The ATPase Pathway That Drives the Kinesin-14 Kar3Vik1 Powerstroke

Kar3, a Saccharomyces cerevisiae microtubule minus-end-directed kinesin-14, dimerizes with either Vik1 or Cik1. The C-terminal globular domain of Vik1 exhibits the structure of a kinesin motor domain and binds microtubules independently of Kar3 but lacks a nucleotide binding site. The only known function of Kar3Vik1 is to cross-link parallel microtubules at the spindle poles during mitosis. In contrast, Kar3Cik1 depolymerizes microtubules during mating but cross-links antiparallel microtubules in the spindle overlap zone during mitosis. A recent study showed that Kar3Vik1 binds across adjacent microtubule protofilaments and uses a minus-end-directed powerstroke to drive ATP-dependent motility. The presteady-state experiments presented here extend this study and establish an ATPase model for the powerstroke mechanism. The results incorporated into the model indicate that Kar3Vik1 collides with the microtubule at 2.4 μm⁻¹ s⁻¹ through Vik1, promoting microtubule binding by Kar3 followed by ADP release at 14 s⁻¹. The tight binding of Kar3 to the microtubule destabilizes the Vik1 interaction with the microtubule, positioning Kar3Vik1 for the start of the powerstroke. Rapid ATP binding to Kar3 is associated with rotation of the coiled-coil stalk, and the postpowerstroke ATP hydrolysis at 26 s⁻¹ is independent of Vik1, providing further evidence that Vik1 rotates with the coiled coil during the powerstroke. Detachment of Kar3Vik1 from the microtubule at 6 s⁻¹ completes the cycle and allows the motor to return to its initial conformation. The results also reveal key differences in the ATPase cycles of Kar3Vik1 and Kar3Cik1, supporting the fact that these two motors have distinctive biological functions.     

Cik1 targets the minus-end kinesin depolymerase kar3 to microtubule plus ends

Kar3, a Saccharomyces cerevisiae Kinesin-14, is essential for karyogamy and meiosis I but also has specific functions during vegetative growth. For its various roles, Kar3 forms a heterodimer with either Cik1 or Vik1, both of which are noncatalytic polypeptides. Here, we present the first biochemical characterization of Kar3Cik1, the kinesin motor that is essential for karyogamy. Kar3Cik1 depolymerizes microtubules from the plus end and promotes robust minus-end-directed microtubule gliding. Immunolocalization studies show that Kar3Cik1 binds preferentially to one end of the microtubule, whereas the Kar3 motor domain, in the absence of Cik1, exhibits significantly higher microtubule lattice binding. Kar3Cik1-promoted microtubule depolymerization requires ATP turnover, and the kinetics fit a single exponential function. The disassembly mechanism is not microtubule catastrophe like that induced by the MCAK Kinesin-13s. Soluble tubulin does not activate the ATPase activity of Kar3Cik1, and there is no evidence of Kar3Cik1(.)tubulin complex formation as observed for MCAK. These results reveal a novel mechanism to regulate microtubule depolymerization. We propose that Cik1 targets Kar3 to the microtubule plus end. Kar3Cik1 then uses its minus-end-directed force to depolymerize microtubules from the plus end, with each tubulin-subunit release event tightly coupled to one ATP turnover.     

