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. 2020 Jan 2;9:e50556. doi: 10.7554/eLife.50556

Figure 3. Inhibition of Pef1 kinase in G1-arrested mis4-367 cells allows neo-synthesized Rad21 to bind chromatin.

(A) The tetracycline (TET) inducible tet07-rad21-FLAG construct can substitute for the endogenous rad21 gene. The last two lanes show that pef1Δ suppresses mis4-367 ts phenotype when tet07-rad21-FLAG is the sole source of Rad21. (B) Experimental scheme. Cells cultured in EMM2 medium were arrested in G1 by the cdc10-129 mutation. After 4.5 hr tet07-rad21-FLAG was induced by the addition of TET or left un-induced (DMSO). Pef1-as was inhibited 30 min later and samples collected after 30 min. (C) DNA content analysis. (D) Western blot analysis of Rad21-FLAG in the chromatin (P) and soluble (S) fractions. (E) Fractionation controls. Anti-tubulin and anti-Histone H3 antibodies were used as markers for the soluble (S) and chromatin (P) fractions, respectively. (F) Rad21-FLAG signals were quantified for the TET samples from the long and short exposure blots shown in (D). The bars represent the mean relative band intensities + /- SD.

Figure 3.

Figure 3—figure supplement 1. Pef1 acts independently from the Psm3/Rad21 interface.

Figure 3—figure supplement 1.

(A) Deletion of wpl1 or a psm3-rad21 gene fusion allows cell survival in the absence of the otherwise essential eso1 gene. (B) The psm3-rad21 gene fusion does not suppress the mis4-367 thermosensitive growth defect. However deletion of the pef1 gene improves growth in this genetic setup. (C) Deletion of the wpl1 gene does not suppress mis4-367 growth defect.