., 1998; Maekawa et al., 2003). This raises the question of how proteins may have functions in both pathways but seem genetically to be essential in only one.

The orientation of the mitotic spindle across the bud neck is coordinated with differences between the two SPBs. The old SPB is translocated into the bud and the new SPB is retained in the mother cell (Pereira et al., 2001). During G₁, Kar9p localizes to the unduplicated SPB (Maekawa et al., 2003). After SPB duplication, Kar9p remains associated with the older SPB. This asymmetric restriction involves the cell cycle-dependent phosphorylation of Kar9p by Cdc28p (Liakopoulos et al., 2003; Maekawa et al., 2003). From the daughter-bound SPB, Kip2p is thought to transport Kar9p toward the cMT plus end where it establishes contacts with the cortex through Myo2p (Beach et al., 2000; Yin et al., 2000; Hwang et al., 2003; Maekawa et al., 2003). By linking microtubules (MTs) from the older SPB to the bud cortex, Kar9p directs only that pole to the daughter cell.

Two B-type cyclins have been implicated in spindle positioning and the Cdc28p-dependent phosphorylation of Kar9p. Clb4p regulates the stability of astral MT interactions with the bud tip (Maekawa and Schiebel, 2004). Kar9p is required for Clb4p localization at the SPBs and MTs (Maekawa and Schiebel, 2004). Deletion of CLB4 reduces the level of Kar9p phosphorylation and disturbs the restriction of Kar9p to the daughter-bound SPB (Liakopoulos et al., 2003; Maekawa and Schiebel, 2004). A second B-type cyclin, CLB5, interacts with KAR9 by two-hybrid analysis (Maekawa et al., 2003) and in vitro phosphorylation assays suggest that Kar9p is a specific substrate of the Clb5p-Cdc28p kinase (Loog and Morgan, 2005). A clb5 cdc28-4 double mutation also alters Kar9p localization on SPBs and at the cMT plus end (Maekawa et al., 2003; Maekawa and Schiebel, 2004). Mutations in clb5 affect spindle pole polarity and can result in both spindle poles being transported into the bud (Segal et al., 1998, 2000b).

Previously, Kar9p has been shown to interact with three MT-associated proteins: Bim1p, Kip2p, and Stu2p (Korinek et al., 2000; Lee et al., 2000; Miller et al., 2000; Maekawa et al., 2003). In this study, we identify Bik1p as a fourth MT-associated protein (MAP) that interacts with Kar9p. Bik1p promotes MT stability and is a member of the CLIP-170 family of MT plus end-tracking proteins (Berlin et al., 1990; Schuyler and Pellman, 2001). Mutations in either BIK1 or KIP2 result a similar phenotype of short cMTs, presumably because Kip2p can recruit Bik1p to cMT plus ends (Cottingham and Hoyt, 1997; Miller et al., 1998; Carvalho et al., 2004). The molecular explanation for the placement of Bik1p in the dynein genetic pathway is based on the finding that Bik1p functions in concert with Kip2p and the LIS1 homologue Pac1p to recruit dynein to the MT plus end (Lee et al., 2003; Sheeman et al., 2003; Carvalho et al., 2004).

Here, we show a new role for Bik1p. Our data suggest that Bik1p is important for accurate SPB inheritance by directing the localization of Kar9p onto cMTs emanating from the older SPB, thus enabling them to interact with the bud cortex. Mechanistically, we propose that Bik1p influences Kar9p localization through a phosphorylation mechanism by regulating the interaction between Kar9p and the cyclin Clb5p. This represents a novel mechanism of regulation in which a MT binding protein affects the localization of a binding partner by modulating its phosphorylation.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION ACKNOWLEDGMENTS REFERENCES

 
Yeast Strains and Growth Conditions
S. cerevisiae strains and plasmids used in this study are listed in Table 1. Primers and oligonucleotides used for strain constructions can be found in the online supplement (Table 1-S). Cells were grown in yeast peptone dextrose (YPD) or synthetic complete (SC) media as described previously (Miller et al., 1999).

Two-Hybrid Assay
The two-hybrid system of James et al. (1996) was used. The GAL4 DNA binding domain (DBD) plasmid pGBDU-C3 (James et al., 1996) was modified by replacing the BglII site with a SacI site. PGBDU-C3 was cut with BglII and oligonucleotides #38 and #39 containing the SacI site were ligated in, creating pRM2345. This destroyed the original BglII site. Truncations of KAR9 were amplified by PCR with terminal SalI and SacI sites using the oligonucleotides listed in Table S1 and pRM381 as a template. These were cloned into the SalI and SacI sites of pRM2345 and verified by sequencing. The truncation of KAR9 containing amino acids 1–316 was generated by digestion of the full-length construct (pRM1493) with SwaI and PstI to yield pRM2432. Full-length KAR9 fused to the DBD (pRM1493/pMR4150) was generated as described previously (Miller et al., 2000).

To generate a BIK1 fusion with the GAL4-activation domain (AD), BIK1 was synthesized by PCR with terminal EcoRI and BamHI restriction sites using primers #59 and #60 and pDP65/B3102/pRM 493 as template. This product was cloned into the EcoRI and BamHI sites of pGAD-C3/pRM1153 and verified by sequencing, generating pRM2627.

CLB5 was fused to the GAL4 AD by ligating a 2.1-kb fragment excised with BamHI and PstI from pRM2340/BDU-C5.1 (a generous gift from Fred Cross, Rockefeller University, New York, NY) to pRM1151/pGAD-C1 cut with BamHI and PstI. This generated pRM2721, which lacks the first four amino acids of Clb5p.

CLB4 was amplified from the chromosomal locus by PCR with terminal EcoRI and BamHI restriction sites using primers #344 and #345 and then cloned into the EcoRI and BamHI sites of pGAD-C3/pRM1153 to generate pRM 5041 (wild-type AD-CLB4). Mutant CLB4 was synthesized by PCR and cloned into the SalI and SacI sites of pRM2603 to generate pRM4949, which contains the CLB4 amino acid substitutions Q36R and I244M. These fusions were verified by sequencing.

DBD and AD plasmids were transformed into the yeast reporter strain PJ69-4A (yRM1757) (James et al., 1996) and selected for growth on plates lacking uracil and leucine. Interactions were assayed by transferring cells with a multiprong transfer device to SC plates lacking uracil and leucine (–uraleu) or histidine (–his). Growth was scored after incubation at 30°C for 2–3 d.