Mechanistic analysis of the Saccharomyces cerevisiae kinesin Kar3

Kar3 is a minus-end-directed microtubule motor that is implicated in meiotic and mitotic spindle function in Saccharomyces cerevisiae. To date, the only truncated protein of Kar3 that has been reported to promote unidirectional movement in vitro is GSTKar3. This motor contains an NH2-terminal glutathione S-transferase (GST) tag followed by the Kar3 sequence that is predicted to form an extended alpha-helical coiled-coil. The alpha-helical domain leads into the neck linker and COOH-terminal motor domain. Kar3 does not homodimerize with itself but forms a heterodimer with either Cik1 or Vik1, both of which are non-motor polypeptides. We evaluated the microtubule-GSTKar3 complex in comparison to the microtubule-Kar3 motor domain complex to determine the distinctive mechanistic features required for GSTKar3 motility. Our results indicate that ATP binding was significantly faster for GSTKar3 than that observed previously for the Kar3 motor domain. In addition, microtubule-activated ADP release resulted in an intermediate that bound ADP weakly in contrast to the Kar3 motor domain, suggesting that after ADP release, the microtubule-GSTKar3 motor binds ATP in preference to ADP. The kinetics also showed that GST-Kar3 readily detached from the microtubule rather than remaining bound for multiple ATP turnovers. These results indicate that the extended alpha-helical domain NH2-terminal to the catalytic core provides the structural transitions in response to the ATPase cycle that are critical for motility and that dimerization is not specifically required. This study provides the foundation to define the mechanistic contributions of Cik1 and Vik1 for Kar3 force generation and function in vivo.     

Neck Rotation and Neck Mimic Docking in the Noncatalytic Kar3-associated Protein Vik1

It is widely accepted that movement of kinesin motor proteins is accomplished by coupling ATP binding, hydrolysis, and product release to conformational changes in the microtubule-binding and force-generating elements of their motor domain. Therefore, understanding how the Saccharomyces cerevisiae proteins Cik1 and Vik1 are able to function as direct participants in movement of Kar3Cik1 and Kar3Vik1 kinesin complexes presents an interesting challenge given that their motor homology domain (MHD) cannot bind ATP. Our crystal structures of the Vik1 ortholog from Candida glabrata may provide insight into this mechanism by showing that its neck and neck mimic-like element can adopt several different conformations reminiscent of those observed in catalytic kinesins. We found that when the neck is α-helical and interacting with the MHD core, the C terminus of CgVik1 docks onto the central β-sheet similarly to the ATP-bound form of Ncd. Alternatively, when neck-core interactions are broken, the C terminus is disordered. Mutations designed to impair neck rotation, or some of the neck-MHD interactions, decreased microtubule gliding velocity and steady state ATPase rate of CgKar3Vik1 complexes significantly. These results strongly suggest that neck rotation and neck mimic docking in Vik1 and Cik1 may be a structural mechanism for communication with Kar3.     

The ATPase cross-bridge cycle of the Kar3 motor domain. Implications for single head motility

Kar3 is a minus-end directed microtubule motor involved in meiosis and mitosis in Saccharomyces cerevisae. Unlike Drosophila Ncd, the other well characterized minus-end directed motor that is a homodimer, Kar3 is a heterodimer with a single motor domain and either the associated polypeptides Cik1 or Vik1. Our mechanistic studies with Ncd showed that both motor domains were required for ATP-dependent motor domain detachment from the microtubule. We have initiated a series of experiments to compare the mechanistic requirements for Kar3 motility in direct comparison to Ncd. The results presented here show that the single motor domain of Kar3 (Met(383)-Lys(729)) exhibits characteristics similar to monomeric Ncd. The microtubule-activated steady-state ATPase cycle of Kar3 (k(cat) = 0.5 s(-1)) is limited by ADP release (0.4 s(-1)). Like monomeric Ncd, Kar3 does not readily detach from the microtubule with the addition of MgATP. These results show that the single motor domain of Kar3 is not sufficient for ATP-dependent microtubule dissociation, suggesting that structural elements outside of the catalytic core are required for the cyclic interactions with the microtubule for force generation.     

