Characterization of the deubiquitinating activity of USP19 and its role in endoplasmic reticulum-associated degradation

Abstract

Deubiquitinating enzymes (DUBs) regulate various cellular processes ranging from protein degradation to cellular signaling. USP19, the only DUB containing a carboxyl-terminal transmembrane domain, was proposed to function in endoplasmic reticulum-associated degradation (ERAD). Here we characterize the function and regulation of USP19. We identify Hsp90 as a specific partner that binds the catalytic domain of USP19 to promote substrate association. Intriguingly, although overexpressed USP19 interacts with Derlin-1 and other ERAD machinery factors in the membrane, endogenous USP19 is mostly in the cytosol where it binds Hsp90. Accordingly, we detect neither interaction of endogenous USP19 with Derlin-1 nor significant effect on ERAD by USP19 depletion. The USP19 transmembrane domain appears to be partially stabilized in the cytosol by an interaction with its own catalytic domain, resulting in auto-inhibition of its deubiquitinating activity. These results clarify the role of USP19 in ERAD and suggest a novel DUB regulation that involves chaperone association and membrane integration. Moreover, our study indicates that the localization of tail-anchored membrane proteins can be subject to regulation in cells.

Keywords: Deubiquitination; ER-associated Degradation; Hsp90; Membrane Proteins; Tail-anchored Protein Biogenesis; USP19; Ubiquitin.

FIGURE 1.

Interaction of USP19 with Hsp90. A, cells transfected with either a control or FLAG-USP19-expressing vector were lysed. USP19 was purified using beads conjugated with FLAG antibodies. The purified samples were analyzed by SDS-PAGE and silver staining. B, fraction of the purified samples in A was analyzed by immunoblotting (IB). C, endogenous interaction of USP19 with Hsp90. D, FLAG-USP19 purified from mammalian cells in A was subject to another round of immunoprecipitation with either control IgG or Hsp90 specific antibodies.

FIGURE 2.

Hsp90 binds USP19 specifically via its catalytic domain. A, schematic representation of the USP19 mutant constructs used in the interaction studies. B, Hsp90 binds USP19 via the catalytic domain. Cells expressing the indicated FLAG-tagged USP19 variants were lysed, and the extracts were subject to immunoprecipitation by FLAG antibodies. cont, control. C, as in B, except that cells expressing the indicated DUBs were used.

FIGURE 3.

Hsp90 regulates USP19 activity. A, deubiquitinating activity of USP19 was measured using 0.4 μm Ub-AFC as the substrate in the absence or presence of the indicated inhibitor. GA, geldanamycin. B, in vitro ubiquitinated Ub-V-GFP was incubated with FLAG-USP19 purified from mammalian cells in the absence or presence of 1 mm DTT (−/+). Where indicated, USP19 was pre-incubated with GA for 1 h before the DUB activity was measured. C, GA did not affect USP7 activity. As in A, except that purified USP7 was used. D, cells treated with GA (2.5 or 10 μm) for 16 h were lysed. USP19 immunoprecipitated from cell extracts was analyzed by immunoblotting. E, GA inhibits USP19 activity at low substrate concentrations. USP19 activity was measured as in A using Ub-AFC at the indicated concentrations. The plot shows the relative inhibition of the USP19 activity.

FIGURE 4.

Hsp90 and USP19 are not involved in ERAD. A, cells transiently expressing the ERAD substrate NHK-GFP were treated with GA (10 μm) or MG132 (10 μm) for 15 h. Whole cell extracts (WCE) were analyzed by immunoblotting. B, cells expressing the ERAD substrates TTR D18G-GFP or TCRα-YFP were treated with the indicated inhibitors for 15 h. Shown are the relative fluorescence intensities measured by flow cytometry. C and D, USP19 knockdown does not affect ERAD of NHK-GFP and TTR D18G-GFP. E, interaction of overexpressed USP19 with a retrotranslocation complex. ER membrane fractions isolated from either control or USP19-expressing cells were solubilized. Protein extracts were subject to immunoprecipitation with FLAG beads. A fraction of the input and the precipitated samples were analyzed by immunoblotting. F, As in E, except that protein extracts from both the ER membrane (memb) and the cytosol (cyto) fractions were subject to immunoprecipitation with anti-USP19 antibodies. # indicates IgG.

FIGURE 5.

Endogenous USP19 is primarily localized in the cytosol. A, subcellular fractionation shows that overexpressed USP19 is mainly localized in the membrane. B, shown is a representative HeLa cell expressing FLAG-USP19 stained with FLAG antibody in green. DAPI stains the nucleus in blue. Scale bar, 10 μm. C, endogenous USP19 is primarily localized in the cytosol. D, cell fractionation shows that USP19 is primarily localized in the cytosol in different kinds of cells. E, As in D, except that HEK293 cells treated at 37 °C or 42 °C for 16 h were used. F, as in E, except that cells treated with an ER stress inducer tunicamycin (Tm) or expressing the non-glycosylated ERAD substrate NHK-QQQ were used. cont, control. G, as in E, except that cells treated with the indicated inhibitors were used. H, as in E, except that cells treated with GA as indicated were used.

FIGURE 6.

The USP19 transmembrane domain inhibits its catalytic activity. A, purified USP19 mutant lacking transmembrane domain was incubated with Lys48-linked di-Ub in the presence of increased concentration of a transmembrane (TMD) peptide at 37 °C for 1 h. B, as in A, except that the purified USP19 catalytic domain was used.