Introduction

Top Abstract Introduction Materials & Methods Results Discussion References

MICROTUBULES are a component of all eukaryotic cytoskeletons and are essential for many diverse cellular and intracellular movements, such as flagellar motility, organelle positioning, vesicle trafficking, mitotic spindle orientation, and chromosome segregation. Two families of chemomechanical motor proteins, dynein and kinesin-related proteins, are crucial for these microtubule functions (Vale et al., 1985; Schnapp and Reese, 1989; Steuer et al., 1990; reviewed by Vale and Fletterick, 1997; Hirokawa, 1998). These proteins possess highly conserved motor domains responsible for movement along microtubules (Yang et al., 1990). The nonmotor portion of these proteins links the motor domain to a vast array of cargoes or localizes the motor to various subcellular sites (Yang et al., 1989; Afshar et al., 1995; Wittmann et al., 1998).

The family of proteins sharing a region of homology with the motor domain of conventional kinesin-heavy chain (KHC)¹ has grown enormously in recent years (reviewed by Hirokawa, 1996; Moore and Endow, 1996). These kinesin-related proteins (KRPs) have been identified in all eukaryotes from yeast to humans. KRPs vary in directionality and the position of their motor domains. Most KRPs, as well as conventional kinesin, have NH₂-terminal motor domains and travel toward the more dynamic plus ends of microtubules. Others have internally located motor domains and may also have plus-end polarity. Another subfamily of KRPs have COOH-terminal motors and travel toward the less dynamic minus ends of microtubules, the end generally associated with microtubule-organizing centers. Whereas KRPs share a high degree of sequence similarity within their motor domains, the remainder of their primary sequences are highly divergent. These variable regions are thought to interact with different proteins to mediate the functional specificity of the motor (Page et al., 1994). Recent studies have unveiled many details about the molecular structure and function of kinesin-like motor domains (Hirose et al., 1995, 1996; Arnal et al., 1996; Kull et al., 1996; Sablin et al., 1996; Gulick et al., 1998), but far less is known about how these proteins are regulated and what other components are involved in their function.

Aside from the light chains of conventional KHC, few other kinesin-associated proteins (KAPs) have been identified. In many organisms, KHCs associate with various isoforms of kinesin-light chains (KLCs) to form complexes involved in transport of organelle and vesicle populations (Cyr et al., 1991; Beushausen et al., 1993; Gauger and Goldstein, 1993; Wedaman et al., 1993; Khodjakov et al., 1998; Rahman et al., 1998). Additionally, KHC has been shown to tightly associate with both a KLC kinase and a KLC phosphatase that putatively regulate motor function (McIlvain et al., 1994; Lindesmith et al., 1997). Two KAPs that interact with specific KRPs have also been identified. The KIF3 heterodimeric class of KRPs, also known as kinesin-II, interact with a single KAP to form a heterotrimeric complex (Cole et al., 1993, 1998; Scholey, 1996; Wedaman et al., 1996; Yamazaki et al., 1996). Finally, a Saccharomyces cerevisiae KRP, Kar3p, has been shown to associate with the Cik1 protein, which is essential for Kar3p localization and function during mating (Page et al., 1994).

The Kar3 protein is one of six KRPs encoded by the S. cerevisiae genome (Meluh and Rose, 1990). KAR3 encodes a protein containing a kinesin-motor domain at its COOH terminus (Meluh and Rose, 1990). The Kar3p-motor domain possesses minus-end directionality and microtubule-depolymerizing activity in vitro (Endow et al., 1994). In addition to an essential role in nuclear fusion during mating, or karyogamy, Kar3p has been implicated in several microtubule functions during the vegetative cell cycle. These putative functions include spindle assembly, mitotic chromosome segregation, microtubule depolymerization, kinetochore-motor activity, spindle positioning, and as a force opposing the action of other KRPs (Meluh and Rose, 1990; Roof et al., 1991; Saunders and Hoyt, 1992; Hoyt et al., 1993; Endow et al., 1994; Middleton and Carbon, 1994; Cottingham and Hoyt, 1997; DeZwaan et al., 1997; Saunders et al., 1997a,b; Huyett et al., 1998). This presents an interesting problem: how can one motor protein perform such a diverse array of functions within a single cell?

The role of Cik1p during mating is to target Kar3p to cytoplasmic microtubules (Meluh and Rose, 1990; Page et al., 1994). Kar3p and Cik1p are interdependent for their localization to the SPBs and cytoplasmic microtubules of cells treated with mating pheromone (Page et al., 1994). Expression of KAR3 and CIK1 is increased upon exposure to pheromone, but both genes are also expressed during vegetative growth (Meluh and Rose, 1990; Page and Snyder, 1992; Kurihara et al., 1996). Cik1p is also involved in a subset of Kar3p's vegetative functions. kar3 and cik1 mutants share several vegetative phenotypes, including a growth defect at 37°C, enhanced cytoplasmic microtubules, very short mitotic spindles, and an accumulation of large budded cells indicative of a mitotic cell-cycle checkpoint delay (Meluh and Rose, 1990; Page and Snyder, 1992; Page et al., 1994). They also share genetic interactions with several genes (Manning et al., 1997). Furthermore, Cik1p requires Kar3p for its mitotic spindle localization (Page et al., 1994), and the two proteins coimmunoprecipitate from vegetative cell lysates (Barrett, J.G., B.D. Manning, and M. Snyder, unpublished data).

However, unlike during mating, Kar3p does not require Cik1p for its localization to the spindle poles in mitosis (Page et al., 1994; this study). This suggests that Kar3p has some Cik1p-independent functions. Genetic studies support this hypothesis. Kar3p is believed to oppose the force generated by two other S. cerevisiae KRPs, Cin8p and Kip1p, which are involved in spindle pole separation both during spindle assembly and during anaphase B spindle elongation (Hoyt et al., 1992, 1993; Roof et al., 1992; Saunders and Hoyt, 1992; Saunders et al., 1995). Disruption of KAR3 function partially rescues the temperature-sensitive growth defect and spindle collapse phenotype of cin8^ts kip1 mutants (Saunders and Hoyt, 1992; Hoyt et al., 1993). In contrast, disruption of CIK1 does not rescue this mutant (Page et al., 1994; this study). Together, these results suggest that Kar3p may perform some of its vegetative functions alone or in association with a different KAP.

In this study we describe a Cik1p-homologous protein in S. cerevisiae that acts as a second KAP for Kar3p. We demonstrate that this protein, Vik1p (vegetative interaction with Kar3p), is present in vegetatively growing cells but absent from mating-pheromone treated cells. Vik1p forms a complex with Kar3p that is distinct from that between Kar3p and Cik1p. Furthermore, we show that Kar3p and Vik1p are interdependent for their concentration at the poles of the mitotic spindle. Phenotypic and genetic comparisons of cik1 and vik1 mutants demonstrate that Cik1p and Vik1p are likely to mediate distinct subsets of Kar3p functions. Our data suggest that Cik1p and Vik1p regulate Kar3p function, at least in part, by targeting the motor to various sites of action within the cell. This is the first example of two distinct associated proteins differentially regulating a single KRP.

