USP19-Mediated Deubiquitination Facilitates the Stabilization of HRD1 Ubiquitin Ligase

Abstract

In the endoplasmic reticulum (ER), misfolded and unfolded proteins are eliminated by a process called ER-associated protein degradation (ERAD) in order to maintain cell homeostasis. In the ERAD pathway, several ER-localized E3 ubiquitin ligases target ERAD substrate proteins for ubiquitination and subsequent proteasomal degradation. However, little is known about how the functions of the ERAD ubiquitin ligases are regulated. Recently, USP19, an ER-anchored deubiquitinating enzyme (DUB), has been suggested to be involved in the regulation of ERAD. In this study, HRD1, an ERAD ubiquitin ligase, is shown to be a novel substrate for USP19. We demonstrate that USP19 rescues HRD1 from proteasomal degradation by deubiquitination of K48-linked ubiquitin chains. In addition, the altered expression of USP19 affects the steady-state levels of HRD1. These results suggest that USP19 regulates the stability of HRD1 and provide insight into the regulatory mechanism of the ERAD ubiquitin ligases.

Keywords: ERAD; deubiquitinating enzyme; endoplasmic reticulum; membrane protein; ubiquitin ligase.

Figure 1

USP19 interacts with HRD1. The 293T cells were transfected with HRD1-FLAG (A) or FLAG-Nixin (B) along with either a pcDNA3 vector (lanes 1 and 3) or Myc-USP19 (lanes 2 and 4). The cells were lysed and then subjected to immunoprecipitation (IP) with an anti-Myc antibody. The lysates (10% of the input; lanes 1 and 2) and the immunoprecipitates (50% of the eluates; lanes 3 and 4) were analyzed by Western blotting with antibodies against FLAG (top panels), Myc (middle panels) and α-tubulin (an internal loading control; bottom panels).

Figure 2

USP19 deubiquitinates HRD1. (A) HRD1-FLAG was transfected into 293T cells along with either pcDNA3 (lanes 1, 4 and 7), Myc-USP19 (lanes 2, 5 and 8) or Myc-USP19^C548S (lanes 3, 6 and 9). After epoxomicin treatment for 7.5 h, the cells were lysed and then subjected to immunoprecipitation (IP) with an anti-FLAG antibody under denaturing conditions. The immunoprecipitates (50% of the eluates) were analyzed by Western blotting with antibodies against K48-linked ubiquitin (lanes 1–3) and FLAG (lanes 4–6). The lysates (50 μg of protein; lanes 7–9) were analyzed by Western blotting with antibodies against Myc (top panel), α-tubulin (middle panel) and NPT II (the Neo^r gene product as a transfection control; bottom panel); (B) 293T cells were transfected with control siRNA (lanes 1, 3 and 5) or USP19-specific siRNA duplexes (lanes 2, 4 and 6). Two days after transfection, the cells were further transfected with HRD1-FLAG. After epoxomicin treatment for 6 h, the cells were lysed and then subjected to immunoprecipitation (IP) with an anti-FLAG antibody under denaturing conditions. The immunoprecipitates (50% of the eluates) were analyzed by Western blotting with antibodies against K48-linked ubiquitin (lanes 1 and 2) and FLAG (lanes 3 and 4). The lysates (50 μg of protein; lanes 5 and 6) were analyzed by Western blotting with antibodies against USP19 (top panel), α-tubulin (middle panel) and NPT II (bottom panel).

Figure 3

Both overexpression and knockdown of USP19 affect endogenous HRD1 expression. (A) The 293T cells were transfected with either a pcDNA3 vector (lane 1), Myc-USP19 (lane 2) or Myc-USP19^C548S (lane 3). Whole cell lysates (20 μg of protein) were analyzed by Western blotting with antibodies against HRD1 (top panel), Myc (second top panel), α-tubulin (third top panel) and NPT II (bottom panel); (B) the 293T cells were transfected with control siRNA (lane 1) or USP19-specific siRNA duplexes (lanes 2 and 3). Three days after transfection, whole cell lysates (30 μg of protein) were analyzed by Western blotting with antibodies against HRD1 (top panel), USP19 (middle panel) and α-tubulin (bottom panel). The bar graphs show the relative expression of HRD1 normalized to α-tubulin expression from at least three independent experiments (mean ± SEM). **, *** statistically significant (one-way ANOVA, post-hoc test, ** p < 0.01 and *** p < 0.001, respectively).

Figure 4

USP19 stabilizes HRD1. HRD1-FLAG (A) and FLAG-Nixin (B) were transfected into 293T cells along with either a pcDNA3 vector, Myc-USP19 (wild type; WT) or Myc-USP19^C548S (C548S). The cells were pulse labeled with ³⁵S for 30 min and chased for the indicated periods of time. The cell lysates were immunoprecipitated with an anti-FLAG antibody and then analyzed by SDS-PAGE followed by autoradiography. The data were plotted as a percentage of the remaining proteins relative to time zero from at least three independent experiments (mean ± SEM). ** statistically significant (Student’s t-test, ** p < 0.01).