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. 2010 Nov 1;191(3):493–503. doi: 10.1083/jcb.201006092

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© 2010 Moore et al.

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Figure 6. — Elm1 is necessary for Kin4 activation. (A) Alignment of the activation-loop regions of Kin4 and three known substrates of the Elm1 kinase. Phosphorylation of each highlighted threonine residue is necessary for kinase activation. Boxes indicate the majority sequence identity at a given residue position. (B) The presence of slow-migrating species of Kin4 depends on Elm1. Immunoblots of lysates from wild-type and elm1Δ cells expressing Kin4 tagged with a 13myc epitope, either grown asynchronously or arrested at metaphase by treatment with nocodazole. Arrow denotes slower-migrating species of Kin4-13myc. Strains: wild type (WT), yJC6382 and elm1Δ, yJC6396. (C) Phosphomimetic kin4-T209D is functional in the absence of Elm1. High-copy plasmids containing KIN4, kin4-T209D, or kin4-T209A under the control of a galactose-inducible promoter were transformed into either wild-type, bfa1Δ, or elm1Δ strain backgrounds. 10-fold dilution series were spotted onto media selective for plasmid retention and containing either galactose to induce expression or glucose to inhibit expression. Strains containing empty vector are shown as controls. Strains: wild type (wt), yJC2295; bfa1Δ, yJC6447; and elm1Δ, yJC5254. Plasmids: pGAL-KIN4, pBJ1651; pGAL-kin4-T209D, pBJ1896; pGAL-kin4-T209A, pBJ1840; and vector, pBJ216. (D) Phosphomimetic Kin4-T209D rescues the SPC defect of elm1Δ mutants. Indicated strains were arrested by treatment with α factor and released into fresh media at 14°C for 20 h. Cells were then briefly fixed in formaldehyde, and the percentage of cells exhibiting intact anaphase spindles within the mother compartment (light gray bars) or multiple MTOCs resulting from checkpoint failure (dark gray bars) was determined based on observation of GFP-tubulin. Values are the means of 10 counts of at least 50 cells from two separate experiments. Error bars are the standard error of the means. Strains: dyn1Δ, yJC5603; dyn1Δ kin4-T209D, yJC7080; dyn1Δ elm1Δ, yJC7079; and dyn1Δ elm1Δ kin4-T209D, yJC7081.