	Materials and Methods

Top Abstract Introduction Materials & Methods Results Discussion References

Strains, Media, and Standard Methods

S. cerevisiae strains used in this study are listed in Table I. Yeast growth media, molecular biological techniques, and genetic manipulations were as described previously (Sambrook et al., 1989; Guthrie and Fink, 1991). Yeast transformation procedures were performed using the lithium acetate method (Ito et al., 1983). Where indicated, rich medium, consisting of yeast extract, peptone, and dextrose, was supplemented with benomyl (DuPont), dissolved in DMSO, to a final concentration of 10, 20, or 30 µg/ml. Sensitivity of wild-type yeast strains to these concentrations of benomyl varied between preparations of benomyl containing agar plates and, hence, growth comparisons were always performed on the same plate. Sensitivity also varies dramatically between growth temperatures (i.e., at 23°C strains are much more benomyl sensitive than at 30°C).

Construction of VIK1::3XHA Strain

A strain containing the VIK1::3XHA allele (Y1744) was constructed by the PCR-epitope tagging method described previously (Schneider et al., 1995). The primers 5'-TATTAACGATTTCAGAAGAAGTTCAAACACAACTTTGTAAAAGAAAGAAAAAGCTCACTAGGGAACA-AAAAGCTGG-3' and 5'-CTTATTTGTTTCATATCTAAATGGCTGTG TTAAGAAAGACGATAATG TGACCGAGC TTAC TATAGG-GCGAATTGG-3' were used in a PCR reaction with pMPY-3XHA as the template. The resulting 1.5-kb PCR product contains the URA3 gene flanked by direct repeats encoding three copies of the hemagglutinin (HA) epitope and contains 59 bp of sequence from the 3' end of the VIK1 gene at one end and 59 bp of sequence downstream of, and including, the VIK1 translation termination codon at the other end. This fragment was used to transform yeast strain Y1731, and transformants were selected on synthetic complete medium lacking uracil. Correct integration into the 3' region of the VIK1 locus was confirmed by PCR analysis with primers to sequences flanking the site of insertion. Transformants with a correct 3XHA-URA3-3XHA integration were then incubated on plates containing 5-fluoroorotic acid to select for loss of the URA3 marker by recombination between the repeated 3XHA regions. The resulting VIK1::3XHA allele was confirmed by PCR and immunoblot analysis. This allele does not display any vik1 phenotypes; growth of VIK1::3XHA strains in the presence of benomyl is identical to wild-type strains, and the temperature-sensitive growth defect of cik1 mutants is the same in a VIK1 or VIK1:: 3XHA background.

Immunoprecipitations and Immunoblot Analysis

Cells were grown in rich liquid medium to mid-logarithmic phase (OD₆₀₀ = 0.5-0.8), and a total of 10 OD₆₀₀ units of cells were collected by centrifugation, washed, and resuspended in 100 µl lysis buffer (1 M NaCl, 10 mM EDTA, 2 mM EGTA, 5% glycerol, 40 mM Tris-HCl, pH 7.5, containing 1 µl yeast protease inhibitor cocktail (Sigma Chemical Co.) and 200 µM PMSF). When indicated, cells were first washed twice with fresh medium, resuspended in medium containing 5 µg/ml -factor mating pheromone (Sigma Chemical Co.), and incubated for 2 h before harvesting. Cell lysates were prepared in Eppendorf tubes using zirconia/silica beads (Biospec Products) with 40-s pulses of vortexing separated by incubations on ice; this procedure was repeated 6-8 times. Lysates were then centrifuged for 10 min at 6,500 g, and a 10-µl aliquot was removed for immunoblot analysis.

For immunoprecipitations, the remaining cell lysate and beads were washed with 500 µl lysis buffer containing detergents (1% NP-40, 0.5% sodium deoxycholate, and 0.1% SDS) without NaCl for 20 min on a roller drum at 4°C. Lysates were then cleared of unlysed cells and cell debris by centrifugation for 10 min at 6,500 g and ~500 µl of supernatant was transferred to a new tube. The 500 µl lysate was brought to a 1-ml vol with the same lysis buffer plus detergents (100 mM final NaCl concentration). The cell lysate was then precleared for 1 h by incubation with 20 µl of a 1:1 slurry of protein A/G-agarose (Pierce) and TBS (150 mM NaCl, 50 mM Tris-HCl, pH 8.0). Immunoprecipitations were performed by incubation of cell lysates with either 2 µl mouse monoclonal anti-HA antibodies (12CA5 from BABCO), 10 µl rabbit polyclonal anti-Cik1p antibodies (Page and Snyder, 1992), or 5 µl rabbit polyclonal anti-Kar3p antiserum (gift of L.L. Satterwhite, P.B. Meluh, and M.D. Rose, Princeton University, Princeton, NJ; Meluh and Rose, 1990; Page et al., 1994) for 2 h. 20 µl protein A/G-agarose was then added and incubated for 1 h before collection by centrifugation at 2,000 g for 1 min. The protein A/G-agarose antibody complexes were then washed twice with 1 ml TBS containing detergents and protease inhibitors (1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS, 1 µl Sigma yeast protease inhibitor cocktail, 200 mM PMSF) and once with 1 ml TBS plus protease inhibitors. The final pellet was resuspended in 30 µl Laemmli sample buffer (Laemmli, 1970).

Proteins from cell lysates and immunoprecipitations were denatured by incubation at 90°C for 10 min before electrophoretic separation in either 8% or 10% SDS-polyacrylamide gels. For attempts to detect Vik1p-3XHA in mating-pheromone treated cells, a threefold larger volume of cell lysate was loaded than for vegetative cell lysates (data not shown). Proteins were then transferred to Immobilon-P membranes (Millipore) for immunoblot analysis with either mouse monoclonal anti-HA antibodies (12CA5 from BABCO), rabbit polyclonal anti-Cik1p antibodies (Page and Snyder, 1992), or a crude IgG fraction of the rabbit polyclonal anti-Kar3p antiserum (Meluh and Rose, 1990; Page et al., 1994). Reactive protein bands were then detected with alkaline phosphatase-conjugated secondary antibodies (Amersham) and the CDP Star detection reagent (Boehringer Mannheim). Overexposure of all blots failed to detect Vik1p-3XHA in cell lysates from mating-pheromone treated cells.

