Figure 3. The E3 ubiquitin ligase Mot2 negatively affects the levels of the Mmi1 inhibitor Mei2.

(A) Western blot showing total Mei2-3xHA levels in wt and mot2∆ cells grown at 30°C to mid-log phase in rich (YE) and minimal (EMM0.5X) media. An anti-tubulin antibody was used as a loading control. (B) Western blot showing total TAP-Mei2 levels expressed from the P41nmt1 promoter in wt and mot2∆ cells grown in minimal medium (EMM0.5X). An anti-tubulin antibody was used as a loading control. (C) Quantification of TAP-Mei2 protein levels, normalized to tubulin and expressed relative to wt cells. Error bars represent the standard deviation from five independent experiments. Stars denote statistical significance (t-test p-value=1.38E-3). (D) RT-qPCR analysis of mei2+, ssm4+ and mcp5+ transcripts in wt and mot2∆ strains expressing Mei2 from the P41nmt1 promoter. Cells were grown in minimal medium (EMM0.5X). Signals were normalized to act1+ mRNA levels and expressed relative to the P41nmt1-TAP-Mei2 strain. Error bars represent the standard deviation from four independent experiments. Stars denote statistical significance (t-test p-values: mei2+ = 8.5E-3; ssm4+ = 1.53E-3; mcp5+ = 1.49E-3).

Figure 3—figure supplement 1. The E3 ubiquitin ligase Mot2 negatively affects the levels of the Mmi1 inhibitor Mei2.

(A) RT-qPCR analysis of mei2+ mRNAs levels in wt and mot2∆ cells grown in minimal medium (EMM0.5X). Signals were normalized to act1+ mRNA levels and expressed relative to the wild type strain. Error bars represent the standard deviation from at least three independent experiments. The star denotes statistical significance relative to wild type cells (t-test p-value=1.1E-2). (B) Western blot showing total TAP-Mei2 levels expressed from the P41nmt1 promoter in minimal medium (EMM0.5X) in wild type cells and strains deleted for non-essential subunits of the Ccr4-Not complex (i.e. all but Not1). An anti-tubulin antibody was used as a loading control.