Kar3 interaction with Cik1 alters motor structure and function

Kar3, a kinesin-14 motor of Saccharomyces cerevisiae required for mitosis and karyogamy, reportedly interacts with Cik1, a nonmotor protein, via its central, predicted coiled coil. Despite this, neither Kar3 nor Cik1 homodimers have been observed in vivo. Here we show that Kar3 is a dimer in vitro by analytical ultracentrifugation. The motor domains appear as paired particles by rotary-shadow electron microscopy (EM) and circular dichroism (CD) spectroscopy of the nonmotor region shows characteristics of helical structure, typical of coiled coils. Remarkably, the Kar3/Cik1 nonmotor region shows greater helicity by CD analysis and rotary-shadow EM reveals a stalk joined to one large or two smaller particles. The highly helical Kar3/Cik1 nonmotor region and visible stalk indicate that dimerization with Cik1 causes structural changes in Kar3. The Cik1 and Kar3 stalk regions preferentially associate with one another rather than forming homodimers. Kar3/Cik1 moves on microtubules at 2-2.4 microm min(-1), 2-5-fold faster than Kar3, and destabilizes microtubules at the lagging ends. Thus, structural changes in Kar3 upon dimerization with Cik1 alter the motor velocity and likely regulate Kar3 activity in vivo.     

Large conformational changes in a kinesin motor catalyzed by interaction with microtubules

Kinesin motor proteins release nucleotide upon interaction with microtubules (MTs), then bind and hydrolyze ATP to move along the MT. Although crystal structures of kinesin motors bound to nucleotides have been solved, nucleotide-free structures have not. Here, using cryomicroscopy and three-dimensional (3D) reconstruction, we report the structure of MTs decorated with a Kinesin-14 motor, Kar3, in the nucleotide-free state, as well as with ADP and AMPPNP, with resolution sufficient to show alpha helices. We find large structural changes in the empty motor, including melting of the switch II helix alpha4, closure of the nucleotide binding pocket, and changes in the central beta sheet reminiscent of those reported for nucleotide-free myosin crystal structures. We propose that the switch II region of the motor controls docking of the Kar3 neck by conformational changes in the central beta sheet, similar to myosin, rather than by rotation of the motor domain, as proposed for the Kif1A kinesin motor.     

The loop 5 element structurally and kinetically coordinates dimers of the human kinesin-5, Eg5

Eg5 is a homotetrameric kinesin-5 motor protein that generates outward force on the overlapping, antiparallel microtubules (MTs) of the mitotic spindle. Upon binding an MT, an Eg5 dimer releases one ADP molecule, undergoes a slow (∼0.5 s⁻¹) isomerization, and finally releases a second ADP, adopting a tightly MT-bound, nucleotide-free (APO) conformation. This conformation precedes ATP binding and stepping. Here, we use mutagenesis, steady-state and pre-steady-state kinetics, motility assays, and electron paramagnetic resonance spectroscopy to examine Eg5 monomers and dimers as they bind MTs and initiate stepping. We demonstrate that a critical element of Eg5, loop 5 (L5), accelerates ADP release during the initial MT-binding event. Furthermore, our electron paramagnetic resonance data show that L5 mediates the slow isomerization by preventing Eg5 dimer heads from binding the MT until they release ADP. Finally, we find that Eg5 having a seven-residue deletion within L5 can still hydrolyze ATP and move along MTs, suggesting that L5 is not required to accelerate subsequent steps of the motor along the MT. Taken together, these properties of L5 explain the kinetic effects of L5-directed inhibition on Eg5 activity and may direct further interventions targeting Eg5 activity.     