Fluorescence Microscopy

Indirect immunofluorescence was performed as described previously (Gehrung and Snyder, 1990; Pringle et al., 1991). Due to sensitivity of the HA-epitope to formaldehyde fixation, mid-logarithmic phase cells were fixed with 3.7% formaldehyde for only 15 min. They were then washed twice with solution A (1.2 M sorbitol, 50 mM potassium phosphate buffer, pH 6.8), and spheroplasts were prepared by incubating cells in solution A containing 5 µg/ml Zymolyase 100T, 0.03% glusulase, and 0.2% 2-mercaptoethanol at 37°C for 15 to 30 min. Spheroplasted cells were then washed and resuspended in solution A and placed onto poly(L-lysine)- coated slides. Vik1p-3XHA and Kar3p-HAT were detected by incubation overnight at 4°C with preabsorbed mouse monoclonal anti-HA primary antibodies (16B12 from BABCO) diluted in PBS/BSA. Bound mouse anti-HA antibodies were then detected by incubation for 90 min at room temperature with preabsorbed CY3-conjugated goat anti-mouse secondary antibodies (Jackson ImmunoResearch Laboratory, Inc.). Microtubules were detected by incubation with rabbit anti-yeast -tubulin (Tub2p) primary antibodies (gift of F. Solomon, Massachusetts Institute of Technology; Bond et al., 1986) followed by incubation with FITC-conjugated goat anti-rabbit secondary antibodies. After both primary and secondary antibody incubations, slides were washed twice with PBS/BSA and twice with PBS/BSA plus 0.1% NP-40. Finally, slides were mounted in 70% glycerol, 2% n-propyl gallate, and 0.25 µg/ml Hoechst 33258 to preserve the preparation and stain DNA for localization of nuclei.

Photographs of representative cells stained with anti-HA antibodies, anti-Tub2p antibodies, and/or Hoechst 33258 were taken, and composite figures were produced and processed using Adobe Photoshop version 3.0 (Adobe Systems, Inc.). Processing procedures were identical for each photo of a particular staining method within a composite figure.

Construction of KAR3::HAT Strains

A strain containing the KAR3::HAT allele was constructed using a transposon insertion technique described previously (Ross-MacDonald et al., 1997). The entire KAR3 coding region was cloned into the vector pHSS6 and subjected to transposon mutagenesis in Escherichia coli as described (Ross-MacDonald et al., 1997). Plasmid DNA was prepared from selected strains and digested with NotI, producing a fragment containing the mTn-3xHA/lacZ transposon inserted randomly into the KAR3 gene. These fragments were then used to transform yeast strain Y1869, using the URA3 marker encoded by the transposon for selection. Transformants with in-frame transposon insertions into the KAR3 genomic locus were selected by detection of -galactosidase activity as described (Burns et al., 1994). Cre recombinase-mediated excision, leaving only the 93 amino acid HA/transposon tag (HAT) inserted, was induced by growth on galactose (pB227 contains cre under control of a galactose-inducible promoter). Recombinants were selected on 5-fluoroorotic acid. Strains with in-frame HAT insertions were then tested for kar3 mutant phenotypes, such as defects in karyogamy and temperature sensitivity. PCR and sequence analysis was performed on DNA from fully complementing strains in order to determine the site of insertion within the KAR3 gene.

Strain Y1870 contains a KAR3 allele with a HAT insertion after the codon for S⁶⁸, and does not exhibit any kar3 phenotypes. Y1870 was then crossed with Y1864 to yield Y1871. Y1871 was then sporulated and a MAT KAR3::HAT cik1::LEU2 segregant (Y1751) was isolated. The VIK1 gene was disrupted with the HIS3 marker in Y1870 yielding Y1752 (see below). Y1752 was then crossed with Y1751 yielding Y1753. Y1753 haploid segregants were used to analyze Kar3p-HAT localization in wild-type, cik1, vik1, and cik1 vik1 strains.

Cloning of VIK1 and Disruption of VIK1, CIK1, and KAR3

Sequence of the VIK1 gene was acquired from the Saccharomyces Genome Database (ORF designation YPL253c; Stanford University). A 2,920-bp region, from the MscI site 5' of the predicted VIK1 translation start site to the SpeI site 3' of the VIK1 translation termination site, was PCR amplified from yeast genomic DNA and cloned into pBluescript SK (Stratagene), replacing its EcoRV-SpeI fragment (pSK-VIK1). The SalI-SacII fragment from this plasmid, containing the VIK1 gene and flanking sequences, was cloned into the SalI-SacII site of the CEN plasmid pRS316 (Sikorski and Hieter, 1989). This plasmid was linearized by deleting the NruI-AflII fragment containing the entire VIK1 ORF, then gap repaired in a wild-type yeast strain (Guthrie and Fink, 1991). This CEN plasmid encoding wild-type VIK1 was used for subsequent phenotypic analysis.

VIK1 disruption constructs were made by replacing the NruI-AflII fragment of pSK-VIK1 with the SmaI fragment from either pJA50 (HIS3-Km^r) or pJA53 (URA3-Km^r) (Elledge and Davis, 1988). SalI-XbaI fragments from these plasmids, containing either the HIS3 or URA3 selectable markers flanked by the 5' and 3' noncoding regions of VIK1, were used to transform yeast strains Y270, Y818, and Y1870 yielding vik1 strains Y1733, Y1748, and Y1752, respectively.

CIK1 and KAR3 disruptions were made as previously described (Page and Snyder, 1992; Page et al., 1994). The cik1-3::LEU2 construct was used to transform strains Y817 and Y1744 yielding cik1 strains Y1758 and Y1745, respectively. The kar3-4::URA3 construct was used to transform strains Y818 and Y1744 yielding kar3 strains Y1759 and Y1750, respectively.

	Results

Top Abstract Introduction Materials & Methods Results Discussion References

Vik1p: A Cik1p Homologue Present in Vegetative Cells but Not in Cells Treated with Mating Pheromone

A database homology search with the Cik1p amino acid sequence identified an open reading frame (ORF) on chromosome XVI of the S. cerevisiae genome (BLAST search of predicted translation products of S. cerevisiae ORFs; Saccharomyces Genome Database ORF designation YPL253c) predicted to encode a protein with significant homology to Cik1p. This gene was named VIK1, for vegetative interaction with Kar3p (see below). VIK1 is predicted to encode a protein of 647 amino acids, with an overall sequence identity of 24%, and similarity of 37%, to Cik1p. The Vik1p sequence also shares structural similarity to Cik1p. It contains a predicted -helical coiled-coil domain (amino acids 80-385) of the same length and location as that found in Cik1p (amino acids 81-388; Page and Snyder, 1992; Fig. 1; predicted with COILS version 2.2; Lupas et al., 1991; Lupas, 1996); both proteins contain a short break of 40 amino acids in their coiled-coil regions. This break and an 80 residue NH₂-terminal globular domain are the most divergent regions of the two proteins (16% identity). One notable difference in the amino terminal domain is that Vik1p lacks a recognizable nuclear localization signal found in Cik1p (amino acids 24 to 33; Fig. 1; Barrett, J.G., and M. Snyder, manuscript in preparation). Finally, the COOH-terminal globular domains of the two proteins share two regions of 134 and 43 amino acids in length with 25% identity each. Therefore, Vik1p shares sequence and structural homology to Cik1p which is not confined to their coiled-coil domains.

