(A) Flow cytometric measurement of the activity of RPN4 reporter variants in untreated cells. Data are normalized to the reporter containing the native RPN4 promoter. The other reporters contain mutations in the two Pdr1/3 response elements (PDREm), the Yap1 response element (YREm) or the heat shock element (HSEm). Mean ±SEM, n = 4. (B) As in panel A, but after treatment with 5 µg/ml tunicamycin for the times indicated. Mean ±SEM, n = 3. (C) Flow cytometric measurement of the activity of the native RPN4 reporter in tpk1/2/3-as and tpk1/2/3 ∆msn2/4 cells treated with the ATP analog 1NM-PP1 for the times indicated. The analog-sensitive tpk1/2/3-as alleles enable specific inhibition of protein kinase A with 1NM-PP1. Mean ±SEM, n = 3. (D) RPN4 mRNA levels in wild-type (WT) and tpk1/2/3-as cells treated with 1NM-PP1 for the times indicated. Data are normalized to WT cells at t = 0. Mean ±SEM, n = 3. (E) Flow cytometric measurement of the activity of RPN4 reporter variants in WT and ∆msn2/4 cells treated with 5 µg/ml tunicamycin. Data are normalized to WT cells containing the reporter with the native RPN4 promoter. Mean ±SEM, n = 3. (F) Model for the cooperation of Rpn4 and the UPR. ER protein misfolding causes increased flux through the ERAD pathway, which strains proteasome capacity and inhibits efficient protein degradation (1). In addition, the UPR is activated (2). Severe ER stress leads to translocation defects, causing mislocalization of secretory proteins to the cytosol, where they cannot fold properly (3). These cytosolic misfolded proteins further burden the proteasome (4). As a result, Rpn4 is stabilized, the Rpn4 regulon is activated and proteasome biogenesis is enhanced. If cytosolic misfolded proteins persist, the RPN4 gene is induced (5), providing a second mechanism to increase Rpn4 abundance and augment proteasome biogenesis.