Slk19 enhances cross-linking of microtubules by Ase1 and Stu1

The Saccharomyces cerevisiae protein Slk19 has been shown to localize to kinetochores throughout mitosis and to the spindle midzone in anaphase. However, Slk19 clearly also has an important role for spindle formation and stabilization in prometaphase and metaphase, albeit this role is unresolved. Here we show that Slk19’s localization to metaphase spindles in vivo and to microtubules (MTs) in vitro depends on the MT cross-linking protein Ase1 and the MT cross-linking and stabilizing protein Stu1. By analyzing a slk19 mutant that specifically fails to localize to spindles and MTs, we surprisingly found that the presence of Slk19 amplified the amount of Ase1 strongly and that of Stu1 moderately at the metaphase spindle in vivo and at MTs in vitro. Furthermore, Slk19 markedly enhanced the cross-linking of MTs in vitro when added together with Ase1 or Stu1. We therefore suggest that Slk19 recruits additional Ase1 and Stu1 to the interpolar MTs (ipMTs) of metaphase spindles and thus increases their cross-linking and stabilization. This is in agreement with our observation that cells with defective Slk19 localization exhibit shorter metaphase spindles, an increased number of unaligned nuclear MTs, and most likely reduced ipMT overlaps.     

The microtubule-based motor Kar3 and plus end-binding protein Bim1 provide structural support for the anaphase spindle

In budding yeast, the mitotic spindle is comprised of 32 kinetochore microtubules (kMTs) and ~8 interpolar MTs (ipMTs). Upon anaphase onset, kMTs shorten to the pole, whereas ipMTs increase in length. Overlapping MTs are responsible for the maintenance of spindle integrity during anaphase. To dissect the requirements for anaphase spindle stability, we introduced a conditionally functional dicentric chromosome into yeast. When centromeres from the same sister chromatid attach to opposite poles, anaphase spindle elongation is delayed and a DNA breakage-fusion-bridge cycle ensues that is dependent on DNA repair proteins. We find that cell survival after dicentric chromosome activation requires the MT-binding proteins Kar3p, Bim1p, and Ase1p. In their absence, anaphase spindles are prone to collapse and buckle in the presence of a dicentric chromosome. Our analysis reveals the importance of Bim1p in maintaining a stable ipMT overlap zone by promoting polymerization of ipMTs during anaphase, whereas Kar3p contributes to spindle stability by cross-linking spindle MTs.     

Mechanistic analysis of the mitotic kinesin Eg5

Eg5 is a slow, plus-end-directed microtubule-based motor of the BimC kinesin family that is essential for bipolar spindle formation during eukaryotic cell division. We have analyzed two human Eg5/KSP motors, Eg5-367 and Eg5-437, and both are monomeric based on results from sedimentation velocity and sedimentation equilibrium centrifugation as well as analytical gel filtration. The steady-state parameters were: for Eg5-367: k(cat) = 5.5 s(-1), K(1/2,Mt) = 0.7 microm, and K(m,ATP) = 25 microm; and for Eg5-437: k(cat) = 2.9 s(-1), K(1/2,Mt) = 4.5 microm, and K(m,ATP) = 19 microm. 2'(3')-O-(N-Methylanthraniloyl)-ATP (mantATP) binding was rapid at 2-3 microm(-1)s(-1), followed immediately by ATP hydrolysis at 15 s(-1). ATP-dependent Mt.Eg5 dissociation was relatively slow and rate-limiting at 8 s(-1) with mantADP release at 40 s(-1). Surprisingly, Eg5-367 binds microtubules more effectively (11 microm(-1)s(-1)) than Eg5-437 (0.7 microm(-1)s(-1)), consistent with the steady-state K(1/2,Mt) and the mantADP release K(1/2,Mt). These results indicate that the ATPase pathway for monomeric Eg5 is more similar to conventional kinesin than the spindle motors Ncd and Kar3, where ADP product release is rate-limiting for steady-state turnover.     