View larger version (70K): [in this window] [in a new window]   Fig. 1.   Vik1p is a homologue of Cik1p. (A) Schematic representation of Cik1p and Vik1p. Regions with the same shading pattern between the two proteins share at least 25% amino acid sequence identity. The two proteins have homologous regions predicted to form -helical coiled-coil structures (wavy line shading), as well as two other stretches of homology in their carboxyl termini (horizonal and vertical line shading). Cik1p contains a nuclear localization signal at its NH2 terminus (black box). (B) Amino acid sequence alignment of Vik1p and Cik1p. Identical and similar residues are indicated by dark and light shading, respectively. The predicted coiled-coil regions of the two proteins are denoted by a black bar over the sequence, whereas the nuclear localization signal of Cik1p is underlined. The alignment was performed with BCM Search Launcher: Multiple Sequence Alignments (Baylor College of Medicine, Houston, TX). Shading was performed by the Boxshade 3.21 program (ISREC Bioinformatics Group).

To characterize the Vik1 protein by immunoblot and immunofluorescence analysis, DNA encoding a triple HA-epitope tag was integrated into the COOH-terminal coding region of the VIK1 genomic locus (see Materials and Methods). The resulting VIK1::3XHA fusion allele fully complements VIK1 function (see Materials and Methods). Immunoblot analysis using anti-HA monoclonal antibodies detects a protein of 92-kD in cell lysates and anti-HA immunoprecipitations from VIK1::3XHA strains (Fig. 2 A). This molecular mass is close to the predicted 76-kD of Vik1p plus the triple-HA epitope tag. This fusion protein is not detected in cell lysates or anti-HA immunoprecipitations from VIK1 untagged strains (Fig. 2 A). Therefore, the VIK1::3XHA allele is expressed during vegetative growth and produces a functional protein of the expected size.

View larger version (66K): [in this window] [in a new window]   Fig. 2.   Vik1p is not present in mating pheromone-treated cells and does not coimmunoprecipitate with Cik1p. Cell lysates were prepared from a VIK1::3XHA strain (Y1744) and a VIK1 untagged strain (Y1731) that were either grown vegetatively (-F ) or first exposed to the -factor mating pheromone (-F +). Proteins from these lysates were either separated by SDS-PAGE using a 10% gel (Lysate) or first immunoprecipitated with anti-HA antibodies (HA IP) or anti-Cik1p antibodies (Cik1 IP) before SDS-PAGE separation. (A) Immunoblot analysis with anti-HA antibodies detects a protein of 92 kD in VIK1::3XHA cell lysates and anti-HA immunoprecipitations from vegetative cells, but not from -factor treated cells, anti-Cik1p immunoprecipitations, or VIK1 anti-HA immunoprecipitations. (B) Immunoblot analysis with anti-Cik1p antibodies detects the 73-77-kD Cik1 protein in VIK1::3XHA cell lysates and anti-Cik1p immunoprecipitations from both vegetative and -factor treated cells, but not from VIK1::3XHA or VIK1 anti-HA immunoprecipitations. The mass of molecular mass markers are shown in kilodaltons.

The Vik1p-3XHA protein is not detected in cell lysates or anti-HA immunoprecipitations from cultures first exposed to the -factor mating pheromone (Fig. 2 A; see Materials and Methods). In contrast, Cik1p greatly increases in abundance upon exposure to -factor (Fig. 2 B; Page and Snyder, 1992). Consistent with this result, the region upstream of the VIK1 ORF does not contain any predicted pheromone-response elements (Kronstad et al., 1987). However, the pheromone-inducible CIK1 and KAR3 genes each contain multiple pheromone-response elements in their 5' noncoding regions (Meluh and Rose, 1990; Page and Snyder, 1992). Thus, since Vik1p is not present during mating-pheromone induced differentiation, it is not likely to have a significant role during mating.

Vik1p Coimmunoprecipitates with Kar3p

Cik1p physically associates with Kar3p (Page et al., 1994), and Vik1p shares sequence and structural similarity to Cik1p; we therefore tested whether Vik1p could also interact with Kar3p. Cell lysates were prepared from wild-type, kar3, and cik1 strains expressing either the VIK1:: 3XHA allele or untagged VIK1. Proteins from these cell lysates were separated by SDS-PAGE (Fig. 3 A) or first immunoprecipitated with anti-Kar3p polyclonal antibodies (Fig. 3 B; Meluh and Rose, 1990; Page et al., 1994). Immunoblot analysis using anti-HA antibodies detects Vik1p-3XHA in cell lysates from wild-type, kar3, and cik1 strains containing the VIK1::3XHA allele but not in untagged strains (Fig. 3 A). Therefore, Vik1p stability is not affected by the absence of either Kar3p or Cik1p. Anti-HA immunoblots of proteins immunoprecipitated with anti-Kar3p antibodies detects Vik1p-3XHA from a wild-type VIK1::3XHA strain but not from a kar3 VIK1:: 3XHA strain or an untagged strain (Fig. 3 B). Therefore, immunoprecipitation data indicate that Kar3p and Vik1p physically interact. Furthermore, the Kar3p-Vik1p complex is quite stable, as cell lysates were prepared in a 1 M NaCl solution (see Materials and Methods).

View larger version (57K): [in this window] [in a new window]   Fig. 3.   Vik1p coimmunoprecipitates with Kar3p. Cell lysates were prepared from vegetatively growing cultures of a wild-type (WT) VIK1 untagged strain (Y1731; HA-tag ) and wild-type (Y1744), kar3 (Y1750), and cik1 (Y1745) VIK1::3XHA strains (HA-tag +). Proteins from these lysates were either separated by SDS-PAGE using an 8% gel (A; Lysates) or first immunoprecipitated with anti-Kar3p antibodies (B; -Kar3p IPs) before SDS-PAGE separation. (A) Immunoblot analysis with anti-HA antibodies detects the 92 kD Vik1-3XHA protein in cell lysates from the wild-type, kar3, and cik1 VIK1::3XHA strains, but not from the VIK1 untagged strain. (B, top) Immunoblot analysis with anti-HA antibodies detects Vik1p-3XHA in anti-Kar3p immunoprecipitations from the wild-type and cik1 VIK1::3XHA strains, but not from the VIK1 untagged strain or the kar3 VIK1::3XHA strain (B, bottom). Immunoblot analysis with anti-Kar3p antibodies detects the 88 kD Kar3 protein in anti-Kar3p immunoprecipitations from the wild-type VIK1 untagged strain and wild-type and cik1 VIK1::3XHA strains, but not from the kar3 VIK1::3XHA strain (B, bottom). The mass of molecular mass markers are shown in kilodaltons.

