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. 2020 Jun 24;16(6):e1008840. doi: 10.1371/journal.pgen.1008840

Fig 6. Stabilization of an endogenous Isr1 phosphodegron recapitulates overexpression phenotypes.

Fig 6

(A) Diagram of Pho85 and Cdc4 consensus sites in Isr1 that comprise a phosphodegron. Six sites (T3, T4, S8, T85, T86, S92) are mutated to alanine at the endogenous locus in the ISR1-PD mutant. (B) An Isr1 phosphodegron mutant is stable. Cycloheximide-chase assay of Isr1-13xmyc in wild-type, pho85Δ, or cells expressing ISR1-PD. Cycloheximide was added for the indicated number of minutes. Levels of Isr1-13xMyc and PSTAIR (loading control) are shown. PSTAIR is a monoclonal antibody that recognizes the PSTAIR sequence in Cdc28. (C) An Isr1 phosphodegron mutant is resistant to calcofluor white (CFW). Strains of the indicated genotypes were diluted onto YPD with or without 40 μg/ml CFW. (D) An Isr1 phosphodegron mutant is sensitive to tunicamycin. Experiment was performed as in C, except strains were spotted on YPD with or without 0.25 μg/ml tunicamycin. (E) Diploid strains of the indicated genotypes were diluted onto YPD with or without 0.25 μg/ml tunicamycin.