To generate a two-hybrid reporter strain disrupted for BIK1, pVB17 (B2134/pRM494) (Berlin et al., 1990) was digested with SnaBI and EcoRI and transformed into PJ69-4A (yRM1757) (James et al., 1996). Prototrophs were selected on SC plates lacking tryptophan. The disruption in yRM2258 was confirmed biochemically by Western blotting with -Bik1p and phenotypically by scoring defects in nuclear positioning as described previously (Miller and Rose, 1998).

Affinity Chromatography
The glutathione S-transferase (GST) vector pGEX-4T-2 (GE Healthcare, Little Chalfont, Buckinghamshire, United Kingdom) was modified by replacing the NotI site in the multiple cloning site with SacI, using oligonucleotides #72 and #73 to generate pRM2759. A fragment encoding the carboxy-terminal basic domain of Kar9p (amino acids 391–644) was synthesized by PCR with terminal SalI and SacI ends and cloned into the SalI and SacI of pRM2759 to produce pRM4167. This was verified by sequencing.

Expression of GST-Kar9^391–644aa and GST alone were induced in BL21(DE3) Escherichia coli (Stratagene, La Jolla, CA) using 1 mM isopropyl -D-thiogalactoside (IPTG) at 37°C for 1.5 h. Cells were resuspended in PEM buffer (80 mM Pipes, pH 6.8, 1 mM MgCl₂, 1 mM EGTA, and protease inhibitors) and lysed by sonication. The crude bacterial lysates were centrifuged at 16,000 x g at 4°C for 25 min. The supernatant was transferred to a fresh tube and centrifuged at 16,000 x g at 4°C for 20 min. Bacterial lysate (1 mg) was added to a Microspin GST purification column (GE Healthcare) containing a 50-µl bed of glutathione agarose and mixed for 30 min at 4°C. Unbound protein was removed from the beads by centrifugation at 837 x g for 1 min. The beads were washed 20 times with 350 µl of cold PEM buffer.

His₆-Bik1p expressed in BL21(DE3) bacteria was purified by nickel column chromatography, eluted with a step gradient of imidazole. The 350 mM imidazole fraction was dialyzed into PEM buffer, pH 6.8. Purified Bik1p (10 µg) was added to spin columns containing bound with either GST-Kar9^391–644aa or GST alone and mixed for 30 min at 4°C. Unbound protein was cleared from the column by centrifugation at 837 x g, followed by 25 washes with 600 µl of cold PEM buffer. Bound proteins were eluted by the addition of 100 µl of 10 µM reduced glutathione and centrifugation.

TAP-tagging KAR9
Chromosomal KAR9 was tagged at its carboxy terminus with a tripartite tandem affinity purification (TAP) tag consisting of 6x-histidine, hemagglutinin (HA), and protein A as described Puig et al. (2001). Briefly, the TAP tag and Kluyveromyces lactis URA3 marker were amplified by PCR using primers #144 and #147 and the template pRM3175/AVA0258 (a generous gift from Eric Phizicky, University of Rochester, Rochester, NY). The amplified region included 50 terminal base pairs of homology to the 3' end of the KAR9 locus and the downstream region, allowing for integration by homologous recombination. Recombinants were selected at 30°C on SC plates lacking uracil. The KAR9-tap fusion was verified by Western blot and PCR of genomic DNA. All KAR9-tap strains described in this study are derived from yRM3221.

To determine whether the KAR9-tap fusion was functional, we assayed for both nuclear positioning defects and synthetic lethality in crosses with mutants of the dynein pathway (Miller et al., 1998). KAR9-tap (yRM3221) was crossed to dyn1 (yRM672), kip2 (yRM666), and bik1 (yRM526/MS4734). KAR9-tap (yRM3379) was also crossed to pac1 (yRM3138/ATCC4002525, a nonisogenic cross). In each case, tetrad dissection produced 100% viable double mutants: KAR9-tap dyn1 (15 of 15 predicted double mutants), KAR9-tap bik1 (11 of 11), KAR9-tap kip2 (17 of 17), and KAR9-tap pac1 (8 of 8). No difference was detected in the growth of the double mutant colonies in comparison to the single mutants or wild type. To test for nuclear positioning defects, KAR9-tap (yRM3221), wild type (yRM 2147), and kar9 (yRM433) strains were grown to mid-log phase in YPD media at 30°C, fixed in 75% methanol/25% acetic acid, and stained with 6-diamidino-2-phenylindole (DAPI). Nuclear postioning defects were scored as described previously (Miller et al., 1998; Miller and Rose, 1998). The KAR9-tap strain exhibited the same percentage of binucleate mothers and unmigrated mitotic nuclei (1.8% of large-budded cells, n = 228) as wild type (1%, n = 202). This percentage was significantly lower than in kar9 (21%, n = 229). Combined, these results suggest that the function of the Kar9p-tap fusion protein has not been compromised.

Fluorescence Microscopy
To visualize Kar9p at endogenous protein levels, genomic KAR9 was fused to three copies of green fluorescent protein (GFP) at its carboxy terminus. The pBS-3xGFP-TRP1 vector (Lee et al., 2003) was modified to include a SalI-XmaI-SacI linker at the BamHI site using the oligonucleotides #188 and #189, generating pRM3634. A fragment of KAR9 (349–1932 bp) was synthesized by PCR to include a 3' triple Gly Ala linker and terminal SalI and SacI sites using the primers #69 and #194. This was cloned into the SalI-SacI sites of pRM3634 and verified by sequencing to create pRM3662. To integrate the 3xGFP tag at the KAR9 locus, pRM3662 was linearized at the ClaI site of KAR9 and transformed into the wild-type strain, MS52/yRM2146 to generate yRM3681. Recombinants were selected on SC plates lacking tryptophan at 30°C. The functionality of KAR9-3GFP fusion was assayed by scoring for nuclear migration defects and synthetic lethality with mutants in the dynein pathway. KAR9-3GFP did not exhibit any obvious spindle positioning defects, as the frequency of incorrectly migrated mitotic nuclei was similar to nontagged wild-type control (1.4%; n = 215). Additionally, KAR9-3GFP (yRM3681) was viable in double mutant combinations with dyn1 (yRM425, 18 of 18 predicted double mutants), bik1 (yRM565, 18 of 18), and pac1 (yRM3138/ATCC4002525, 14 of 14 in a nonisogenic cross). We conclude that KAR9 tagged with 3xGFP tag is functional. All Kar9p-3GFP described in this study were derived from yRM3681.

To visualize MTs, CFP-Tub1 (pAFS125C; a gift from J. Cooper, Washington University Medical School, St. Louis, MO) was cut with StuI and integrated at the URA3 locus, generating yRM 3886 and other strains. To identify the ends of cMTs, images above and below the Z-plane of the apparent MT end were examined.

