We have developed microtubule binding and motility assays for Cin8p, a kinesin-related mitotic spindle motor protein from Saccharomyces cerevisiae. The methods examine Cin8p rapidly purified from crude yeast cell extracts. We created a recombinant form of CIN8 that fused the biotin carrying polypeptide from yeast pyruvate carboxylase to the carboxyl terminus of Cin8p. This form was biotinated in yeast cells and provided Cin8p activity in vivo. Avidin-coated glass surfaces were used to

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... ly bind biotinated Cin8p from crude extracts. Microtubules bound to the Cin8p-coated surfaces and moved at 3.4 ؎ 0.5 m/min in the presence of ATP. Force production by Cin8p was directed toward the plus ends of microtubules. A mutation affecting the microtubule-binding site within the motor domain (cin8-F467A) decreased Cin8p's ability to bind microtubules to the glass surface by >10-fold, but reduced gliding velocity by only 35%. The cin8 -3 mutant form, affecting the ␣2 helix of the motor domain, caused a moderate defect in microtubule binding, but motility was severely affected. cin8-F467A cells, but not cin8 -3 cells, were greatly impaired in bipolar spindle forming ability. We conclude that microtubule binding by Cin8p is more important than motility for proper spindle formation. Eukaryotic chromosome segregation is mediated by the mitotic spindle, a microtubule-based motile structure that undergoes a distinct program of morphological changes. It is now clear that many spindle movements are accomplished by microtubule-based motor proteins. Perhaps the best characterized type of spindle motor is that of the BimC subfamily of kinesinrelated proteins. Members of the BimC family, first discovered in Aspergillus nidulans (1), have been found in numerous eukaryotic species (2-7). These proteins are conserved in amino acid sequence of the motor (force-producing) domain and apparently perform similar roles in many different cell types (8 -10). BimC motors are required for bipolar spindle assembly; elimination of their function blocked this essential early mitotic step in fungal, insect, and mammalian cells. The yeast Saccharomyces cerevisiae expresses two BimC-related motors that overlap in function, Cin8p and Kip1p. Although neither is individually essential, one of the pair is required for viability (3, 4, 11) . Loss of KIP1 causes less severe phenotypes than loss of CIN8, suggesting that Cin8p is more important for successful yeast spindle function (3) . Besides essential roles in spindle assembly, Cin8p and Kip1p are also required for the maintenance of spindle bipolarity following assembly and are responsible for producing most of the spindle-elongating force during anaphase (11, 12) . In addition to genetic experiments, several lines of evidence suggest that BimC motors act by cross-linking and sliding antiparallel microtubules found in the spindle midzone. BimC motors have been localized exclusively to the microtubules that lie between the spindle poles (3, 4, 13-15). In vitro, BimC motors from Drosophila melanogaster and Xenopus laevis have been found to move exclusively toward microtubule plus ends, albeit at a rate much slower than that achieved by kinesin (ϳ2 m/min versus 20 -40 m/min for kinesin) (16). In particular, the "bipolar" molecular structure determined for the Drosophila BimC motor Klp61F suggested a mechanism by which these motors may contribute to spindle structure and elongation. Klp61F is an elongated bipolar homotetramer, with two motor domains positioned at each end of a rodlike structure (17). Such a bipolar molecule would have the ability to cross-link and slide antiparallel microtubules (10). Despite these suggestive observations, the actual molecular role of BimC motors during spindle dynamics has yet to be established. In this study, we developed in vitro assays for the analysis of the S. cerevisiae Cin8p motor. Our purpose was 2-fold. First, the in vitro properties of Cin8p have not been described. Mere analogies to other BimC members are clearly indirect and unsatisfactory. Second, the ease of genetic and cell cycle manipulations in yeast demands a similarly direct method to examine and dissect Cin8p functions in vitro. Although exogenous expression of motors allows initial reconstituted studies, only endogenous expression in yeast allows direct study of Cin8p cell cycle regulation and different mitotic roles. With these goals in mind, we developed a rapid assay for the activity of Cin8p expressed in yeast cells. For a one-step purification and adsorption to avidin-coated surface, the Cin8 protein was fused to a peptide (biotin carrier peptide or BCP) that is endogenously biotinated in yeast. Subsequent analysis could distinguish microtubule binding from translocation activities, and photoactivation of caged-ATP further increased the sensitivity of our assays. Using these assays, the study of two Cin8p mutants revealed the relative importance of binding and motility for Cin8p in vivo functions. MATERIALS AND METHODS Yeast Strains and Media-The S. cerevisiae strains used in these experiments are derivatives of S288C and are listed in Table I . The cin8::HIS3 and kip1::HIS3 alleles were described previously (12). Rich (YPD) and minimal (SD) media were as described by Sherman et al. (18). To derepress galactose-inducible genes, cells were grown in 2% raffinose minimal media at 26°C for 24 h prior to induction by the addition of galactose to 2%. Cycloheximide (Sigma) was added to a final concentration of 5 g/ml. All media for expression of biotinated Cin8p