Hos2p/Set3p Deacetylase Complex Signals Secretory Stress through the Mpk1p Cell Integrity Pathway

The Hos2p/Set3p complex is required for a secretory stress response. (A) Hos2p complex deletion strains were spotted as described in the legend to Fig. 1 onto medium containing 2-deoxyglucose or DTT or medium lacking inositol. (B) UPR activation was monitored by Northern analysis from wild-type (wt) or hos2Δ mutant yeast strains challenged with 1 μg/ml tunicamycin (Tm) for 1 h. Northern analysis was performed with a HAC1-specific probe detecting both the full-length (HAC1^u) and spliced (HAC1ⁱ) isoforms of HAC1. (C) Northern analysis was performed on wild-type and hos2Δ mutant strains treated with tunicamycin for the indicated times with INO1- and KAR2-specific probes. (D) erg6Δ or erg6Δ hos2Δ cells were serially diluted onto complete medium containing 100 μg/ml brefeldin A (BFA), 1 μg/ml bafilomycin A1 (Baf-A1), or 50 μM chlorpromazine (CPZ) for stress sensitivity analysis.

Genetic interactions between hos2Δ and UPR components. Wild-type (wt) and hos2Δ, hac1Δ, and hos2Δ hac1Δ mutant strains were serially diluted, as described in the legend to Fig. 2, onto complete medium at 30 or 37°C or complete medium containing 1 mM hydrogen peroxide (H₂O₂).

Hos2p is not required for the proper repression of ribosomal gene expression during secretory failure. Northern analysis was performed with the wild-type or hos2Δ mutant strain challenged with 1 μg/ml tunicamycin (Tm) for 0, 1, 2, or 3 h. Northern analysis was performed with labeled probes detecting RPL3, RPL28, and RPL30 previously shown to respond to secretory defects. 28S rRNA served as a loading control.

The Hos2p complex is required for Mpk1p activation following secretory stress. The wild-type (wt) and hos2Δ mutant strains were grown to mid-log phase and challenged with 1 μg/ml tunicamycin (Tm) for 1 h. Western analysis was carried out with antibodies against total Mpk1p and activated phospho-Mpk1p.

Ectopic activation of Mpk1p signaling rescues the hos2Δ secretory growth defect. The wild-type (wt) and hos2Δ and hac1Δ mutant strains harboring the control vector, BCK1-20, or MPK1-2μm plasmid were grown to mid-log phase and serially spotted, as described in the legend to Fig. 5, onto selective medium containing 1 μg/ml tunicamycin (Tm). Plates were incubated at 30°C for 48 h.