Vik1p-3XHA is also detected in anti-Kar3p immunoprecipitations from a cik1 VIK1::3XHA strain (Fig. 3 B), indicating that Cik1p is not required for this interaction between Kar3p and Vik1p. Furthermore, an association between Vik1p and Cik1p is not detected by immunoprecipitation experiments. Vik1p-3XHA is not precipitated from VIK1::3XHA cell lysates by anti-Cik1p polyclonal antibodies (Fig. 2 A; Page and Snyder, 1992; Page et al., 1994), and, reciprocally, Cik1p is not precipitated from these cell lysates with anti-HA antibodies (Fig. 2 B). Protein preparations from vegetatively growing cells or -factor-treated cells produce the same results. Thus, Vik1p and Cik1p do not appear to be part of the same complex. Together, these results suggest that Vik1p and Cik1p interact with Kar3p separately, and, therefore, form two different complexes during vegetative growth.

Vik1p Localizes to the Spindle-Pole Bodies

To determine the subcellular localization of Vik1p, asynchronous cultures of VIK1::3XHA and VIK1 untagged strains were fixed briefly with formaldehyde and analyzed by immunofluorescence using anti-HA and anti-tubulin antibodies (see Materials and Methods). VIK1::3XHA cells display anti-HA staining concentrated at the yeast microtubule-organizing centers, or spindle-pole bodies (SPBs), in all stages of the cell cycle (Fig. 4, B and C). The position of the SPBs correspond to the brightest foci of anti-tubulin staining. Anti-HA staining at the SPBs is not detected in VIK1 untagged cells (Fig. 4 A). Moreover, detection of Vik1p-3XHA at the SPBs does not require costaining with anti-tubulin antibodies; staining of dots at the edges of both preanaphase and anaphase nuclei is still observed in control cells not stained with anti-tubulin antibodies (Fig. 4 C). Vik1p-3XHA staining is brightest in preanaphase cells with short spindles, where localization is clearly concentrated at the spindle poles (pattern observed in 100% of preanaphase spindles; n > 200; Fig. 4 B). Cells in the G1 phase of the cell cycle, scored as unbudded cells with a single SPB, display SPB staining that is more faint and not observed in every cell (staining observed in 60% of G1 cells; n = 100). Finally, cells in early or late anaphase, as scored by elongated spindles and/or elongated nuclei penetrating the bud neck, display faint staining concentrated at the spindle poles (staining of at least one spindle pole is observed in 73% of anaphase cells; n = 100; Fig. 4 C, inset). Anti-HA staining is not detected along the lengths of spindle or cytoplasmic microtubules in VIK1::3XHA cells. Short fixation times due to sensitivity of the HA-epitope to formaldehyde leads to a diminished number of cytoplasmic microtubules. However, in cells that contain cytoplasmic microtubules, only staining at the SPBs is observed; microtubule staining is not evident. These staining patterns are independent of ploidy, and the nature and position of the epitope tag. VIK1::3XHA/VIK1::3XHA homozygous diploids, and cells expressing a VIK1::3Xmyc NH₂-terminal fusion allele stained with anti-myc monoclonal antibodies, exhibit identical patterns to those found in VIK1::3XHA haploid cells (data not shown).

View larger version (72K): [in this window] [in a new window]   Fig. 4.   Vik1p localizes to the spindle-pole bodies. Cells from growing cultures of VIK1 untagged strain Y1731 (A) and VIK1::3XHA strain Y1744 (B) were prepared for indirect immunofluorescence microscopy using anti-HA antibodies (A-C, left), anti-Tub2p antibodies (A and B, middle), and Hoechst 33258 staining (A-C, right). Representative cells are pictured: (A) two cells with preanaphase spindles from the VIK1 untagged strain demonstrating background staining; (B) two cells with preanaphase spindles from the VIK1::3XHA strain displaying Vik1p-3XHA staining at the spindle poles; (C) a field of VIK1::3XHA cells from a preparation not stained with anti-Tub2p antibodies, demonstrating that Vik1p-3XHA localization to one or two dots at the nuclear periphery does not depend on costaining of microtubules. Presumptive spindle-pole staining of two preanaphase cells and one cell in anaphase (inset) is indicated (C, arrowheads). The portion of the nucleus that has entered the bud in the inset C cell is out of the plane of focus, but its periphery overlaps with the dot indicated by the arrowhead.

The localization of Vik1p at, or near, the SPBs at various levels throughout the vegetative cell cycle is very similar to that reported for fusion proteins of Kar3p with either -galactosidase or HA (Page et al., 1994; Saunders et al., 1997; see below). However, in pheromone-treated cells, when Kar3p localizes to the SPB and cytoplasmic microtubules (Meluh and Rose, 1990; Page et al., 1994), Vik1p-3XHA staining above background is not detected (data not shown). This is consistent with immunoblot data demonstrating the absence of Vik1p in cell lysates from -factor-treated cells (Fig. 2 A), and further suggests that Vik1p functions in vegetative cells, but not mating cells.

Spindle-Pole Body Localization of Vik1p is Kar3p Dependent

Since Kar3p and Vik1p physically associate and have similar localization patterns, we investigated whether the localization of Vik1p to the SPBs was dependent on the presence of Kar3p. Therefore, Vik1p-3XHA localization was analyzed by anti-HA immunofluorescence in kar3 cells. Whereas Vik1p-3XHA concentrates at the SPBs in wild-type cells, it does not localize to the SPBs at any stage of the cell cycle in the kar3 mutant. Instead, the protein is present in cytoplasmic patches that appear to be excluded from the nucleus (SPB staining was not detected in any cells; n > 300; a representative preanaphase cell is shown in Fig. 5 B). This pattern is also observed with anti-myc staining in kar3 cells containing the VIK1::3Xmyc allele (data not shown). These cytoplasmic patches are not detected in kar3 VIK1 untagged strains (Fig. 5 A). SPB localization of Vik1p-3XHA can be restored by introducing a CEN plasmid containing KAR3 into the kar3 VIK1:: 3XHA strain (Fig. 5 C). This plasmid also restores normal spindle length, since disruption of KAR3 results in accumulation of cells with short spindles compared with wild-type cells (Meluh and Rose, 1990). Therefore, the Kar3p-Vik1p association is required for localization of Vik1p to the SPB.