Modulation of kinesin half-site ADP release and kinetic processivity by a spacer between the head groups

A series of modifications of the junction of the neck linker and neck coil of dimeric Drosophila kinesin were constructed to determine the influence of head orientation and spacing on the ATPase kinetics. Ala(345) is the first residue in the coiled-coil of the neck, and its replacement with glycine or proline produces no significant change in the k(cat) or K(0.5(MT)) values for activation of their ATPase by microtubules (MTs) or in their k(bi(ratio)) value for the average number of ATP molecules hydrolyzed during a processive encounter with a MT. Addition or deletion of a single amino acid at the junction produces only modest changes with less than a 2-fold reduction in kinetic processivity. Insertion of a spacer of 6 or 12 additional amino acids at the neck linker junction increases the K(0.5(MT)) value by 3-4-fold with a corresponding decrease in kinetic processivity. The sliding velocities of all the mutant constructs under multimotor conditions are within 30% of the wild-type value. All the constructs with single residue changes exhibit half-site ADP release on binding to MTs. The constructs with long insertion, however, rapidly release both ADP molecules per dimer on binding to a MT, indicating that the steric constraints that prevent release of ADP from the tethered head of wild-type kinesin have been relieved by the long insertions. The constructs with long inserts have decreased kinetic processivity and dissociate from the MT during ATP hydrolysis 3-fold faster than wild-type.     

Kar3Vik1, a member of the kinesin-14 superfamily, shows a novel kinesin microtubule binding pattern

Kinesin-14 motors generate microtubule minus-end-directed force used in mitosis and meiosis. These motors are dimeric and operate with a nonprocessive powerstroke mechanism, but the role of the second head in motility has been unclear. In Saccharomyces cerevisiae, the Kinesin-14 Kar3 forms a heterodimer with either Vik1 or Cik1. Vik1 contains a motor homology domain that retains microtubule binding properties but lacks a nucleotide binding site. In this case, both heads are implicated in motility. Here, we show through structural determination of a C-terminal heterodimeric Kar3Vik1, electron microscopy, equilibrium binding, and motility that at the start of the cycle, Kar3Vik1 binds to or occludes two αβ-tubulin subunits on adjacent protofilaments. The cycle begins as Vik1 collides with the microtubule followed by Kar3 microtubule association and ADP release, thereby destabilizing the Vik1-microtubule interaction and positioning the motor for the start of the powerstroke. The results indicate that head-head communication is mediated through the adjoining coiled coil.     

The Kar3-interacting protein Cik1p plays a critical role in passage through meiosis I in Saccharomyces cerevisiae.

Meiosis I in Saccharomyces cerevisiae is dependent upon the motor protein Kar3. Absence of Kar3p in meiosis results in an arrest in prophase I. Cik1p and Vik1p are kinesin-associated proteins known to modulate the function of Kar3p in the microtubule-dependent processes of karyogamy and mitosis. Experiments were performed to determine whether Cik1p and Vik1p are also important for the function of Kar3p during meiosis. The meiotic phenotypes of a cik1 mutant were found to be similar to those of kar3 mutants. Cells without Cik1p exhibit a meiotic defect in homologous recombination and synaptonemal complex formation. Most cik1 mutant cells, like kar3 mutants, arrest in meiotic prophase; however, in cik1 mutants this arrest is less severe. These data are consistent with the model that Cik1p is necessary for some, but not all, of the roles of Kar3p in meiosis I. vik1 mutants sporulate at wild-type levels, but have reduced spore viability. This loss in viability is partially attributable to vegetative chromosome loss in vik1 diploids. Cellular localization experiments reveal that Kar3p, Cik1p, and Vik1p are present throughout meiosis and are consistent with Cik1p and Vik1p having different meiotic roles.     

Differential regulation of the Kar3p kinesin-related protein by two associated proteins, Cik1p and Vik1p

