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. 2014 Jul 29;9(7):e103517. doi: 10.1371/journal.pone.0103517

Figure 4. Acm1 proteolysis does not require assembly of polyubiquitin chains.

Figure 4

A) YKA247 cells containing PGAL1-driven plasmids pHLP391 (for Acm1), pESCW-Fin1-Myc, or pHLP309 (for Clb2) and PCUP1-driven ubiquitin (Ub) overexpression plasmids LHP306 (for Ub-K7R mutant) or LHP308 (for wild-type Ub) were grown to early-exponential phase. Cells were arrested at G1 before expression of wild-type or mutant Ub was induced with 100 µM CuSO4 and Acm1, Fin1-myc, and Clb2 with galactose. Stability of Acm1, Fin1-Myc, and Clb2 were monitored with anti-Acm1, anti-Myc, and anti-Clb2 antibodies, respectively. G6PD is a loading control. NC, negative control without galactose induction. B) The same experiment described in panel A was performed in doa4Δ cells to limit the abundance of endogenous ubiquitin, and only glucose was used to terminate expression, allowing continuous synthesis of mutant or wild-type Ub. In this experiment pHLP212 was used to express 3HA-Acm1, which was detected with an anti-HA antibody and Fin1-3HA was used as a control.