The ER-resident ubiquitin-specific protease 19 participates in the UPR and rescues ERAD substrates

Abstract

Ubiquitination regulates membrane events such as endocytosis, membrane trafficking and endoplasmic-reticulum-associated degradation (ERAD). Although the involvement of membrane-associated ubiquitin-conjugating enzymes and ligases in these processes is well documented, their regulation by ubiquitin deconjugases is less well understood. By screening a database of human deubiquitinating enzymes (DUBs), we have identified a putative transmembrane domain in ubiquitin-specific protease (USP)19. We show that USP19 is a tail-anchored ubiquitin-specific protease localized to the ER and is a target of the unfolded protein response. USP19 rescues the ERAD substrates cystic fibrosis transmembrane conductance regulator (CFTR)DeltaF508 and T-cell receptor-alpha (TCRalpha) from proteasomal degradation. A catalytically inactive USP19 was still able to partly rescue TCRalpha but not CFTRDeltaF508, suggesting that USP19 might also exert a non-catalytic function on specific ERAD substrates. Thus, USP19 is the first example of a membrane-anchored DUB involved in the turnover of ERAD substrates.

Figure 1

USP19 is a ubiquitin-specific protease containing a predicted transmembrane domain. (A) Domain overview of the identified human deubiquitinating enzymes with potential transmembrane (TM) domains. (B) Ubiquitin (Ub) conjugates isolated from haemagglutinin (HA)-Ub-expressing cells were incubated with GST-USP19ΔTM. The reaction was carried out for the indicated time in the presence or absence of the cystein protease inhibitor N-ethylmaleimide (NEM). Western blots using an anti-HA antibody are shown. (C) Schematic representation of the Ub/Ubl (Ub-like) reporters. (D) Western blot of samples from bacteria co-expressing the reporter constructs and GST-USP19ΔTM (wt) or GST-USP19MUTΔTM (m). Antibodies were used against the S-tag, to detect the reporters, and against GST to verify USP19 expression. CS, CHORD and SGT1; DUSP, domain in USPs; GST, glutathione S-transferase; MYND, myeloid translocation protein 8, Nervy and Deaf1; p23, domain named after the protein p23; TMD, transmembrane domain; UBL, ubiquitin-like domain; USP, ubiquitin-specific protease.

Figure 2

USP19 is anchored to the endoplasmic reticulum facing the cytoplasm. (A) Human embryonic kidney (HEK)293 cells expressing USP19-Myc-His or USP19ΔTM-Myc-His, stained with a Myc antibody. White squares indicate the zoomed areas. The nucleus was visualized by DAPI. Scale bars, 20 μm. (B) Lysates of HEK293T cells were fractionated into a soluble cytosolic fraction (S) and a microsomal fraction (P). The mitogen-activated protein kinase p38 and the endoplasmic reticulum chaperone Grp78/Bip were used as markers for soluble and membrane proteins, respectively. Equal amounts of proteins were loaded in each lane. (C) HEK293 cells stably expressing USP19-GFP (green) stained against calnexin (red, upper panel) or giantin (red, lower panel). Scale bars, 20 μm. (D) Untransfected HeLa cells stained with a calnexin antibody (upper panel) and Myc-USP19-expressing cells stained anti-Myc (lower panel). The cells were treated with 0.1% Triton X-100 to permeabilize all membranes or 0.15% Digitonin to selectively permeabilize only the outer membranes. (E) Western blot of microsomal fractions from HEK293T cells treated with 0.4 μg/ml proteinase K±1% NP40. BiP, immunoglobulin heavy chain-binding protein; GFP, green fluorescent protein; Grp78, 78 kDa glucose-regulated protein precursor; PNS, post-nuclear supernatant; TM, transmembrane; USP, ubiquitin-specific protease.

Figure 3

USP19 messenger RNA expression is induced by endoplasmic reticulum stress. (A) USP19 mRNA levels were analysed by real-time (RT)–PCR in human embryonic kidney (HEK)293 cells treated with tunicamycin (tm; 5 μg/ml) for the indicated time (black line). Values are expressed as USP19 expression relative to the control GAPDH±s.e.m. Values in grey illustrate the spliced X-box binding protein 1 (XBP1s) relative to total XBP1 as quantified by densitometry of the semi-quantitative RT–PCR shown in (B). Mean±s.d. of experiments in triplicate. (B) Agarose gel of RT–PCR showing the level of XBP1s in the samples shown in (A). (C) Real-time PCR of USP19 mRNA expression in HEK293 cells treated with thapsigargin (tg; 1 μM), tunicamycin (5 μg/ml) or dithiothreitol (DTT; 2 mM) for 6 h. Expression levels relative to GAPDH±s.e.m. of three experiments. Primer sets specific for USP19 containing the transmembrane domain (Tmd isoform) and all predicted USP19 transcripts (all isoforms) were used. (D) Agarose gel of RT–PCR showing XBP1 and XBP1s of samples used in (C). USP, ubiquitin-specific protease.

Figure 4

Membrane-associated USP19 rescues endoplasmic reticulum associated degradation substrates from degradation. (A) Western blot of human embryonic kidney (HEK)293T cells transfected with a constant amount of GFP-CFTRΔF508 and titrated amounts of the various USP19 constructs as indicated. A plasmid expressing the red fluorescent protein (RFP) was co-transfected to control for equal transfection efficiency. (B) Western blot of HEK293T cells transfected with FLAG-TCRα and the various USP19 constructs as indicated. The glycosylation of FLAG-TCRα is indicated and the numbers of glycans marked in grey are predicted on the basis of the earlier literature (Hirsch et al, 2003). RFP was used as a transfection control. (C) Western blot of HEK293T cells transfected with the soluble cytosolic proteasome substrate ubiquitin (Ub)-R-GFP and the various USP19 constructs as indicated. RFP was used as a transfection control. CFTR, cystic fibrosis transmembrane conductance regulator; GFP, green fluorescent protein; TCRα, T-cell receptor α; TM, transmembrane; Untr., untransfected; USP, ubiquitin-specific protease.

Figure 5

Myc-USP19 associates with endoplasmic reticulum-associated degradation substrates. (A) Western blot of human embryonic kidney 293T cells where Myc-USP19 was co-transfected with GFP-CFTRΔF508 or GFP as control. Immunoprecipitations from whole-cell lysates were performed using a GFP antibody. The input is shown in the lower panels. (B) Western blot of co-immunoprecipitation experiments where Myc-USP19 was co-transfected with FLAG-TCRα. Immunoprecipitations were performed using a FLAG antibody or an isotype-matched antibody as control. The input is shown in the lower panels. CFTR, cystic fibrosis transmembrane conductance regulator; GFP, green fluorescent protein; TCRα, T-cell receptor α; USP, ubiquitin-specific protease.