View larger version (50K): [in this window] [in a new window]   Fig. 5.   The spindle-pole body localization of Vik1p depends on Kar3p. Cells from growing cultures of a kar3 VIK1 untagged strain (A), kar3 VIK1::3XHA strain Y1750 (B), Y1750 containing KAR3 on a YCp50-derived CEN plasmid (C), and cik1 VIK1::3XHA strain Y1745 (D) were prepared for indirect immunofluorescence microscopy with anti-HA antibodies (left), anti-Tub2p antibodies (middle), and Hoechst 33258 staining (right). Representative preanaphase cells are pictured: (A) an untagged cell demonstrating background staining; (B) a cell displaying Vik1p-3XHA mislocalization to cytoplasmic patches in the absence of Kar3p; (C) restoration of Vik1p-3XHA spindle-pole localization by introduction of a wild-type copy of KAR3, and; (D) Vik1p-3XHA localization to the poles of a short spindle from a cik1 cell.

Cik1p also localizes to the SPB in a Kar3p-dependent manner (Page et al., 1994), and, like kar3 mutants, cik1 mutants accumulate cells with short mitotic spindles (Page and Snyder, 1992). Therefore, in order to determine if Cik1p was involved in localizing Vik1p to the SPB or if the short spindle phenotype was responsible for Vik1p mislocalization in kar3 mutants, we analyzed Vik1p-3XHA localization in a cik1 mutant. In contrast to kar3 cells, cik1 cells display Vik1p-3XHA staining concentrated at the SPBs during all stages of the cell cycle, as observed in wild-type cells. Furthermore, HA staining is brightest at the poles of the short preanaphase spindles present in cik1 cells (a representative cell is shown in Fig. 5 D), indicating that the short spindle phenotype alone is not the cause of mislocalization of Vik1p. Therefore, these immunolocalization data support the immunoprecipitation experiments; Vik1p does not appear to be part of the same complex as Cik1p. Furthermore, Vik1p localization to the SPBs does not require Cik1p function.

Proper Spindle-Pole Body Localization of Kar3p in Vegetative Cells Is Vik1p Dependent

Cik1p is required to localize Kar3p to the SPB and cytoplasmic microtubules during mating but is not required for Kar3p localization to the mitotic spindle poles (Page et al., 1994). This suggests that in vegetative cells, Kar3p can either target to the SPBs by itself or interacts with another protein that is responsible for its concentration at the SPBs. Based on its localization and association with Kar3p, Vik1p could fulfill this function. Therefore, Kar3p was localized in the absence of Vik1p, Cik1p, and both Vik1p and Cik1p.

The subcellular localization pattern of Kar3p has been determined previously using fusion proteins that do not fully complement all of the kar3 phenotypes (Meluh and Rose, 1990; Page et al., 1994; Saunders et al., 1997). To create a fully complementing HA epitope-tagged version of Kar3p, we used a transposon insertion technique described previously (Ross-MacDonald et al., 1997; see Materials and Methods). Using this technique we created several strains containing in-frame insertions of the HAT into random regions of the KAR3 genomic locus. We then tested these strains for several kar3 phenotypes, such as defects in karyogamy and meiosis, slow growth, temperature-sensitive growth, enhanced cytoplasmic microtubules, and the ability to properly localize Vik1p-3Xmyc to the SPBs (Meluh and Rose, 1990; Page et al., 1994; Bascom-Slack and Dawson, 1997; Saunders et al., 1997a). Fully complementing alleles were sequenced and found to lie within the region encoding the NH₂-terminal globular domain of Kar3p (data not shown). A strain containing one of these KAR3:: HAT alleles (encoding Kar3p with the 93-amino acid HAT insertion at S⁶⁸) was used for immunofluorescence analysis with anti-HA antibodies.

Localization of Kar3p-HAT was examined in wild-type, cik1, vik1, and cik1 vik1 strains. Asynchronous cultures of haploid segregants from a cik1/CIK1 vik1/ VIK1 KAR3::HAT/KAR3::HAT diploid strain were fixed and prepared for immunofluorescence with anti-HA and anti-tubulin antibodies (see Materials and Methods). KAR3:: HAT wild-type cells, in all stages of the cell cycle, display anti-HA staining concentrated at the SPB region, as well as faint patches confined to the nucleus (as determined by colocalization with anti-tubulin and Hoechst staining, respectively). These fluorescence patterns are not observed in KAR3 untagged strains, in which background staining is restricted to the cytoplasm (Fig. 6 A). Consistent with previous Kar3p localization studies (Page et al., 1994; Saunders et al., 1997), Kar3p-HAT staining is brightest at the poles of preanaphase spindles (Fig. 6 B; Table II). Most cells in G1 (80%, 100 G1 cells counted) and anaphase (86%, 100 anaphase cells counted) also display Kar3p-HAT localization at the SPBs, but this staining is fainter than that observed in preanaphase cells (data not shown). In contrast to previous studies, Kar3p-HAT localization along spindle microtubules is not detected in any stage of the cell cycle. When cytoplasmic microtubules are observed, in wild-type cells or the mutant cells described below, Kar3p-HAT localization is not detected along their lengths. Hence, Kar3p is most abundant at the SPBs of cells throughout the cell cycle.

View larger version (60K): [in this window] [in a new window]   Fig. 6.   Concentrated localization of Kar3p to the spindle-pole bodies depends on Vik1p. Cells from growing cultures of KAR3 untagged strain Y1869 (A) and Kar3::HAT haploid segregants from strain Y1753 (B-E) were prepared for indirect immunofluorescence microscopy with anti-HA antibodies (left), anti-Tub2p antibodies (middle), and Hoechst 33258 staining (right). Representative preanaphase cells are pictured: (A) an untagged cell demonstrating background staining; (B) a wild-type cell with Kar3p-HAT staining primarily at the spindle poles, as well as faint patches confined to the nucleus (as determined by overlapping Hoechst staining); (C) a cik1 cell with Kar3p-HAT remaining at the spindle-poles, the nuclear patch staining is diminished in these cells; (D) a vik1 cell with Kar3p-HAT mislocalized to spindle microtubules and bright nuclear patches, and; (E) a cik1 vik1 cell with Kar3p-HAT mislocalized to bright patches more diffusely distributed throughout the nucleus.

The effect of disrupting Vik1p and Cik1p function on Kar3p-HAT localization was determined. Since wild-type localization is most obvious in preanaphase cells, we quantified Kar3p-HAT staining patterns in cells from asynchronous cultures at this stage (Table II). Similar to wild-type strains, KAR3::HAT cik1 cells display anti-HA staining at the SPBs during all stages of the cell cycle; again, the brightest staining is observed at the poles of the preanaphase spindle (Fig. 6 C; Table II). However, the nuclear patch staining detected in wild-type cells is qualitatively diminished in the cik1 mutant. Therefore, as described previously (Page et al., 1994), Cik1p is not required to localize Kar3p to the SPBs during the vegetative cell cycle.