SPC110 was tagged using the primers (#251 and #252) and templates as described previously (Yoder et al., 2003). The cyan fluorescent protein (CFP) tag was constructed using the pDH3/pRM4340 template, and the red fluorescent protein (DsRed) tag was based on the pTY24/pRM4335 template (both gifts of the Yeast Resource Center, University of Washington, Seattle, WA).

Microscopy was carried out on a motorized Zeiss Axioplan 2 microscope equipped with a 100x Plan-Neofluor lens (1.3 numerical aperture) (Carl Zeiss, Thornwood, NY), a cooled charge-coupled device camera (ORCA-ER; Hamamatsu, Hamamatsu City, Japan), and Chroma and/or Zeiss filter sets. Images were acquired and processed using Openlab 3.5.2 software (Improvision, Lexington, MA).

Preparation of Bik1p Antibodies
A his6x-BIK1 plasmid was generated by synthesizing BIK1 by PCR with terminal BamHI and HindIII sites using primers #80 and #79 and pDP65/pRM493 as template. This fragment was cloned into the BamHI and HindIII sites of pET-30+ (Novagen, Madison, WI) to generate pRM2860. Expression of the his₆-Bik1p protein was induced in BL21(DE3) bacteria (Novagen) using 1 mM IPTG at 37°C for 3 h. Cells were lysed by sonication on ice in binding buffer (20 mM Tris, pH 8.0, 0.5 M NaCl, 5 mM imidazole, and 0.1% Triton). The fusion protein was isolated by nickel-affinity chromatography using a step gradient of imidazole. his₆-Bik1p eluted at 250 and 300 mM imidazole. Fractions were pooled, concentrated to 3.2 mg/ml in phosphate-buffered saline, and injected into New Zealand White rabbits (rabbit #2832; Harlan Bioproducts for Science, Indianapolis, IN). Serum (1:1000 dilution in 5% milk/phosphate-buffered saline) was preabsorbed on three Western blots of bik1 extract for 8 h each. Western blots of wild-type whole cell extracts probed with this serum yielded a single band at 51.1 kDa corresponding to the molecular weight of Bik1p that was not present in extracts from bik1 strains. Alternatively, IgG was purified on Bio-Rad DEAE cartridges (Bio-Rad, Hercules, CA) according to manufacturer's instructions.

Immunoprecipitations and Western Blotting
Protein extracts were prepared from cultures grown to mid-exponential phase. Cells were washed once with water, resuspended in cold B150 buffer (50 mM Tris, pH 7.4, 150 mM NaCl, and 0.2% Triton X-100) with protease inhibitors (Sigma protease inhibitor cocktail, 1 mM phenylmethylsulfonyl fluoride, CLAAP, E64, and bestatin), and lysed by vortexing with glass beads. Crude extracts were then clarified by centrifugation at 16,000 x g for 20 min. The supernatant was transferred to a fresh tube and centrifuged at 16,000 x g for 20 min.

Kar9p-HA was coimmunoprecipitated with Bik1p-V5 (Invitrogen, Carlsbad, CA) from exponentially growing cultures as previously described for Bim1p-V5 (Miller et al., 2000), except that -HA-conjugated agarose beads were from Santa Cruz Biotechnology (Santa Cruz, CA). Bik1p-V5 expression was induced by the addition of 2% galactose for 3 h.

To immunoprecipitate Bik1p, 3 µl of cartridge purified -Bik1p was added to 1 ml of 3 mg/ml protein extract and incubated on a rotisserie at 4°C for 4 h. Protein A-Sepharose (5 µg; GE Healthcare) in 20 µl of B150 buffer was added and incubated on a rotisserie at 4°C for 1 h. The precipitate was then washed five times with cold B150 buffer. Bound proteins were eluted by the addition of 30 µlof3x Laemmli buffer and boiled for 3 min. Then, 15 µl of each sample was analyzed after 8% SDS-PAGE by Western blotting.

For Western blotting, Kar9p-tap was detected with -HA (Santa Cruz Biotechnology) at 1:200. Bik1p-V5 fusions were detected using -V5 (Invitrogen) at 1:5000, and GST fusions were detected using -GST (Sigma-Aldrich, St. Louis, MO) at 1:8000. Cartridge purified -Bik1p was used at 1:750. Chicken -actin was used at 1:20,000.

kar9 Mutants
Point mutations were introduced into the KAR9-3GFP plasmid (pRM3662) by site-directed mutagenesis using the QuikChange mutagenesis kit (Stratagene) to generate kar9-S197A (pRM6048), kar9-S496A (pRM5778), and kar9-S197A S496A (pRM6049) using the oligonucleotide pairs #427/#428 for S496A and #446/#447 for S197A. The absence of errors was confirmed by sequencing. These were integrated at the endogenous KAR9 locus of the wild-type strain MS52/yRM2146 after digestion with the restriction enzyme ClaI. Each integration was confirmed by PCR and Western blotting. TAP-tagged versions of these mutants were created by integrating the TAP tag cassette for KAR9 into the corresponding GFP-tagged strains, replacing the 3GFP tag and TRP+ marker. This generated kar9-S197A-tap (yRM6168), kar9-S496A-tap (yRM6015), and kar9-S197A S496A-tap (yRM6170).

Drug and Phosphatase Treatments
Nocodazole Treatment. Cultures were grown to early exponential phase in YPD media. Nocodazole (Sigma-Aldrich; 1.5 mg/ml stock solution in dimethyl sulfoxide [DMSO]) was added to a final concentration of 15 µg/µl at 30°C for 1.5 h. An equivalent volume of DMSO was added to a separate culture as a mock experiment. Depolymerization of MTs was confirmed by indirect immunofluorescence as described previously (Miller, 2004). Less than 0.5% of treated cells displayed any -tubulin staining.

Hydroxyurea Treatment. Cultures were grown to early exponential phase in YPD media. Hydroxyurea (HU) (Sigma-Aldrich) was added to a final concentration of 100 mM at 30°C for 2 h. Bud morphology was used to demonstrate that >98% of cells were arrested in late S phase.

Phosphatase Treatment. Protein extracts were prepared from asynchronous mid-log cultures. Phosphatase (800 U; New England Biolabs, Beverly, MA) was added to 150 µg of extract (in B150 buffer containing 2 mM MnCl₂ with 1x phosphatase buffer) and incubated at 30°C for 30 min. As a negative control, extracts were incubated without enzyme under identical reaction conditions.

