Coiled-coil mutations without destabilization of coiled-coils.

<p>(A) Rpt tet-off strains were transformed with the pAUR123 yeast expression vector encoding wild-type and CC0 and CC00 mutants of Rpt subunits, grown to early log phase, and individually spotted in duplicate as a ten-fold serial dilutions on plates either without (right panel) or with (left panel) doxycycline. Plates were incubated at 30°C for 2 days and then photographed. (B) Accumulation of polyubiquitinated proteins in the Rpt tet-off yeast cells expressing wild-type and CC0 or CC00 mutants (Rpt1CC00, Rpt2CC00, Rpt3CC00, Rpt4CC00, Rpt5CC0, and Rpt6CC0) were analyzed using western blot with an anti-polyubiquitin antibody. After the yeast cell at early log phase were treated with 20 μg/mL Dox for 3 h, cells were harvested and lysed with glass beads in the presence of 10% TCA. PGK1 was used as a loading control.