Rfp2 is an unstable protein that becomes stabilized with proteasomal inhibition. (A) HEK293 cells were transfected with GST-tagged Rfp2, Dltet, and empty vector, and exogenous protein expression was detected with monoclonal antibodies against GST. (B) Steady state levels of transfected FLAG-tagged Rfp2 in HEK293 cells after treatment with 30 μM MG-132 for 4 h. Rfp2 protein was detected with FLAG antibodies. Asterisks indicate Rfp2 conjugated with single ubiquitin molecules (see below). Actin and tubulin were used as loading controls as indicated. Numbers indicate protein size in kDa.

Specificity of a novel polyclonal Rfp2 antibody. (A) To monitor the specificity of the Rfp2 antibody, plasmids expressing FLAG-tagged Rfp2 were cotransfected with siRNA oligos targeting RFP2 and DLTET, respectively. Blots were developed using Rfp2-specific polyclonal antibodies to detect exogenous Rfp2 protein. (B) Extracts of HEK293 cells treated with RFP2 siRNA oligos, and siRNA oligos targeting green fluorescent protein (GFP) were analyzed for endogenous Rfp2 expression by Western blotting with Rfp2 antibody. Numbers indicate protein size in kDa. Detection of tubulin was used as loading control.

Rfp2 is monoubiquitinated in cells. (A) Analysis of endogenous protein levels of Rfp2 in a panel of human cell lines. Protein lysates were prepared from neuroblastoma (SHSY5Y), neuroepithelioma (SK-N-MCIXC), Burkitt's lymphoma (Daudi), acute lymphoblastic leukemia (RS-4), keratinocyte (Rhek-1), and human diploid fibroblast (HDF) cells and subjected to Western blot analysis with Rfp2 antibodies. Nonubiquitinated Rfp2 is marked with a solid arrowhead, and asterisks indicate Rfp2 conjugated with single ubiquitin molecules. Tubulin was used as a loading control. (B) Identification of Rfp2 bands with higher molecular weight as ubiquitinated forms of the protein. GST-tagged Rfp2 and an empty vector were expressed in HEK293 cells, precipitated with Rfp2 antibodies, separated by SDS-PAGE, and visualized with Coomassie. Each Rfp2 band was excised, subjected to in-gel tryptic digestion, and analyzed by mass spectrometry. The bands analyzed are marked with numbers (1–3) and solid arrowheads. High-molecular-mass Rfp2 bands are marked with asterisks (monoubiquitinated Rfp2). Boxed text summarizes the mass spectrometric data and indicates the number of peptides identified and corresponding sequence coverage of the isolated nonubiquitinated (band 1) and monoubiquiti-nated Rfp2 protein bands (bands 2 and 3). (C) GST-Rfp2 was overexpressed together with HA-tagged ubiquitin in HEK293 cells and precipitated with glutathione beads. This was followed by Western blot analysis with HA antibodies to detect ubiquitinated Rfp2 (left) and Rfp2 antibodies to detect both unconjugated and ubiquitinated Rfp2 (panel). Molecular size markers are shown on either side in kDa.

Rfp2 is polyubiquitinated in cells in a RING-dependent manner. (A) Polyubiquitination of Rfp2. Left panel, cotransfection of GST-Rfp2 and HA-ubiquitin into HEK293 cells was followed by cell lysis under denaturing conditions to disrupt noncovalent interactions. Rfp2 was precipitated using glutathione beads, and Western blot analysis with HA antibodies was used to detect high-molecular-weight smears indicative of ubiquitinated Rfp2 conjugates. In cells treated with MG-132 (30 μM) ubiquitinated Rfp2 conjugates accumulate. Right panel, Western blot of whole cell lysate using 5% of the input used for glutathione precipitation. Rfp2 antibodies detect both unubiquitinated and polyubiquitinated forms of Rfp2. Nonubiquitinated Rfp2 is marked with a solid arrowhead, and asterisks indicate Rfp2 conjugated with single ubiquitin molecules. Numbers indicate protein size in kDa. (B) RING-dependent polyubiquitination of Rfp2. Top panel, HEK293 cells were transfected with indicated constructs and immunoprecipitated with Rfp2 antibodies. Where indicated MG-132 (30 μM) was added for 4 h before harvesting. Ubiquitinated proteins were visualized with HA antibodies. Polyubiqutinated smears (marked with brackets) are seen in samples transfected with full-length Rfp2 but not with the RING deletion mutant Rfp2-ΔRING. Solid arrowhead indicates immuno-globulin heavy chain from the immunoprecipitation. Bottom panel, Western blot of whole cell lysate using 10% of the input used for immunoprecipitation showing stabilization of the Rfp2-ΔRING mutant. Rfp2 and Rfp2-ΔRING are marked with solid arrowheads. Numbers indicate protein size in kDa. (C) Stabilization of the Rfp2[¹³] mutant. Left panel, Western blot analysis of HEK293 cells ectopically expressing wild-type Rfp2 or Rfp2[¹³] with Rfp2 antibodies showing stabilization of the mutant protein. Actin was used as a loading control. Right panel, steady state levels of transfected GST-tagged Rfp2 and Rfp2[¹³] in HEK293 cells at various indicated time points after cycloheximide treatment in the presence or absence of MG-132. Western blot with GST antibodies shows stabilization of the Rfp2 point mutant irrespective of proteasomal inhibition. Tubulin was used as a loading control.