	RESULTS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION ACKNOWLEDGMENTS REFERENCES

 
Kar9p Physically Interacts with Bik1p
Because Kar9p interacts with several MAPs, we tested whether Kar9p might also interact with the CLIP-170 homologue Bik1p. To assay for this interaction, Kar9p was immunoprecipitated from a strain in which Kar9p was tagged with the HA epitope and Biklp-V5 was expressed under the control of the GAL1 promoter as described previously (Miller et al., 2000). As shown in Figure 1A, Bik1p-V5 was present in the precipitate, indicating that the two proteins interact physically. Because Bik1p also interacts with Bim1p (Ito et al., 2001) (our unpublished observations), we asked whether Bik1p-V5 could be associating indirectly with Kar9p through a tertiary complex with Bim1p. To test this possibility, we repeated the Kar9p immunoprecipitation in bim1 cells. Bik1p-V5 again coimmunoprecipitated specifically with Kar9p-HA (Figure 1A). Thus, the Kar9p–Bik1p interaction is not dependent on Bim1p.

View larger version (30K): [in this window] [in a new window]   Figure 1. Bik1p binds directly with the basic domain of Kar9p. (A) Coimmunoprecipitation. Kar9p-HA was immunoprecipitated with -HA-conjugated agarose beads from extracts of wild-type (yRM2147) and bim1 (yRM2060) strains. Each strain contained the indicated plasmids: Kar9p-HA on a 2µ plasmid (pRM516), non-tagged KAR9 (pRM514), pGAL-BIK1-V5 (pRM2149), or empty vector (pRS426). The presence of Bik1p in the precipitate was detected using -V5. The two forms of Kar9p seen here are not comparable with the three forms seen in later experiments because a different percentage of gel and a different epitope tag were used for this experiment. (B) Two-hybrid analysis. Full-length KAR9 (pRM1493) and three truncations of KAR9 fused to the GAL4-DNA binding domain. 1–316 aa (pRM 2432), 200–399 aa (pRM2634), and 391–644 aa (pRM2632) were assayed for interaction with BIK1 fused to the GAL4-activation domain (pRM2627) in the two-hybrid reporter strain PJ69-4A. Three independent colonies were spotted onto plates lacking histidine (–his) and scored for growth at 30°C after 3 d (see Materials and Methods). Growth was equivalent for all spots on plates lacking uracil and leucine to demonstrate the presence of both plasmids (our unpublished data). (C) Direct binding. Either GST alone (pRM2759) or the basic domain of Kar9p (391–644aa) fused to glutathione S-transferase (pRM4167) was harvested from bacteria and bound in glutathione agarose columns (see Materials and Methods). Bacterially expressed his6-Bik1p was purified by nickel column chromatography, and 10 µg was added to columns of either GST-Kar9391–644aa (lane 2) or GST alone (lane 3). Bound proteins were eluted by the addition of reduced glutathione (lanes 2 and 3) and prepared for Western blotting. Lane 1 represents 1/5 of the his6-Bik1p that was loaded onto the GST-Kar9p beads.

We next wanted to determine whether the interaction between Bik1p and Kar9p was direct. The basic domain of Kar9p (391–644 aa) was fused to GST, expressed in E. coli, and purified on glutathione agarose. his₆-Bik1p was expressed in E. coli, purified by nickel column chromatography, and applied to columns of either the GST-Kar9p^391–644aa fusion (lane 2) or GST alone (lane 3). As shown in Figure 1C, elution with reduced glutathione revealed that his₆-Bik1p bound to GST-Kar9p^391–644aa but not GST alone. Thus, the interaction between Kar9p and Bik1p occurs through the basic domain of Kar9p and does not require an intervening protein.

Bik1p and Kip2p Are Not Required for Kar9p Localization at the Plus End
Bik1p plays a role in recruiting dynein to the plus ends of cMTs (Sheeman et al., 2003; Carvalho et al., 2004). To test whether Bik1p might act similarly to focus Kar9p at plus ends, endogenous levels of a functional Kar9p-3GFP (see Materials and Methods) were visualized in both wild-type and bik1 cells containing cMTs visualized with CFP-labeled -tubulin. We assayed whether Kar9p was present at three sites within the cell, at the SPB, along the microtubule, and at the plus end of the cMT. Because Kar9p can exist at multiple locations simultaneously, cells were scored for combinations of these localizations (Table S2). When the localization of Kar9p at the plus end of the MT was analyzed (Figure 2B), irrespective of its localization at other sites, 74% of wild-type preanaphase cells were found to display a dot of Kar9p localization at the plus end (n = 251). When localization at the SPB was analyzed, irrespective of whether it was present at the plus end, 64% of wild-type cells displayed Kar9p at the SPB (Figure 2B). Forty-three percent of wild type displayed Kar9p at both sites (Table S2). Because bik1 strains contain shorter MTs, we scored the subset that contained cMTs long enough to clearly permit the distinction between the SPB and the plus end. For this assay, however, the average length of cMTs remained shorter in bik1 than in wild type. The selected cMTs in preanaphase bik1 cells had a mean length of 1.24 ± 0.034 (SEM) µm (n = 103), whereas the mean length of all cMTs in preanaphase bik1 cells was 0.84 ± 0.03 (SEM) µm (n = 173). The average length of microtubules in wild type was 1.57 ± 0.055 (SEM) µm (n = 110), a statistically significant difference. Kar9p-3GFP was present at the plus ends of cMTs in 85% of bik1 cells (n = 250), (Figure 2, A and B). Thus, Bik1p is not required for the localization of Kar9p at the plus end.

View larger version (53K): [in this window] [in a new window]   Figure 2. Localization of Kar9p-3GFP on cMTs in mutants. (A) Examples of Kar9p-3GFP localization in preanaphase wild-type, bik1, and kip2 cells expressing Tub1p-CFP-labeled MTs. Arrowheads point to the Kar9p dot at the plus end of the cMT in kip2 cells. (B) Quantification of Kar9p-3GFP localization. "Plus end" represents the cells in which any Kar9p-3GFP was detected at the plus ends of cytoplasmic microtubules. "Along" represents cells in which Kar9p-3GFP could be seen along the length of the microtubule. "SPB" represents cells in which Kar9p-3GFP was observed at the minus end of cytoplasmic microtubules, near the spindle pole body. Cells in which Kar9p-3GFP was observed at multiple locations were scored in multiple categories. Kar9p-3GFP localization was scored in wild type (yRM3886; n = 252), bik1 (yRM4261; n = 250), kip2 (yRM4371; n = 250), bik1 kip3 (yRM5301; n = 251), and kip3 (yRM5293; n = 250) strains. Cells were grown in SC media–TRP at 30°C. Error bars denote the SEM of the percentages calculated in five separate experiments.

