FIGURE 3.

The Asf1-30 mutant protein was degraded via the ubiquitin-proteasome system. A, proteasome-dependent degradation of mutant Asf1-30. Wild-type and mts2-1 ts mutants harboring pREP41-asf1-30-13myc were pregrown in EMM medium at 26 °C, followed by a temperature shift to 36 °C. After incubation for 1 h at 36 °C, 2 μm thiamine and 100 μg/ml CHX were added to the medium, and whole cell lysates were prepared at the indicated time points at 36 °C. Asf1-30-13myc was detected by immunoblotting with an anti-Myc antibody (top). Tubulin was used as a loading control (bottom). B, quantification of Asf1-30 protein stability. Band intensities of Asf1-30-13Myc in A were quantified by normalizing relative intensity to tubulin signals. Relative intensity at time 0 was set up as 100% in each case. C, Asf1-30 was polyubiquitinated in vivo, but wild-type Asf1 was not. His₆-ubiquitin (pREP1-His₆-Ubi) was expressed in asf1⁺-13myc and asf1-30-13myc strains, and polyubiquitinated proteins were purified with Ni²⁺-NTA beads in the presence of 6 m guanidine HCl. Immunoblotting was performed with an anti-Myc antibody to examine the polyubiquitination of Asf1 (top). Ubiquitin was also detected with an anti-ubiquitin antibody as a loading control (bottom).