The mechanisms by which kinesin-related proteins interact with other proteins to carry out specific cellular processes is poorly understood. The kinesin-related protein, Kar3p, has been implicated in many microtubule functions in yeast. Some of these functions require interaction with the Cik1 protein (Page, B.D., L.L. Satterwhite, M.D. Rose, and M. Snyder. 1994. J. Cell Biol. 124:507-519). We have identified a Saccharomyces cerevisiae gene, named VIK1, encoding a protein with sequence and structural similarity to Cik1p. The Vik1 protein is detected in vegetatively growing cells but not in mating pheromone-treated cells. Vik1p physically associates with Kar3p in a complex separate from that of the Kar3p-Cik1p complex. Vik1p localizes to the spindle-pole body region in a Kar3p-dependent manner. Reciprocally, concentration of Kar3p at the spindle poles during vegetative growth requires the presence of Vik1p, but not Cik1p. Phenotypic analysis suggests that Cik1p and Vik1p are involved in different Kar3p functions. Disruption of VIK1 causes increased resistance to the microtubule depolymerizing drug benomyl and partially suppresses growth defects of cik1Delta mutants. The vik1Delta and kar3Delta mutations, but not cik1Delta, partially suppresses the temperature-sensitive growth defect of strains lacking the function of two other yeast kinesin-related proteins, Cin8p and Kip1p. Our results indicate that Kar3p forms functionally distinct complexes with Cik1p and Vik1p to participate in different microtubule-mediated events within the same cell.     

Kinetic studies of dimeric Ncd: evidence that Ncd is not processive

Ncd is a kinesin-related motor protein which drives movement to the minus-end of microtubules. The kinetics of Ncd were investigated using the dimeric construct MC1 (Leu(209)-Lys(700)) expressed in Escherichia coli strain BL21(DE) as a nonfusion protein [Chandra, R., Salmon, E. D., Erickson, H. P., Lockhart, A., and Endow, S. A. (1993) J. Biol. Chem. 268, 9005-9013]. Acid chemical quench flow methods were used to measure directly the rate of ATP hydrolysis, and stopped-flow kinetic methods were used to determine the kinetics of mantATP binding, mantADP release, dissociation of MC1 from the microtubule, and binding of MC1 to the microtubule. The results define a minimal kinetic mechanism, M.N + ATP M.N.ATP M.N.ADP.P N. ADP.P N.ADP + P M.N.ADP M.N + ADP, where N, M, and P represent Ncd, microtubules, and inorganic phosphate respectively, with k(+1) = 2.3 microM(-1) s(-1), k(+2) =23 s(-1), k(+3) =13 s(-1), k(+5)= 0.7 microM(-)(1) s(-)(1), and k(+6) = 3.7 s(-)(1). Phosphate release (k(+4)) was not measured directly although it is assumed to be fast relative to ADP release because Ncd is purified with ADP tightly bound at the active site. ATP hydrolysis occurs at 23 s(-)(1) prior to Ncd dissociation at 13 s(-)(1). The pathway for ATP-promoted detachment (steps 1-3) of Ncd from the microtubule is comparable to kinesin's. However, there are two major differences between the mechanisms of Ncd and kinesin. In contrast to kinesin, mantADP release for Ncd at 3.7 s(-)(1) is the slowest step in the pathway and is believed to limit steady-state turnover. Additionally, the burst amplitude observed in the pre-steady-state acid quench experiments is stoichiometric, indicating that Ncd, in contrast to kinesin, is not processive for ATP hydrolysis.     

Quantitative analysis of an anaphase B switch: predicted role for a microtubule catastrophe gradient

Anaphase B in Drosophila embryos is initiated by the inhibition of microtubule (MT) depolymerization at spindle poles, which allows outwardly sliding interpolar (ip) MTs to drive pole-pole separation. Using fluorescence recovery after photobleaching, we observed that MTs throughout the preanaphase B spindle are very dynamic and display complete recovery of fluorescence, but during anaphase B, MTs proximal to the poles stabilize and therefore display lower recovery than those elsewhere. Fluorescence microscopy of the MT tip tracker EB1 revealed that growing MT plus ends localize throughout the preanaphase B spindle but concentrate in the overlap region of interpolar MTs (ipMTs) at anaphase B onset. None of these changes occurred in the presence of nondegradable cyclin B. Modeling suggests that they depend on the establishment of a spatial gradient of MT plus-end catastrophe frequencies, decreasing toward the equator. The resulting redistribution of ipMT plus ends to the overlap zone, together with the suppression of minus-end depolymerization at the poles, could constitute a mechanical switch that initiates spindle elongation.     