Whereas Kar9p was only seen along the MT in 13% of wild type, in 44% of bik1 cells Kar9p was distributed along the MT. To further confirm this mislocalization in bik1 cells, we sought to increase MT length in the bik1 background by using a kip3 mutation (DeZwaan et al., 1997; Miller et al., 1998). In a kip3 bik1 double mutant, the average length of cMTs analyzed was 1.77 ± 0.064 (SEM) µm(n = 109). In 48% of these cells, Kar9p-3GFP was also distributed along cMTs (Figure 2B). In the kip3 single mutant, Kar9p localization was found along the MT in 32% of cells. Because microtubules can be extremely long in kip3, cells were selected for this analysis in which the MTs did not curl along the inside of the bud cortex. The average length of the MTs analyzed in kip3 was 2.08 ± 0.061 (SEM) µm (n = 146). Thus, the localization of Kar9p is more dispersed along the MT in bik1 mutants. In a converse experiment, we sought to determine whether Kar9p affected the plus end localization of Bik1p. Bik1p-3GFP localization was examined in strains deleted for KAR9. In 99% of preanaphase kar9 cells (n = 157), Bik1p-3GFP was found as a dot at the plus end of cMTs, compared with 94% in wild-type cells (n = 152) (Figure S2A). Therefore, Kar9p is not required for the plus end binding of Bik1p.

Because mutations in KIP2 also result in short cMTs and Kip2p is thought to be responsible for the transport of Kar9p to the plus end of the cMT, we examined Kar9p localization in kip2 mutants. For this assay, the average length of cMTs scored was 1.19 ± 0.036 µm (mean ± SEM; n = 107). The average length of all MTs was 0.68 ± 0.03 (SEM) µm (n = 192). Consistent with previous reports (Maekawa et al., 2003), 87% of kip2 cells displayed a concentrated focus of Kar9p at the SPB. However, 60% of kip2 cells also showed some Kar9p-3GFP localized at the plus end of MTs (Figure 2, A and B). Forty-seven percent displayed Kar9p localizations at both sites (Table S2). Typically, the dot at the SPB was brighter than the dot at the plus end. Thus, Kip2p enhances the localization of Kar9p at the plus end, but it is not required for its localization there.

Kar9p Localizes to Both SPBs in bik1 Strains
Previous studies have shown that Kar9p associates asymmetrically with the daughter-bound spindle pole (Liakopoulos et al., 2003; Maekawa et al., 2003). Because Bik1p also localizes to SPBs (Lin et al., 2001; Carvalho et al., 2004), we asked whether Bik1p might alter the localization of Kar9p at the SPB. Kar9p-3GFP localization was scored in wild-type and bik1 strains containing the SPB marker Spc110p-CFP. As expected, Kar9p-3GFP localized to one SPB in 69% of wild-type cells with short bipolar spindles (Figures 3 and S3) (Liakopoulos et al., 2003; Maekawa et al., 2003). In contrast, Kar9p-3GFP was localized to one pole in only 29% of bik1 cells. Instead, 70% of these cells display Kar9p on both SPBs (Figures 3 and S3). The majority of these cells displayed a slightly stronger GFP signal at one of the two poles. This suggests that Bik1p acts to restrict Kar9p localization to the daughter-bound SPB.

View larger version (24K): [in this window] [in a new window]   Figure 3. Bik1p restricts Kar9p to one SPB. (A) Kar9p-3GFP was localized in wild-type and bik1 strains containing SPBs labeled with Spc110p-CFP. Composites are shown of differential interference contrast with either GFP or CFP single images. (B) Quantification of Kar9p-3GFP at SPBs in wild-type (yRM4357; n = 277), bik1 (yRM4358; n = 289), kip2 (yRM4375; n = 252), and bik1kip2 (yRM4598; n = 153) cells with short bipolar spindles. Cells were grown in SC media–TRP at 30°C. Error bars denote the SEM of the percentages calculated in at least three separate experiments.

Bik1p localizes to a region associated with both SPBs in diploid cells (Lin et al., 2001). Therefore, it seemed unlikely that in haploids Bik1p would promote Kar9p asymmetry by localizing to one SPB only. To further discount this possibility, we monitored the localization of Bik1p-3GFP in cells with SPBs labeled with Spc110p-DsRed. Bik1p-3GFP was always localized in a region associated with both SPBs in cells with short bipolar spindles (n = 150; Figure S2B). Thus, the localization of Bik1p itself does not explain the asymmetry of Kar9p, and additional factors are also probably involved.

Because mutations in Kip2p and Bik1p result in several similar phenotypes, we next tested whether Kip2p would have the same effect on Kar9p localization at the SPB. As shown in Figure 3B, Kar9p-3GFP was restricted to one SPB in kip2 cells at nearly the same frequency as seen in wild type. This suggests that Kip2p and Bik1p provide separate functions for Kar9p.

If Bik1p functions to restrict Kar9p localization to the daughter-bound SPB before its transport to MT plus ends by Kip2p, then the localization of Kar9p-3GFP to both SPBs seen in bik1 should be epistatic to the asymmetric localization seen in kip2. Indeed, the localization of Kar9p in bik1 kip2 strains was similar to that seen in bik1, supporting the model that Bik1p functions before Kip2p with respect to Kar9p (Figure 3B).

Bik1p Functions in the Kar9p-dependent Mechanism of SPB Inheritance
In >90% of wild-type cells, the old SPB is inherited by the daughter cell and the new SPB is retained in the mother (Pereira et al., 2001; Yoder et al., 2003). Current models postulate that the asymmetric localization of Kar9p to one SPB is important for accurate SPB inheritance (Liakopoulos et al., 2003). Our finding that Kar9p localized to both SPBs in bik1 cells suggested that either pole may be directed toward the bud, in which case bik1 cells should also display a defect in SPB inheritance.

To test this hypothesis, SPB inheritance was analyzed using methods similar to those described by Pereira et al. (2001), except that we labeled the SPB component SPC110 with a slow-folding form of the DsRed chromophore (Yoder et al., 2003). This allowed the relative age of either SPB to be distinguished in cells with bipolar spindles. The old SPB displays a brighter Spc110p-DsRed signal, resulting from the increased time allowed at this SPB for the DsRed-fluor to mature. The new SPB is therefore dimmer than the old SPB. To visualize the spindle, MTs were labeled with GFP (GFP-TUB1). Cultures were grown to stationary phase to allow for the accumulation of a bright DsRed signal at the unduplicated SPB and then released into fresh media for 2–3 h to allow SPB duplication and progression through the cell cycle. In 91% of wild-type cells with bipolar spindles aligned parallel to the mother-bud axis, the brighter SPB was oriented toward the bud (Figure 4). In cells lacking Kar9p, only 52% of the budproximal SPBs displayed the brighter SPB signal. These findings are in close agreement with previous work suggesting that SPB inheritance is nearly random in kar9 strains (Pereira et al., 2001). In bik1 cells, 74% of the brighter SPBs were directed toward the bud, representing a defect of 26%.

