Abstract
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 specifically bind biotinated Cin8p from crude extracts. Microtubules bound to the Cin8p-coated surfaces and moved at 3.4 +/- 0.5 micrometer/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 alpha2 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.
Highlights
Eukaryotic chromosome segregation is mediated by the mitotic spindle, a microtubule-based motile structure that undergoes a distinct program of morphological changes
A Biotinated Form of Cin8p Provides Activity in Vivo—For this study, we developed an in vitro assay to examine the microtubule binding and motile properties of the Cin8p kinesin-related motor from S. cerevisiae
Fusion proteins that contained the homologous region of E. coli pyruvate carboxylase become endogenously biotinated both in E. coli and S. cerevisiae [19]
Summary
Eukaryotic chromosome segregation is mediated by the mitotic spindle, a microtubule-based motile structure that undergoes a distinct program of morphological changes. Members of the BimC family, first discovered in Aspergillus nidulans [1], have been found in numerous eukaryotic species [2,3,4,5,6,7] 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). 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. 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. Tel.: 410-516-7299; Fax: 410-516-5213; E-mail: hoyt@jhu. edu
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