Differential cellular localization among mitotic cyclins fromSaccharomyces cerevisiae: a new role for the axial budding protein Bud3 in targeting Clb2 to the mother-bud neck

Abstract

The mitotic cyclin Clb2 plays a major role in promoting M-phase in budding yeast, despite its functional redundancy with three closely related cyclins Clb1, Clb3 and Clb4. Here, we further investigate the mechanisms controlling the cellular distribution of Clb2 in living cells. In agreement with observations recently made by Hood et al. [Hood, J. K., Hwang, W. W. and Silver, P. A. ( 2001) J. Cell Sci. 114, 589-597], we find that GFP-tagged Clb2 expressed from its natural promoter localizes to various cellular compartments, including the nucleus, the mitotic spindle, the spindle pole bodies as well as the mother-bud neck. The neck localization is specific to Clb2 as Clb1, Clb3 and Clb4 are never observed there, even when over-expressed. Mutational analysis identifies a central region of Clb2, comprising residues 213-255 and a phylogenetically conserved hydrophobic patch, as an essential cis-acting determinant. Clb2 co-localizes with the bud site selection protein Bud3. Consistent with a role of Bud3 in targeting Clb2 to the bud neck, we report a two-hybrid interaction between these proteins. Furthermore, Clb2 is shown to be specifically delocalized in Δ bud3 cells and in a bud3 mutant deleted for its C-terminal Clb2-interacting domain ( bud3 Δ 1221 ), but not in a Δ bud10 mutant. Correlating with this phenotype, bud3 Δ 1221 cells exhibit a pronounced (15-30 minutes) delay in cytokinesis and/or cell separation, suggesting an unanticipated function of Clb2 in these late mitotic events. Taken together, our data uncover a new role for Bud3 in cytokinesis that correlates with its capacity to target Clb2 at the neck, independently of its well established cell-type-specific function in bud site selection.