View larger version (42K): [in this window] [in a new window]   Figure 4. Bik1p has a function in SPB inheritance. (A) bik1 cells expressing Tub1p-GFP and SPBs labeled with Spc110p-DsRed. Composites are shown of differential interference contrast with either GFP or DsRed single images. Arrowheads point to the brighter and therefore older SPB. (B) Quantification of cells with the older SPB proximal to the bud in wild type (yRM4951; n = 480), kar9 (yRM5031; n = 359), bik1 (yRM4973; n = 409), kip2 (yRM4978; n = 324), and dyn1 (yRM5014; n = 158). Only cells with a bipolar spindle oriented along the long axis of the mother-bud were scored. The origin was set at 50% because this represents random inheritance. Error bars represent the SEM of the percentages obtained from at least three experiments. Fisher's exact test shows that the differences between the following pairs are statistically significant: bik1-kip2 (two-tailed p < 0.0001), dyn1-WT (p = 0.0477), bik1-WT (p < 0.0001), and bik1-dyn1 (p = 0.0036). The kip2-dyn1 (p = 0.565) and kip2-WT (p = 0.1220) pairs are not statistically different.

We also examined kip2 cells for defects in SPB inheritance. In 88% of kip2 cells, the brighter SPB signal was proximal to the bud. This suggests that the SPB inheritance defect in bik1 mutants is not solely the result of short cMTs. Moreover, 85% of dyn1 mutants displayed the correct SPB inheritance. Thus, bik1 mutants have a defect in SPB inheritance that is more severe than that observed in mutants of the dynein pathway but not as severe as kar9 mutants. These data are consistent with a model in which the effect of Bik1p on SPB inheritance is due largely to its effects on Kar9p localization.

Bik1p Binds both Phosphorylated and Unphosphorylated Kar9p
The Kar9p localization defects found in bik1 strains shown here were similar to those observed in cdc28/cyclin mutants and in kar9 alleles with mutated phosphorylation sites (Liakopoulos et al., 2003; Maekawa et al., 2003). This suggested that the pattern of Kar9p mislocalization observed in bik1 could be related to its phosphorylation status. To test whether Bik1p might interact specifically with one isoform of Kar9p, we incorporated a TAP tag at the 3' end of the chromosomal KAR9 locus (see Materials and Methods). Kar9p-tap was observed as a triplet in cellular extracts from asynchronously growing wild-type cultures (Figure 5). Bik1p was immunoprecipitated using a polyclonal Bik1p antibody and the precipitate was probed for Kar9p-tap using -HA. As shown in Figure 5A, lane 2, all three Kar9p-tap isoforms coimmunoprecipitated with Bik1p. An identical immunoprecipitation in a bik1 strain containing Kar9p-tap showed little or no Kar9p-tap in the precipitate (lane 5). Therefore, the Bik1p antibody did not interact significantly with the TAP tag of Kar9p. This suggests that the Bik1p–Kar9p interaction is not dependent on the phosphorylation state of Kar9p.

View larger version (40K): [in this window] [in a new window]   Figure 5. Bik1p promotes Kar9p phosphorylation. (A) Bik1p was immunoprecipitated using rabbit -Bik1p and protein A-Sepharose from exponentially growing wild-type (yRM4366) or bik1 (yRM3442) strains expressing KAR9-tap. The triparate TAP tag consists of his6, HA, and protein A epitopes. The no-antibody control represents a mock immunoprecipitation carried out concurrently using protein A-Sepharose beads alone. The precipitates were analyzed by immunoblotting with -HA to detect Kar9p-tap and rabbit -Bik1p. Extracts from KAR9-tap and bik1 strains were run for comparison. The vertical lines in the control lanes are likely to be the result of the protein A used in the precipitations. (B) Extracts were prepared from wild-type (yRM4366), bik1 (yRM3442), kip2 (yRM4122), bim1 (yRM4114), dyn1 (yRM3383), and kip3 (yRM4888) strains each containing KAR9-tap. Cultures were grown to mid-exponential phase, lysed, and extracts were prepared for immunoblotting with -HA. Actin was used as a loading control. (C) Protein extracts from wild-type and bik1 cells were treated with phosphatase or mock treated with no enzyme (see Materials and Methods) and prepared for immunoblotting. (D) Wild-type, bik1, and kip2 strains expressing KAR9-tap were grown to early exponential phase, treated with HU, and extracts were prepared for immunoblotting. Wild type was also treated with nocodazole. (E) The interaction between Kar9p and Bik1p does not require MTs. Bik1p was immunoprecipitated with -Bik1p from wild-type strains expressing KAR9-tap treated with nocodazole or with the DMSO solvent for 1.5 h. The two highest phosphorylated species of Kar9p-tap were apparent in extracts made from cells treated with nocodazole because Kar9p phosphorylation is increased at G2/M (Maekawa et al., 2003). PPase, phosphatase; nocod, nocodazole; WT, wild type; ext, extract; IP, immunoprecipitate.

Kar9p Is Hypophosphorylated in bik1 Mutants

We next asked whether normal-length MTs were required for Kar9p phosphorylation. We first assayed for the presence of Kar9p isoforms in kip2 strains. As shown in Figure 5B, all three phospho-isoforms of Kar9p were present in kip2, suggesting that short MTs do not affect Kar9p phosphorylation. To eliminate MTs using a different approach, cells were treated with nocodazole. This resulted in Kar9p hyperphosphorylation (Figure 5D). Although the interpretation of this result is confounded by the concomitant M-phase arrest caused by the nocodazole, it nevertheless indicates that the presence of MTs is not required for Kar9p phosphorylation. To determine whether long cMTs might alter Kar9p phosphorylation, we examined strains deleted for KIP3 (DeZwaan et al., 1997; Miller et al., 1998). The isoforms in wild type and kip3 mutants were similar, each containing three isoforms (Figure 5B). This suggests that MT length does not regulate Kar9p phosphorylation.

To further discount the possibility that other spindle positioning mutants might alter Kar9p phosphorylation, we examined strains deleted for dynein and Bim1p. The higher molecular weight species was present in both the dyn1 and bim1 mutants (Figure 5B). Together, these data suggest that the bik1 mutation uniquely affects the phosphorylation state of Kar9p.

