Usa1p is required for optimal function and regulation of the Hrd1p endoplasmic reticulum-associated degradation ubiquitin ligase

Abstract

Usa1p is a recently discovered member of the HRD ubiquitin ligase complex. The HRD pathway is a conserved route of ubiquitin-dependent, endoplasmic reticulum (ER)-associated degradation (ERAD) of numerous lumenal (ERAD-L) and membrane-anchored (ERAD-M) substrates. We have investigated Usa1p to understand its importance in HRD complex action. Usa1p was required for the optimal function of the Hrd1p E3 ubiquitin ligase; its loss caused deficient degradation of both membrane-associated and lumenal proteins. Furthermore, Usa1p functioned in regulation of Hrd1p by two mechanisms. First, Hrd1p self-degradation, which serves to limit the levels of uncomplexed E3, is absolutely dependent on Usa1p and the ubiquitin-like (Ubl) domain of Usa1p. We found that Usa1p allows Hrd1p degradation by promoting trans interactions between Hrd1p molecules. The Ubl domain of Usa1p was required specifically for Hrd1p self-ubiquitination but not for degradation of either ERAD-L or ERAD-M substrates. In addition, Usa1p was able to attenuate the activity-dependent toxicity of Hrd1p without compromising substrate degradation, indicating a separate role in ligase regulation that operates in parallel to stability control. Many of the described actions of Usa1p are distinct from those of Der1p, which is recruited to the HRD complex by Usa1p. Thus, this novel, conserved factor is broadly involved in the function and regulation of the HRD pathway of ERAD.

FIGURE 1.

Usa1p is required for optimal degradation of ERAD-M substrates. Log-phase cultures of either WT, hrd3Δ, or usa1Δ cells expressing normally regulated, catalytically active 1myc-Hmg2p (A), or the unregulated variant 6myc-Hmg2p (B) were subjected to cycloheximide (CHX) chase for the indicated times followed by lysis and immunoblotting to evaluate protein stability. The hrd3Δ strain served as a positive control that stabilizes both substrates. C, cycloheximide chase of Hmg2p-GFP is shown. Log-phase cultures of either WT, hrd3Δ, or usa1Δ cells expressing the normally regulated but catalytically inactive Hmg2p-GFP were subjected to cycloheximide chase. D, degradation of Hmg2p-GFP was evaluated by flow cytometry of live cells at the indicated times after cycloheximide addition using 10,000 cells per point in this and all subsequent flow cytometric experiments. For each strain used (WT (squares), hrd3Δ (triangle), or usa1Δ (circles)) the experiment was run with (open symbols) or without (solid symbols)) 10 μg/ml ZA to evaluate the effect of elevating degradation signal on Hmg2p-GFP degradation. E, shown is ubiquitination of Hmg2p-GFP in WT, hrd3Δ, or usa1Δ strains. Log-phase cultures of the indicated strains expressing Hmg2p-GFP were incubated for 5 min with or without 10 μg/ml ZA followed by lysis, immunoprecipitation (IP), and immunoblotting (WB) for Hmg2p-GFP (bottom row) or ubiquitin immunoreactivity. The − lanes indicate the ubiquitination state of Hmg2p normally present in each strain without drug treatment. F, Hrd1p overexpression suppressed the Hmg2p-GFP degradation defect of usa1Δ strains. Wild type or usa1Δ strains harboring either empty vector or a HRD1-overexpressing plasmid (TDH3-HRD1) were subject to cycloheximide chase for 2 h (gray bars) and compared with strains that were not treated with drug (black bars) by flow cytometry.

FIGURE 2.

Usa1p and Der1p have distinct roles in ERAD. A, shown is UPR caused by usa1Δ or der1Δ null mutants. Otherwise identical WT, usa1Δ, der1Δ, or usa1Δder1Δ strains harboring the 4x UPRE-GFP unfolded protein response reporter were compared by flow cytometry of log-phase cultures for mean fluorescence to evaluate the level of the UPR in each genetic circumstance. B, shown is degradation of Hmg2p-GFP in WT, hrd3Δ, usa1Δ, or der1Δ strains, as measured by flow cytometry after the addition of cycloheximide at the indicated times. C, shown is degradation of 6myc-Hmg2p in the same strains as panel B, measured by cycloheximide (CHX) chase at the indicated times followed by immunoblotting for the myc epitope tag. D, shown is the effect of Der1p overexpression on Hmg2p-GFP levels in wild type, der1Δ, or usa1Δ strains. Each strain type with empty vector or a DER1-overexpressing plasmid (TDH3-DER1) was compared in log phase for Hmg2p-GFP levels by flow cytometry. E, shown is the effect of Der1p overexpression on the lumenal substrate CPY*HA in WT, usa1Δ, or der1Δ strains. Each strain type with empty vector or a DER1-overexpressing plasmid was subjected to cycloheximide chase at the indicated times and immunoblotted for the HA tag. Note that the der1Δ phenotype is suppressed by the DER1-overexpressing plasmid.

FIGURE 3.

