FIGURE 6.

Smc5 stabilizes microtubules in vivo and in vitro. A, the smc5-3KE mutant exhibits a recessive cold-sensitive growth defect in yeast. 5-Fold dilution series of wild-type haploid cells, smc5-6 mutant (22), tubulin mutant tub2-401 (38), wild-type SMC5, or smc5-3KE, were spotted on solid medium to evaluate growth at the indicated temperatures of 18, 23, 30, and 37 °C. Bottom panels, proliferation of diploid yeast heterozygous for SMC5-STH or smc5-3KE-STH on solid medium at various temperatures. The absence of cold-sensitive growth defect in the diploids indicates that the smc5-3KE allele is recessive. B, Smc5 protects microtubules from cold-induced depolymerization. After incubation at the indicated temperature (2 °C or room temperature (RT)), reaction mixtures containing buffer control (−), Smc5-WT, or Smc5-3KE were subjected to ultracentrifugation to separate the dissociated tubulin dimers and the remaining microtubule polymers into supernatant (S) and pellet (P) fractions, respectively. Coomassie Blue-stained gels of the samples are shown. The relative quantity of the Smc5 proteins in arbitrary units (a.u.) in each sample is shown below the corresponding lanes on the gel. C, quantification of the percentage of protection provided by Smc5 proteins (average of three independent experiments; error bars, S.D.). Percentage protection is defined as percentage of excess microtubules retained in the pellet relative to the control at 2 °C. D and E, genetic interactions between smc5-3KE and the tubulin-defective mutant tub2-401 (D) or the spindle assembly checkpoint mutant mad1Δ (E). Growth phenotypes of the indicated yeast strains were evaluated as described in A. F, proliferation of smc5 mutants and wild-type yeast strains on solid medium containing methyl methanesulfonate (MMS) at 0.005%, hydroxyurea (HU) at 100 mm, and 4-nitroquinoline 1-oxide (4NQO) at 0.2 μm.