Fig 1.
Characterization of the novel ubiquitin ligase RNF183.
(A) Expression of RNF183 mRNA in various human and murine tissues. RNF183 mRNA expression in 22 human and 20 murine tissues was analyzed using RT-PCR. (B) Schematic diagrams of the predicted domains of transmembrane ubiquitin ligases. Prediction of domains for E3 ligases were made using SMART [6]. Blue box, transmembrane domain; red triangle, RING-finger domain; purple box, low complexity sequence. The number at right indicates the peptide length. (C) In vitro auto-ubiquitination of wild type and mutant RNF183. In vitro transcribed/translated V5-tagged RNF183 tagged was mixed and incubated with recombinant E1, E2, and HA-ubiquitin. The reaction mixture was immunoprecipitated with an anti-V5 antibody and subjected to Western blotting with anti-polyubiquitin (upper panel) and anti-V5 antibodies (lower panel). WT, wild type; ΔR, RING-finger domain deletion mutant; CS, Cys13-, and Cys16-to-Ser point mutations in the RING domain; IP, immunoprecipitation; Ub, ubiquitin. Asterisk indicates the immunoglobulin heavy chain. (D) In vitro auto-ubiquitination of RNF183 in the absence of each component. (E) Subcellular localization of RNF183. HeLa cells stably transfected with RNF183-V5 (red) were subjected to immunofluorescence staining with various antibodies for organelle makers (Calnexin, GM130, EEA1, Rab7 and LAMP1; green) and DAPI for nuclear staining (blue).
Fig 2.
Interaction and colocalization of RNF183 and Sec16A.
(A) Interaction of RNF183 and Sec16A. Cell lysates from HEK293 cells stably expressing V5-tagged RNF183 were immunoprecipitated using anti-V5 antibody, and the immune complexes were analyzed by Western blotting with anti-Sec16A (upper panel) or anti-V5 (lower panel) antibodies. (B) Colocalization of RNF183 with Sec16A. HeLa cells stably transfected with RNF183-V5 were subjected to immunofluorescence staining with anti-Sec16A (green) and DAPI (blue). (C) Effect of RNF183 knockdown on Sec16A protein. RNF183 expression in HEK293 cells stably transfected with RNF183-V5 was suppressed using siRNA. Endogenous Sec16A protein levels were detected using Western blotting with anti-Sec16A antibody. NC, negative control. (D) Schematic diagram of the predicted domains of Sec16A. (E) Interaction domain of Sec16A with RNF183. Lysates from HEK293 cells stably expressing RNF183 transiently transfected with GFP-tagged full-length Sec16A (Full) or its deletion mutant constructs were subjected to immunoprecipitation with anti-V5 antibody, followed by immunoblotting with anti-GFP antibody.
Table 1.
Proteomics analysis of the RNF183 interacting protein.
Fig 3.
Effects of Sec16 on RNF183 subcellular localization.
(A) Effect of Sec16A downregulation on RNF183 subcellular localization. HeLa cells stably expressing RNF183-V5 were transfected with NC (1st, 3rd, 5th panels) or Sec16A (2nd, 4th, 6th panels) siRNAs. At 48 h after transfection, cells were subjected to immunofluorescence staining with anti-V5 (green) and anti-calnexin, GM130, or LAMP1 (red) antibodies, and DAPI (blue). (B) Effect of proteasome inhibition on RNF183 subcellular localization. HeLa cells stably expressing RNF183-V5 were transfected with NC (top panels) or Sec16A (middle and bottom panels) siRNAs. At 36 h after transfection, cells were incubated with (bottom panels) or without (top and middle panels) 10 μM MG132 for 12 h.
Fig 4.
Effects of Sec16 on RNF183 protein stability.
(A) Effect of Sec16A downregulation on RNF183 protein stability. HeLa cells stably expressing RNF183-V5 were transfected with NC (1st, 3rd, and 5th panels) or Sec16A (2nd, 4th and 6th panels) siRNA. At 44 h after transfection, cells were treated with 30 μg/ml cycloheximide (CHX) and 10 μM MG132 for the indicated periods. Total cell lysates were analyzed by Western blotting with an anti-V5 (1st and 2nd panels), Sec16A (3rd and 4th panels), and β-actin (5th and 6th panels) antibodies. (B) Quantitative curves of data from (A). RNF183 levels at each time point were plotted relative to the level at time 0 (n = 3). Asterisks represent significant differences (Student’s t test with Bonferroni correction, *p < 0.05; NC vs. Sec16A siRNA; #p < 0.05, ##p < 0.01; ##p < 0.001; Sec16A siRNA vs. Sec16A siRNA + MG132).
Fig 5.
Effects of Sec16 on other ubiquitin ligases.
(A) Interactions of RNF152 and HRD1 with Sec16A. Coimmunoprecipitation was performed in HEK293 cells engineered to stably express V5-tagged RNF183, V5-tagged RNF152, or myc-tagged HRD1. Cell lysates were immunoprecipitated with anti-V5 or anti-myc antibodies or normal mouse immunoglobulin G (IgG; negative control). Immune complexes were analyzed by Western blotting with an anti-Sec16A antibody (top panel) and anti-V5 (second panel) or anti-myc antibodies (third panel). (B, D) Effect of Sec16A downregulation on RNF152 and HRD1 protein stability. Stable RNF152-V5- or HRD1-myc-expressing HEK293 cells were transfected with NC or Sec16A siRNA. At 48 h after transfection, cells were subjected to a CHX assay. (C, E) Asterisks represent significant differences (n = 3; Student’s t test with Bonferroni correction, *p < 0.05, ***p < 0.001; NC vs. Sec16A siRNA).