In contrast, Kar3p-HAT localization during vegetative growth is dramatically altered in vik1 cells (Fig 6 D; Table II). Kar3p-HAT is no longer concentrated at the SPBs in these cells; instead, it predominantly displays a bright nuclear patch localization in all vik1 cells. Moreover, staining along the lengths of spindle microtubules is detected in many cells (34% of preanaphase cells; Fig 6 D; Table II). Some vik1 cells also display faint staining in the vicinity of the SPBs, but not along spindle microtubules (22% of preanaphase cells; Table II). Therefore, although vik1 cells may retain some Kar3p at the SPBs, Vik1p is required to concentrate, or restrict, Kar3p localization to the SPBs.

To determine if the spindle and faint SPB localization of Kar3p detected in vik1 cells is due to Cik1p function, we localized Kar3p-HAT in a cik1 vik1 double mutant. Kar3p-HAT localization is even more aberrant in the double mutant, with the nuclear patch staining distributed more diffusely throughout the nucleus in all cells (representative preanaphase cell shown in Fig. 6 E). Detection of Kar3p-HAT along the spindle or at the SPBs in these cells is greatly reduced compared with the vik1 mutant (spindle and faint SPB localization is observed in 7 and 9%, respectively, of cik1 vik1 preanaphase cells; Table II). Introduction of a CEN plasmid encoding VIK1 into either vik1 or cik1 vik1 mutants completely restores Kar3p-HAT concentration at the SPBs (data not shown). Therefore, during vegetative growth, Vik1p is primarily responsible for SPB targeting, or retention, of Kar3p. Cik1p may mediate Kar3p localization to less concentrated sites within the nucleus, such as the spindle and/or nuclear patches.

CIK1 and VIK1 Disruptions Result in Distinct Phenotypes

Cik1p and Vik1p exhibit differences in their expression patterns, and deletion of CIK1 or VIK1 results in unique effects on Kar3p localization. Therefore, the phenotypes resulting from disruption of VIK1 and CIK1 were compared. Suprisingly, vik1 mutants grow similar to wild-type strains at all temperatures. Using a sectoring assay described previously (Spencer et al., 1990), we determined that, unlike cik1 mutants that have a severe chromosome loss defect (Page and Snyder, 1992), vik1 mutants display only a threefold increase in frequency of chromosome loss per cell division relative to wild-type strains (data not shown). Furthermore, consistent with its absence in mating-pheromone treated cells, vik1 mutants are not defective in karyogamy, as determined by qualitative mating assays. However, compared with wild-type strains, vik1 mutants are resistant to the microtubule-depolymerizing drug benomyl (on plates containing 10, 20, or 30 µg/ml benomyl; 10 µg/ml in Fig. 7 C). The benomyl resistance phenotype always segregates with the vik1 mutation (11 tetrads analyzed).

View larger version (29K): [in this window] [in a new window]   Fig. 7.   Phenotypic analysis of isogenic wild-type, cik1, vik1, and cik1 vik1 strains. Isogenic haploid segregants from a tetratype tetrad of the cik1/ CIK1 vik1/VIK1 double heterozygous diploid strain Y1738 were grown overnight to stationary phase. 10-fold serial dilutions with a starting concentration of 1 × 106 cells/ml were spotted in 5 µl drops onto agar plates containing either rich medium (A and B) or rich medium with 10 µg/ml benomyl (C). Plates were incubated for four days at either 23°C (A and C) or 37°C (B). (A) Growth on rich medium at 23°C was similar for all four strains. (B) Wild-type and vik1 growth is indistinguishable at 37°C, whereas cik1 growth is temperature sensitive. The cik1 vik1 double mutant grows better at the elevated temperature than cik1 mutants. (C) Compared with growth of the wild-type strain on benomyl, the vik1 mutant is more benomyl resistant, and the cik1 and cik1 vik1 mutants are more benomyl sensitive. (D) Cells from mid-logarithmically growing cultures of wild-type, cik1, vik1, and cik1 vik1 haploid segregants of Y1738 were stained with Hoechst 33258 and large budded cells with a single preanaphase nucleus were counted. The percentage of these cells present in the total population counted is shown. The results represent the average of two experiments in each of which greater than 300 total cells were counted for each strain.

To analyze whether CIK1 and VIK1 are functionally redundant, we tested whether expression of VIK1 from a high copy plasmid could suppress the temperature-sensitive growth defect of a cik1 mutant. cik1 strains containing either the VIK1 plasmid or vector alone are equally affected by growth at the restrictive temperature (data not shown). A cik1 mutant was then mated with a vik1 mutant, and the resulting heterozygous diploid strain was sporulated. Haploid segregants from tetratype tetrads were then analyzed (eight tetrads analyzed; results from a representative tetrad are shown in Fig. 7). Wild-type, cik1, vik1, and cik1 vik1 segregants all display similar growth on rich medium at 23°C (Fig. 7 A). However, cik1 mutants are temperature sensitive for growth at 37°C (Page and Snyder, 1992), whereas vik1 mutants grow like wild-type strains (Fig. 7 B). Suprisingly, cik1 vik1 double mutants grow substantially better at 37°C than cik1 mutants (Fig. 7 B). This result indicates that disruption of VIK1 partially suppresses the temperature-sensitive growth defect of cik1 mutants. The temperature-sensitive growth defect can be restored to a cik1 vik1 double mutant by introduction of a CEN plasmid encoding VIK1 (data not shown). Deletion of VIK1 also partially suppresses the mitotic delay of cik1 mutants, as scored by the percentage of large budded cells with a single preanaphase nucleus in logarithmic phase cultures growing at 30°C (Fig. 7 D). 54% of cells from cik1 cultures exhibit the mitotic delay phenotype, whereas only 36% of cells from cik1 vik1 cultures have this phenotype. Wild-type and vik1 cultures each have 22% large budded cells. These strains were also analyzed for growth differences in the presence of benomyl (Fig. 7 C). Unlike vik1 mutants, which display increased resistance to benomyl, cik1 and cik1 vik1 mutants are slightly more sensitive than wild-type strains. Therefore, the phenotypic differences of cik1 and vik1 mutants, combined with their different effects on Kar3p localization, suggest that Cik1p and Vik1p are functionally distinct.

