Fig. 3. ZYA robustly stimulates gate-opening in yeast and mammalian proteasomes.

a Dose response of ZYA with mammalian 20S proteasomes (0.5 nM) and nLPnLD-AMC as substrate. Proteasome activity is normalized to DMSO control. Means were fit to the Hill equation, and equilibrium binding coefficients are shown. b WT and gateless (α3∆N) yeast 20S proteasomes (0.5 nM) incubated with or without ZYA (2.5 mM) and nLPnLD-amc. c Mammalian 20S proteasomes (0.5 nM) alone or with ZYA (2.5 mM), or PA26 (55 nM), or both (see graph key). Proteasome activity measured using three different fluorogenic substrates preferentially cleaved by different 20S protease sites (LLVY-amc, chymotrypsin-like, nLPnLD-amc, caspase-like; LRR-amc, trypsin-like). Data (means) are representative of three or more independent experiments each performed in triplicate. Error bars represent ± standard deviation.