The Kar3p kinesin-related protein forms a novel heterodimeric structure with its associated protein Cik1p

Proteins that physically associate with members of the kinesin superfamily are critical for the functional diversity observed for these microtubule motor proteins. However, quaternary structures of complexes between kinesins and kinesin-associated proteins are poorly defined. We have analyzed the nature of the interaction between the Kar3 motor protein, a minus-end-directed kinesin from yeast, and its associated protein Cik1. Extraction experiments demonstrate that Kar3p and Cik1p are tightly associated. Mapping of the interaction domains of the two proteins by two-hybrid analyses indicates that Kar3p and Cik1p associate in a highly specific manner along the lengths of their respective coiled-coil domains. Sucrose gradient velocity centrifugation and gel filtration experiments were used to determine the size of the Kar3-Cik1 complex from both mating pheromone-treated cells and vegetatively growing cells. These experiments predict a size for this complex that is consistent with that of a heterodimer containing one Kar3p subunit and one Cik1p subunit. Finally, immunoprecipitation of epitope-tagged and untagged proteins confirms that only one subunit of Kar3p and Cik1p are present in the Kar3-Cik1 complex. These findings demonstrate that the Kar3-Cik1 complex has a novel heterodimeric structure not observed previously for kinesin complexes.     

Arp2/3 phosphorylation kickstarts cells

The kinesin-13 motor protein family members drive the removal of tubulin from microtubules (MTs) to promote MT turnover. A point mutation of the kinesin-13 family member mitotic centromere-associated kinesin/Kif2C (E491A) isolates the tubulin-removal conformation of the motor, and appears distinct from all previously described kinesin-13 conformations derived from nucleotide analogues. The E491A mutant removes tubulin dimers from stabilized MTs stoichiometrically in adenosine triphosphate (ATP) but is unable to efficiently release from detached tubulin dimers to recycle catalytically. Only in adenosine diphosphate (ADP) can the mutant catalytically remove tubulin dimers from stabilized MTs because the affinity of the mutant for detached tubulin dimers in ADP is low relative to lattice-bound tubulin. Thus, the motor can regenerate for further cycles of disassembly. Using the mutant, we show that release of tubulin by kinesin-13 motors occurs at the transition state for ATP hydrolysis, which illustrates a significant divergence in their coupling to ATP turnover relative to motile kinesins.     

Localization of the Kar3 kinesin heavy chain-related protein requires the Cik1 interacting protein

The Kar3 protein (Kar3p), a protein related to kinesin heavy chain, and the Cik1 protein (Cik1p) appear to participate in the same cellular processes in S. cerevisiae. Phenotypic analysis of mutants indicates that both CIK1 and KAR3 participate in spindle formation and karyogamy. In addition, the expression of both genes is induced by pheromone treatment. In vegetatively growing cells, both Cik1::beta-gal and Kar3::beta-gal fusions localize to the spindle pole body (SPB), and after pheromone treatment both fusion proteins localize to the spindle pole body and cytoplasmic microtubules. The dependence of Cik1p and Kar3p localization upon one another was investigated by indirect immunofluorescence of fusion proteins in pheromone-treated cells. The Cik1p::beta-gal fusion does not localize to the SPB or microtubules in a kar3 delta strain, and the Kar3p::beta-gal fusion protein does not localize to microtubule-associated structures in a cik1 delta strain. Thus, these proteins appear to be interdependent for localization to the SPB and microtubules. Analysis by both the two-hybrid system and co- immunoprecipitation experiments indicates that Cik1p and kar3p interact, suggesting that they are part of the same protein complex. These data indicate that interaction between a putative kinesin heavy chain-related protein and another protein can determine the localization of motor activity and thereby affect the functional specificity of the motor complex.