Fig. 4. Degradation of Pan1 and Las17 elicit YCS on Zeocin.

a Exponentially growing isogenic WT (GA-5731) and PAN1-AID (GA-6810) cells were treated ± 0.5 mM IAA for 4 h to deplete Pan1. Cells were then treated with Zeocin (75 μg/ml) alone or in combination with BHS (10 μM) for 70 min. YCS was monitored as in Fig. 1b and quantitation is described in Methods, with B/A values determined as in Fig. 1 (bold values discussed in text). b As a but for isogenic WT (GA-5731) and LAS17-AID (GA-6839) cells. 0.5 mM IAA was added for 3.5 h and cells were then treated with Zeocin (75 μg/ml) ± BHS (10 μM) for 70 min. YCS was monitored, quantified and scanned as in a. Depletion of Las17 elicits efficient YCS in combination with Zeocin but not alone. c Isogenic WT (GA-5731) and LAS17-AID (GA-6839) strains were exponentially cultured and treated for 4 h with 0.5 mM IAA for 3.5 h to trigger Las17-AID degradation as in b. Cells were treated with LatA (20 μM), Zeocin (50 μg/ml), or the combination in the presence of IAA for 50 or 90 min as indicated. YCS was monitored and quantified as in a. d Isogenic WT, GA-4732 (BY4741 WT), GA-10701 (cap2Δ), GA-10905 cap2Δ las17Δ), and GA-9204 (LAS17-AID, TIR1) were cultured in SC overnight, cells were diluted to OD₆₀₀ 0.16 in 20 ml SC and cultured for 3 h with 0.5 mM IAA for 2 h to deplete Las17. Then equal aliquots were treated with 0.25% DMSO (control), 100 µg/ml Zeocin, 1 µM CMB, or the combination of both for 90 min. YCS was monitored, quantified and scanned as in a, with SYBR safe staining. The strain background is S288C, not W303, and therefore slightly higher concentrations of reagents were used. The cap2Δ mutation partially suppresses YCS provoked by Las17 degradation or by TORC2 inhibition.