Kar9p phosphorylation is increased during late S phase (Liakopoulos et al., 2003; Maekawa et al., 2003). To determine whether the highest molecular weight species would be enhanced in bik1 mutants during S phase, we arrested wild-type and bik1 strains with HU. As shown in Figure 5D, the majority of Kar9p-tap was present in the highest molecular weight isoform in extracts made from HU-treated wild-type cells. In the bik1 mutant, the highest molecular weight band was also enhanced by HU treatment but not to the extent seen in wild type or kip2. This suggests that the phosphorylation event resulting in the highest molecular weight band is not absolutely dependent upon Bik1p but that Bik1p contributes to this event.

The Interaction between Kar9p and Bik1p Does Not Require cMTs
To determine whether the Kar9p–Bik1p interaction requires the MT association of Bik1p, Bik1p was immunoprecipitated from KAR9-tap strains treated with nocodazole. The loss of MTs was confirmed by indirect immunofluorescence. As shown in Figure 5E, Kar9p-tap was present in the -Bik1p precipitate of nocodazole-treated cells but not in the no-antibody control.

BIK1 Is Required for the Interaction between CLB5 and Full-Length KAR9
One explanation for the decreased level of Kar9p phosphorylation in bik1 mutants is that Bik1p promotes Kar9p phosphorylation by facilitating an interaction between Kar9p and a cyclin. To test this, we asked whether the two-hybrid interaction between KAR9 and cyclins CLB5 or CLB4 required BIK1. As shown in Figure 6A, the CLB5-KAR9 interaction was detected in the wild-type two-hybrid reporter strain but not in the reporter strain deleted for BIK1. We did not detect a two-hybrid interaction between KAR9 and wild-type CLB4 (Figure 6B). However, KAR9 interacted strongly with a mutant form of CLB4 containing two amino acid substitutions (Q36R and I244M), consistent with the possibility that these mutations may stabilize a weak or transient interaction. This interaction was not affected by the deletion of BIK1 from the reporter strain (Figure 6B). This suggests that Bik1p is required for the Kar9p–Clb5p interaction but not the Kar9p–Clb4p interaction. These data provide additional evidence that the role of the S-phase cyclin CLB5 is distinct from that of the mitotic cyclin CLB4 with respect to the regulation of spindle positioning.

View larger version (70K): [in this window] [in a new window]   Figure 6. The interaction between CLB5 and full-length KAR9 requires BIK1 by two-hybrid analysis. (A) Full-length KAR9 fused to the GAL4-DBD (pRM1493) was assayed for interaction with CLB5 fused to the GAL4-AD (pRM2721) in a wild-type two-hybrid reporter strain (PJ69-4A) and in a two-hybrid reporter strain in which BIK1 was disrupted (yRM2258). Three independent colonies were spotted onto SC plates lacking histidine (–his). BIM1-AD was used as a positive control in both strains. Growth was equivalent for all spots on plates lacking uracil and leucine to demonstrate the presence of both plasmids (our unpublished data). (B) The wild-type CLB4-AD (pRM5041) and mutant clb4*-AD (pRM4949) plasmids were analyzed for interaction with KAR9 fused to the DBD as described for A. CLB4 in the clb4*-AD plasmid contains two amino acid substitutions, Q36R and I244M. (C) CLB5 interacts with amino acids 471–613 encoded by KAR9. The CLB5-activation domain fusion was tested for interaction against a series of KAR9 truncations fused to the GAL4-DBD in both the wild-type and bik1 reporter strains.

Another way in which Bik1p could facilitate the interaction between Kar9p and Clb5p would be for Bik1p to make Kar9p accessible to Clb5p, perhaps by relieving a steric hindrance present in full-length Kar9p. We reasoned that truncating Kar9p to the minimal region that supports an interaction with Clb5p might relieve this putative steric hindrance. Therefore, we used a two-hybrid mapping strategy to identify a region of KAR9 corresponding to amino acids 471–613 that was sufficient for interaction with CLB5 (Figure 6C). This interaction, unlike full-length KAR9, was apparent in both wild-type and bik1 reporter strains, supporting a model in which Bik1p makes Kar9p accessible to Clb5p.

Clb5p Affects Kar9p Localization
The minimal Clb5p binding site that we have identified on Kar9p contains the serine 496 phosphorylation site identified by Liakopoulos et al. (2003). This suggests that Clb5p could affect the restriction of Kar9p to one SPB by promoting phosphorylation of this residue. Consistent with this idea, we observed that Kar9p-3GFP mislocalized to both SPBs in clb5 at a frequency that was similar to that seen in the clb5bik1 mutant. This was also similar to the mislocalization frequency observed in the kar9-S496A mutant, in which the phosphorylation of serine 496 is prohibited by replacement of this residue with an alanine (Figure 7, A and B). Furthermore, the kar9-S496A mutation also eliminated the highest molecular weight band of Kar9p-tap normally seen in both asynchronous and hydroxyurea-arrested cells (Figure 7, C and D). Thus, the highest molecular weight band is dependent upon phosphorylation at serine 496.

View larger version (37K): [in this window] [in a new window]   Figure 7. Bik1p, Clb5p, and serine 496 function in the restriction of Kar9p to one SPB. (A) Kar9p-3GFP was localized in clb5 strains containing SPBs labeled with Spc110p-CFP. Composites are shown of differential interference contrast with either GFP or CFP single images. (B) Quantification of Kar9p-3GFP at SPBs in wild-type (yRM4357; n = 201), clb5 (yRM5119; n = 200), and bik1clb5 (yRM5647; n = 251) cells with short bipolar spindles, and Kar9p-S496A-3GFP in the wild-type background (yRM6007; n = 250). Cells were grown in SC media–TRP at 30°C. Error bars denote the SEM of the counts in at least four separate experiments. (C) Extracts were prepared from asynchronous cultures of wild-type strains expressing KAR9-tap (yRM4366), kar9-S197A-tap (yRM6168), kar9-S496A-tap (yRM6015), and kar9-S197A S496A-tap (yRM6170). Cultures were grown to mid-exponential phase, lysed, and extracts were prepared for immunoblotting with -HA. (D) Extracts were prepared from either asynchronous or hydroxyurea-arrested cultures expressing KAR9-tap (yRM4366) and kar9-S496A-tap (yRM6015).

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION ACKNOWLEDGMENTS REFERENCES

Although phosphorylation by other kinases is possible, two Cdc28p consensus sites have been identified in Kar9p at serines 197 and 496 (Liakopoulos et al., 2003). The presence of a faint slower migrating band in the kar9-S197A S496A mutant and the presence of the highest molecular weight band in the S197A mutant supports the premise that at least one additional phosphorylation site exists in Kar9p (Figure 7C) (Maekawa and Schiebel 2004). The three bands of Kar9p that we observe are consistent with the highest band being phosphorylated at two sites, whereas the middle band represents phosphorylation at a single site. Thus, the lack of the highest molecular weight isoform in bik1 (Figure 5) suggests that Bik1p facilitates only one phosphorylation event. Furthermore, the absence of the highest molecular weight band in the kar9-S496A mutant in both asynchronous and hydroxyurea-treated cultures suggests that phosphorylation at serine 496 is required for the formation of the highest band.

