Mutational analysis of Scm3. (A) Amino acid sequence of Scm3 showing potential functional motifs and site-directed mutations. The nuclear export signal (NES) and heptad repeat (HR) domain are boxed with the heptad-spaced hydrophobic residues shown in boldface italic. Potential nuclear localization sequences are underlined (BR1, BR2), and acidic residues in the C-terminal acidic (D/E-rich) region are shown in boldface. Deletion endpoints of the scm3-26 and scm3-27 alleles are indicated. (B) Schematic diagrams of HA-tagged WT SCM3 and site-directed scm3 mutants. (C) Plasmid shuffle complementation tests of mutants shown in B. Growth on 5-fluoroorotic acid (FOA) medium indicates the respective mutant allele provides Scm3 function. (D) Immunoblot of protein extracts from the indicated transformants detected with anti-HA antibody.

Scm3 heptad repeat domain is evolutionarily conserved. (A) Alignment of 38 fungal protein sequences homologous to the heptad repeat domain of S. cerevisiae Scm3, shown in Logo format (42). The alignment requires insertion of one to three gaps at positions 20–22. The complete alignment in Clustal format is provided in SI Fig. 7. (B) Maximum likelihood phylogeny of the Scm3 homology domain of 29 representative ascomycetes derived by Bayesian inference (details in SI Materials and Methods). Numbers on the branches are the percentage of times that branch was present in the posterior distribution of trees. All members of the clade defined by the branch marked with the asterisk (∗) have point centromeres.

Scm3p contains a functional NES. (A) The NES consensus sequence (15) and other known NESs are shown in comparison with the Scm3 NES and the mutated sequence of scm3-3. At the bottom is shown an alignment, in Logo format (42), of the NES region found in Scm3 orthologs of 11 close relatives of S. cerevisiae. (B) Results of the NES assay. Positive controls are the NESs of Gle1 and Rev. Negative controls are empty vector as well as a fusion containing a nonfunctional Rev sequence (Rev NESmut). Scm3mut is the fusion containing the mutated NES region of scm3-3.

Scm3 copurifies with Cse4 and cross-links to CEN3. (A) Subcellular fractionation and immunoblot analysis of HA-tagged Scm3 and Cse4. (B) Immunoblot of proteins copurifying with Cse4 from extracts of cells coexpressing Protein A-tagged Cse4 (Cse4SpA) and HA-tagged Scm3 or Scm3 mutant proteins. Purification was performed with IgG-agarose beads and eluted proteins were analyzed by immunoblotting with anti-HA antibody. Cse4SpA protein was detected by its binding to the enzyme-conjugated secondary antibody. T, total cell extract; P, affinity eluate. (C) Formaldehyde cross-linked, solubilized chromatin was immunoprecipitated with anti-HA monoclonal antibody. Total (T) and coprecipitated (P) DNA was used for multiplex PCR with the primer sets shown above the gel image.

Scm3 depletion leads to G₂/M arrest and aberrant Cse4 localization. (A) Strain R421 (scm3^deg CSE4^GFP) was arrested with α-factor for 2 h after which half of the culture was transferred to a separate flask and CuSO₄ added to a final concentration of 0.5 mM to induce the degron and deplete Scm3. After an additional 60 min, cells from the control and Scm3-depleted cultures were rapidly filtered, washed, and resuspended in medium lacking α-factor with (Scm3-depleted) or without (control) CuSO₄. Ninety minutes after release from the α-factor arrest, cells were processed for fluorescence microscopy. Images of the Scm3-depleted cells (Upper) are from a contiguous microscopic field moved digitally to minimize empty space. (Scale bars: 2 μm.) Circles, mask used for quantitating fluorescence. (B) Fluorescence intensity of Cse4-GFP dots. Filled circles, measurements of distinguishable kinetochore dots; open circles, measurements from cells lacking a distinguishable kinetochore dot. The latter were excluded from the statistical analysis. Lines show median (“Dots”) or mean (Background) values; significance of differences was assessed by Mann–Whitney or unpaired t tests, respectively. (C) Bud morphologies of samples from A. UB, unbudded; SB, small-budded; LB, large-budded; DB, dumbbell. Black shading in the LB + DB bars indicates the proportion of DB.