Usa1p is required for Hrd1p self-degradation. A, the effect of a usa1Δ on Hrd1p stability was evaluated in both wild type and hrd3Δ strains. The indicated strains were evaluated for Hrd1p stability by cycloheximide (CHX) chase followed by Hrd1p immunoblotting. B, shown is the effect of usa1Δ on Hrd1p self-ubiquitination observed in the absence of Hrd3p. The indicated strains were treated with the proteasome inhibitor MG132 for 2.25 h and subjected to lysis, immunoprecipitation (IP) of Hrd1p, and immunoblotting (IB) for Hrd1p (bottom panel) or ubiquitin (top panel) to evaluate Hrd1p self-ubiquitination. A hrd1Δ null strain (left lane) was included as a specificity control. Equal amounts of Hrd1p were loaded in all other lanes to allow direct comparison of ubiquitination state. All strains harbored the pdr5Δ null mutation to allow the effective use of MG132. C, comparison of the effect of usa1Δ or der1Δ on the hrd3Δ-dependent degradation of Hrd1p is shown. The indicated strains were compared for Hrd1p stability by cycloheximide chase for the indicated times. Note that the der1Δ still allowed significant degradation of Hrd1p. D, Hrd1p self-ubiquitination in the indicated strains (with added pdr5Δ) is shown. The identical Hrd1p self-ubiquitination assay as described in panel B was employed. E, degradation of TDH3-driven Hrd1p was evaluated in strains containing either empty vector or TDH3-USA1 by cycloheximide chase followed by immunoblotting for Hrd1p. F, degradation of TDH3-driven RING Hrd1p-5myc was evaluated in strains containing either empty vector or TDH3-USA1 by cycloheximide chase followed by immunoblotting for the myc epitope to detect RING-Hrd1p.

FIGURE 4.

Role of the Ubl domain in ERAD and Hrd1p self-degradation. A, shown is a schematic of USA1 and USA1ΔUBL. Two versions of USA1ΔUBL were made, one with a clean deletion of 100 amino acids (USA1ΔUBL) and one with 5 copies of the myc epitope tag in place of the deleted sequence (USA1ΔUBL-myc). B, Hmg2p-GFP degradation in wild type, usa1Δ, USA1ΔUBL, and USA1ΔUBL-myc strains (left) was analyzed by flow cytometry of log-phase cultures treated with cycloheximide for the indicated times. Sec61-2p degradation (right) was analyzed by dilution assay in wild type, usa1Δ, USA1ΔUBL, and USA1ΔUBL-myc strains. 5-Fold serial dilutions were plated and grown at the indicated temperatures. C, CPY* degradation (left) and KWW degradation (right) in wild type, usa1Δ, USA1ΔUBL, and USA1ΔUBL-myc strains was analyzed by cycloheximide (CHX) chase for the indicated time points followed by lysis and immunoblotting for the HA epitope tag. D, Hrd1p degradation was evaluated by cycloheximide chase. Hrd1p degradation was compared in wild type, hrd3Δ, USA1ΔUBL hrd3Δ, and usa1Δhrd3Δ strains. E, Hrd1p self-ubiquitination was evaluated in the same strains as in D. Log-phase cultures were treated with MG132 to allow ubiquitinated Hrd1p to accumulate followed by immunoprecipitation (IP) of the Hrd1p and immunoblotting (WB) for ubiquitin or Hrd1p. Equal amounts of Hrd1p were loaded to directly compare ubiquitination levels. F, degradation of overexpressed Hrd1p (TDH3-Hrd1p) in strains with empty vector (EV), overexpressed USA1 (TDH3-USA1), and overexpressed USA1ΔUBL (TDH3-USA1ΔUBL) was analyzed by cycloheximide chase followed by immunoblotting for Hrd1p. G, degradation of overexpressed RING Hrd1p-5myc in strains with empty vector, overexpressed USA1 (TDH3-USA1), and overexpressed USA1ΔUBL (TDH3-USA1ΔUBL) was analyzed by cycloheximide chase followed by immunoblotting for the myc epitope.

FIGURE 5.

Hrd1p undergoes Usa1p-dependent self-ubiquitination in trans. A, degradation of C399S Hrd1p-5myc was analyzed by cycloheximide chase. Strains containing overexpressed (TDH3) HRD1 and either empty vector (EV), TDH3-USA1, TDH3-USA1ΔUBL, or a strain containing only TDH3-USA1 were subjected to cycloheximide (CHX) chase and immunoblotting for the myc epitope tag on C399S Hrd1p. B, trans-degradation of C399S Hrd1p is proteasome-dependent. A C399S Hrd1p-5myc strain containing overexpressed HRD1 and USA1 was subjected to cycloheximide chase with the addition of the proteasome inhibitor MG132. Log-phase cells were pretreated for 30 min with 25 μg/ml MG132 (or DMSO vehicle) for 15 min before addition of cycloheximide. C, trans-degradation of C399S requires catalytically active Hrd1p. A C399S Hrd1p-5myc strain containing either TDH3-HRD1 or TDH3-C399S HRD1 was subjected to cycloheximide chase for the indicated time points followed by lysis and immunoblotting for the myc epitope on C399S Hrd1p-5myc.

FIGURE 6.

Usa1p abolishes toxicity of overexpressed Hrd1p. A, growth of strains was compared by making 5-fold serial dilutions and incubating at the indicated temperatures. Wild type was compared against strains containing overexpressed Hrd1p with the addition of either empty vector (EV), overexpressed USA1, or overexpressed USA1ΔUBL. B, Hmg2p-GFP degradation was evaluated in strains containing overexpressed Hrd1p with the addition of empty vector, overexpressed USA1, or overexpressed USA1ΔUBL. Log-phase cultures were treated with cycloheximide for the indicated times and analyzed by flow cytometry. C, CPY* degradation was evaluated in strains containing overexpressed Hrd1p and either overexpressed USA1 or overexpressed USA1ΔUBL and compared with a wild type strain. Log-phase cultures were treated with cycloheximide for the indicated times followed by lysis and immunoblotting for the HA epitope on CPY*HA.