- Kinesin-5 motor proteins are evolutionarily conserved and perform essential roles in mitotic spindle assembly and spindle elongation during anaphase. Previous studies demonstrated a specialized homotetrameric structure with two pairs of catalytic domains, one at each end of a dumbbell-shaped molecule. This suggests that they perform their spindle roles by cross-linking and sliding antiparallel spindle microtubules. However, the exact kinesin-5 sequence elements that are important for formation of the tetrameric complexes have not yet been identified. In addition, it has not been demonstrated that the homotetrameric form of these proteins is essential for their biological functions. Thus, we investigated a series of Saccharomyces cerevisiae Cin8p truncations and internal deletions, in order to identify structural elements in the Cin8p sequence that are required for Cin8p functionality, spindle localization, and multimerization. We found that all variants of Cin8p that are functional in vivo form tetrameric complexes. The first coiled-coil domain in the stalk of Cin8p, a feature that is shared by all kinesin-5 homologues, is required for its dimerization, and sequences in the last part of the stalk, specifically those likely involved in coiled-coil formation, are required for Cin8p tetramerization. We also found that dimeric forms of Cin8p that are nonfunctional in vivo can nonetheless bind to microtubules. These findings suggest that binding of microtubules is not sufficient for the functionality of Cin8p and that microtubule cross-linking by the tetrameric complex is essential for Cin8p mitotic functions.