The microtubules of the different mutant strains were examined in fixed cells from asynchronous or hydroxyurea-arrested cultures by immunofluorescence with anti-tubulin antibodies. Under these conditions, the microtubules of vik1 mutants are indistinguishable from those of wild-type strains. As described previously, cik1 mutants have very short spindles compared with wild-type strains and have longer, more abundant cytoplasmic microtubules (Page and Snyder, 1992). This phenotype is identical to that reported for kar3 mutants (Meluh and Rose, 1990; Saunders et al., 1997). cik1 vik1 double mutants display microtubules with no significant difference in length or number to those of cik1 mutants (data not shown). Therefore, if disruption of VIK1 results in defects in microtubule structure, these defects are not detected under these conditions. Finally, consistent with the hypothesis that Cik1p and Vik1p function in complexes with Kar3p, the growth rate and microtubule phenotypes of kar3, kar3 cik1, kar3 vik1, and kar3 cik1 vik1 mutants are all very similar (data not shown).

Loss of Vik1p Function Suppresses the cin8-3 kip1 Mutant

One vegetative function of Kar3p may be to oppose the action of two other S. cerevisiae KRPs, the redundant Cin8p and Kip1p motors (Hoyt et al., 1992, 1993; Saunders and Hoyt, 1992; Saunders et al., 1997b). This Kar3p function has been proposed primarily due to genetic interactions between mutations in the genes encoding these KRPs. Disruption of Cin8p and Kip1p function, using the conditional cin8-3 kip1 mutant, results in a temperature-sensitive growth defect at 35°C that is partially suppressed by disruption of Kar3p function (Saunders and Hoyt, 1992; Hoyt et al., 1993; see below). However, disruption of CIK1, encoding a Kar3p KAP, does not suppress cin8-3 kip1 (Fig. 8; Page et al., 1994), suggesting that the Kar3p activity that opposes Cin8p and Kip1p is independent of Cik1p.

View larger version (59K): [in this window] [in a new window]   Fig. 8.   The temperature-sensitive growth defect of the cin8-3 kip1 mutant is suppressed by disruption of KAR3 or VIK1, but not CIK1. Overnight cultures were grown to stationary phase, diluted down to 5 × 106 cells/ml, and 5-µl spots were plated to rich medium then incubated for three days at 23 or 35°C. Pictured are wild-type strain Y1731 and cin8-3 kip1 mutant strains Y818, vik1 Y1748, cik1 Y1758, and kar3 Y1759.

Since Vik1p was identified as a second KAP for Kar3p during vegetative growth, we tested whether disruption of Vik1p function could suppress the temperature-sensitive growth defect of the cin8-3 kip1 mutant. VIK1, CIK1, and KAR3 were deleted individually from the cin8-3 kip1 strain, and the resulting mutants were examined for growth defects at 23 and 35°C (Fig. 8). As demonstrated previously, the cin8-3 kip1 kar3 mutant grows significantly better than the original cin8-3 kip1 mutant at the restrictive temperature of 35°C (Saunders and Hoyt, 1992; Hoyt et al., 1993). Likewise, disruption of VIK1 also partially suppresses the cin8-3 kip1 temperature-sensitive growth defect, as the cin8-3 kip1 vik1 mutant grows at 35°C. Introduction of a CEN plasmid encoding VIK1 into the cin8-3 kip1 vik1 mutant restores temperature sensitivity to levels identical to the cin8-3 kip1 mutant (data not shown). In contrast, growth of the cin8-3 kip1 cik1 mutant is significantly diminished at all temperatures compared with the cin8-3 kip1 mutant, suggesting a possible added defect in the triple mutant. Therefore, like Kar3p, Vik1p functions antagonistically to Cin8p and Kip1p. This result further indicates that Cik1p and Vik1p are involved in distinct Kar3p functions.

	Discussion

Top Abstract Introduction Materials & Methods Results Discussion References

Cik1p is a previously described kinesin-associated protein that interacts with the yeast KRP Kar3p (Page and Snyder, 1992; Page et al., 1994). We have characterized a Cik1p-homologous protein called Vik1p that is present in vegetatively growing cells but, unlike Cik1p, is not detected in mating-pheromone treated cells. Coimmunoprecipitation experiments demonstrate that Vik1p also physically associates with Kar3p and that the Kar3p-Vik1p complex is separate from that of Kar3p and Cik1p. Therefore, Kar3p interacts with two different KAPs to form distinct complexes within the same cell.

Kar3p and Vik1p Are Interdependent for Proper Localization to the SPBs of Vegetatively Growing Cells

Vik1p requires Kar3p function for its SPB localization. This suggests that Kar3p-Vik1p complex formation and, presumably, the minus-end directed microtubule-motor activity of Kar3p are required to deliver Vik1p to the SPB. In the absence of Kar3p, Vik1p mislocalizes to cytoplasmic patches and is excluded from the nucleus. In contrast, Cik1p mislocalizes throughout the nucleus in the absence of Kar3p during vegetative growth (Page et al., 1994). Two possible models for Vik1p localization can be invoked. First, Vik1p may require association with Kar3p for its nuclear import. In support of this idea, Vik1p does not have sequences predicting a nuclear localization signal. Moreover, Kar3p can target to the nucleus independent of both Cik1p and Vik1p (Page et al., 1994; this study). This nuclear import of Vik1p by association with Kar3p would be analogous to the nuclear import of the yeast -tubulin complex. The import of this complex, and its subsequent binding to the nuclear face of the SPB, requires the nuclear localization signal of one of its components, Spc98p (Pereira et al., 1998). Alternatively, the Kar3p-Vik1p complex may be associated primarily with the cytoplasmic face of the SPB. Therefore, absence of Kar3p would result in release of Vik1p to the cytoplasm specifically, as observed. At this point, we cannot distinguish between these two possibilities, but future studies will address whether the Kar3p-Vik1p complex is on the nuclear, cytoplasmic, or both faces of the SPB.

We demonstrate that, whereas Cik1p is required to localize Kar3p to the SPBs and cytoplasmic microtubules of mating pheromone-treated cells (Page et al., 1994), Vik1p is required for proper concentration of Kar3p at the SPBs of vegetatively growing cells. In the absence of Vik1p, Kar3p mislocalizes to nuclear patches, and can be seen along spindle microtubules in many cells. Therefore, Vik1p is required for proper targeting and/or maintenance of Kar3p at the SPB. Vik1p might mediate interactions between Kar3p and other proteins that tether the complex to the SPB. Alternatively, Vik1p could prevent release of Kar3p from the minus-ends of microtubules, where its motor activity would cause it to accumulate.

In the absence of both Cik1p and Vik1p, Kar3p again mislocalizes but is more diffuse throughout the nucleus. This suggests that Cik1p may be partially redundant with Vik1p for targeting Kar3p to the SPB. However, Cik1p's primary vegetative function may be to direct localization of Kar3p to other sites within the nucleus, such as nuclear patches and the spindle. This is supported by the observation t