We propose a model in which Bik1p promotes the phosphorylation of Kar9p by facilitating its interaction with Clb5p (Figure 6C). The Clb5p binding site on Kar9p contains serine 496 (Figure 6C), which is adjacent to the binding site of Bik1p (our unpublished data). Therefore, the binding of Bik1p could alter the conformation of Kar9p in such a way as to allow Clb5p-Cdc28p access to this site. We also cannot rule out the possibility that Bik1p could be protecting Kar9p from dephosphorylation, perhaps by limiting access to a phosphatase.

Several possibilities could account for the mechanism by which Bik1p promotes the asymmetric localization of Kar9p at one SPB. The asymmetry could be the result of just the interaction itself between Bik1p and Kar9p. However, this is unlikely because Bik1p-3GFP is localized to regions associated with both poles (Figure S2). Although it is also possible that the altered localization of Kar9p could affect its phosphorylation, we instead favor a model in which absence of Bik1p changes the phosphorylation of Kar9p, which in turn causes its aberrant localization. Consistent with this model, mutations in cdc28 and several cyclins result in a similar alteration in the asymmetric localization of Kar9p (Liakopoulos et al., 2003; Maekawa et al., 2003; Maekawa and Schiebel, 2004). It is also possible that the mislocalization of Kar9p might be indirect through an effect of the bik1 mutation on the localization of Bim1p, which also interacts with Kar9p. Distinguishing between these possibilities will require an analysis of bik1 alleles that separate these activities.

It seems unlikely that the hypophosphorylation of Kar9p-tap in bik1 mutants is due to a cell cycle defect (in which a smaller proportion of bik1 cells are in S phase) for two reasons. First, the majority of cells in both wild type and bik1 are in S phase in the HU arrest and Kar9p is under-phosphorylated in bik1 cells in comparison with wild type (Figure 5D). Second, kip2 mutants are expected to show a similar cell cycle profile as bik1 mutants. Kar9p-tap is more heavily phosphorylated in kip2 mutants than in bik1 mutants. In kip2 mutants, the Kar9p-tap isoforms were similar to those seen in wild type for both HU-arrested cells and asynchronous cells (Figure 5, B and D). Moreover, the cell cycle distribution of bik1 cells was not obviously different from that observed in kip2 or dyn1 when assayed by DAPI staining and bud size (our unpublished data).

Four MAPs and Kar9p
Before this work, three MAPs (Bim1p, Stu2p, and Kip2p) were known to interact with Kar9p (Korinek et al., 2000; Lee et al., 2000; Miller et al., 2000; Maekawa et al., 2003). The identification of Bik1p as a fourth MAP adds complexity to the model of how Kar9p associates with MTs. Our model shown in Figure 8 depicts Kar9p interacting with a successive series of MAPs. Bik1p interacts with Kar9p, changing its conformation. This facilitates the phosphorylation of Kar9p at serine 496 by Clb5p-Cdc28p, and this acts to restrict Kar9p to one SPB. Bim1p is involved in loading Kar9p onto the SPB (Liakopoulos et al., 2003) and is responsible for tethering Kar9p to cMTs (Lee et al., 2000; Korinek et al., 2000; Miller et al., 2000). The observation that Kar9p-3GFP fluorescence is less intense, but still present, at the tip of the MT in kip2 strains suggests that both Kip2p-dependent and -independent mechanisms are responsible for the localization of Kar9p at the plus end. In both kip3 and bik1 kip3 cells, we also observed that more cells displayed Kar9p-3GFP localization at plus ends and along the lengths of cMTs (Figure 2B). This could be due to either a possible direct interaction between Kar9p and Kip3p or to the increased stability of MTs in kip3, allowing Kar9p to remain at the plus end.

View larger version (25K): [in this window] [in a new window]   Figure 8. A proposed mechanism for directing Kar9p to one SPB and MT plus end. In this model, Kar9p interacts with a succession of MAPs. 1) Bik1p-facilitated phosphorylation by Clb5p-Cdc28p restricts Kar9p to one SPB. 2) Bim1p loads Kar9p onto the SPB. 3) Bim1p tethers Kar9p to the MT and Kip2p transports a portion of Kar9p to the plus end. Clb4p also plays an important role in directing the localization of Kar9p (Liakopoulos et al. 2003; Maekawa et al. 2004).

Bik1p Has Functions in Both the Kar9p and Dynein Spindle Positioning Pathways
Deletion mutations between KAR9 and BIK1 are synthetically lethal with each other, suggesting that each protein carries out a vital function in one of the two partially redundant pathways. Whereas the placement of Bik1p in the dynein genetic pathway is explained by its recruitment of dynein to the cMT plus end (Sheeman et al., 2003), our analyses here show that Bik1p also conducts a novel function in the Kar9p pathway. This function was not detected by genetic analysis (Miller and Rose, 1998). Although Bik1p and Kip2p act in both pathways, it is not surprising that these genes are not essential because Kar9p remains localized at the plus end in both mutants, albeit with a less intense fluorescence signal. Thus, one explanation for this apparent paradox is that residual amounts of Kar9p at the plus end are adequate for Kar9p to function effectively and link the plus ends of cMT to Myo2p, orienting the spindle. Nevertheless, the finding that Bik1p physically interacts with proteins of both the Kar9p and the dynein pathways provides new insight into how the two pathways may be coregulated.

Our data suggest that the role of Bik1p in SPB inheritance is to help restrict Kar9p localization to one SPB and thus one set of MTs. In this case, the incorrect inheritance of the new SPB should be proportional to the amount of Kar9p mislocalization at that SPB. In bik1, we find that 70% of cells display Kar9p at both SPBs (Figure 3B). If spindle pole body inheritance were random in this 70%, then only half of the 70% (or 35% of the total cells) would be expected to display an SPB inheritance defect. Indeed, our findings show that 26% of bik1 cells do inherit the wrong pole (Figure 4). This is a more severe defect than is seen in other mutants of the dynein pathway, including kip2. These data support the idea that the asymmetric localization of Kar9p to one SPB and corresponding set of MTs designates that SPB as daughter bound. It further suggests that Bik1p acts in that designation. The involvement of Bik1p in the mechanism of SPB inheritance provides further insight into a mechanism