Homotetrameric Form of Cin8p, a Saccharomyces cerevisiae Kinesin-5 Motor, Is Essential for Its in Vivo Function

Abstract

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.Mitotic chromosome segregation is the mechanism by which duplicated genomic information is transmitted to daughter cells during cell division. This essential process is mediated by the mitotic spindle, a highly dynamic, microtubule-based structure that undergoes a distinct set of morphological changes. Many of these changes are achieved by the action of molecular motors from the kinesin-5 (BimC) family, which use ATP hydrolysis to unidirectionally move along microtubules. Members of the kinesin-5 family are conserved in the amino acid sequence of the motor (forceproducing) domain and apparently perform similar roles in many different cell types (1-7). Kinesin-5 motors are required for bipolar spindle assembly, and elimination of their function blocks this essential early mitotic step in fungal, insect, and mammalian cells (8 -11). The yeast Saccharomyces cerevisiae expresses two kinesin-5 motors that overlap in function, Cin8p and Kip1p. Although neither is individually essential, one of the pair is required for viability (2, 3, 12). Loss of KIP1 function causes less severe phenotypes than loss of CIN8, suggesting that Cin8p is more important for successful yeast spindle function (2). Aside from their essential role 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 anaph...

Deletion of the Tail Domain of the Kinesin-5 Cin8 Affects Its Directionality

“…3C, supplemental Movie M2). Individual molecules appeared (42). The SV40 NLS sequence (PKKKRKV) (drawn in light blue) was added to Cin8, prior to the sequence of TEV-GFP-His 6 .…”

Prime movers: the mechanochemistry of mitotic kinesins

“…show that tetramerisation of kinesin-5 (Cin8p) -and presumably the anti-parallel sliding activity -is required for the in vivo function of the motor 58 .…”

To step or not to step? How biochemistry and mechanics influence processivity in Kinesin and Eg5

“…To determine the mechanical and kinetic requirements for Eg5-promoted spindle assembly [47][48][49], a stable Eg5 dimer was developed [11••]. Eg5-513 promoted robust plus-end-directed microtubule gliding at a rate similar to that of native tetramers [11••,50].…”