RING-dependent E3 ligase activity of Rfp2 in vitro. (A) In vitro ubiquitination assay with UbcH5b or UbcH5b [C⁸⁵A], together with GST-tagged Rfp2, Dltet or empty vector, respectively. GST-tagged proteins, expressed in HEK293 cells, were precipitated with glutathione beads, extensively washed, and incubated together with E1, the E2 enzyme UbcH5b, biotinylated ubiquitin, and ATP (top panel). Polyubiquitination, as detected by Western blot with biotin antibodies, is exclusively observed in the presence of catalytically active UbcH5b, and Rfp2 (top panel). Polyubiquitinated proteins are marked with a bracket. The indicated band appearing at about 135 kDa represents ubiquitinated E1. Bands below Rfp2 (indicated with a solid arrowhead) represent proteolytic fragments of Rfp2 as well as ubiquitinated E2 conjugates. (B) In vitro reaction with wild-type Rfp2 and Rfp2[C¹³A] showing extensive polyubiquitination with Rfp2, but not with Rfp2[C¹³A] (top panel). Lower panels, Western blot with GST antibodies showing amount of GST-tagged product in each lane. Numbers indicate protein size in kDa.

Rfp2 is a membrane-bound protein. (A) Schematic representation of selected RBCC proteins showing the varied C-terminal ends. (B) Top, U2OS cells were transfected with wild-type Rfp2 and with Rfp2-ΔTM, respectively. Forty-eight hours after transfection, cells were fixed in 4% formaldehyde, permeabilized with 0.2% Triton X-100 in PBS, and stained with Rfp2 antibodies. Primary antibodies were visualized with FITC-conjugated anti-rabbit IgG, and F-actin fibers were visualized with Texas Red–conjugated phalloidin. DAPI was used to stain the nucleus. Bottom panel, HEK293 cell lysates transfected with Rfp2 and Rfp2-ΔTM were fractionated as described in Material and Methods, and fractions were analyzed by Western blotting for Rfp2 and a set of indicated marker proteins. Wild-type Rfp2 is localized to the membranous and nuclear fraction, whereas Rfp2-ΔTM is relocalized to the cytosol. Schematic figures represent the Rfp2 variants used in the transfections. (C) Subcellular fractionation of HEK293 followed by Western blotting for endogenous Rfp2 protein confirms the subcellular localization observed with ectopically expressed protein.

Rfp2 colocalizes with the ER. Top panel, U2OS cells were cotransfected with Rfp2 and Myc-mmUbc6, processed as described in Material and Methods, and costained with Rfp2 and Myc antibodies. Rfp2 is visualized with FITC-conjugated anti-rabbit IgG, whereas mmUbc6 is visualized with Texas Red–conjugated anti-mouse IgG. DAPI was used to stain nuclei. Merged pictures (far right) show considerable overlap between Rfp2 and mmUbc6 staining. Bottom panel, immunofluorescence staining for endogenous Rfp2 in HEK293 cells using Rfp2 antibodies. Texas Red–conjugated phalloidin visualizes F-actin fibers.

Rfp2[C¹³A] stabilizes the ERAD substrate CD3-δ. (A) Cycloheximide chase analysis of CD3-δ turnover. HEK293 cells were transfected with CD3-δ-YFP together with the indicated GST constructs. Twenty-four hours after transfection cells were subjected to cycloheximide treatment and harvested at indicated time points. CD3-δ abundance was monitored by Western blot analysis using GFP antibodies (top panel). The GFP antibody recognizes the YFP tag fused to CD3-δ. Cells expressing Rfp2[C¹³A] exhibit marked stabilization of CD3-δ, compared with control cells. Middle panel, Western blot with GST antibodies showing amount of GST-tagged product in each lane. Tubulin was used as a loading control (bottom panel). (B) Colocalization of Rfp2 and CD3-δ. U2OS cells were cotransfected with Rfp2 and CD3-δ-YFP, processed as described in Material and Methods, and stained with Rfp2 antibodies. Rfp2 is visualized with Alexa Fluor 594–conjugated anti-rabbit IgG. DAPI was used to stain nuclei. Merged pictures (far right) show considerable overlap between Rfp2 staining and CD3-δ-YFP.

Rfp2 degrades the ERAD substrate CD3-δ. (A) Metabolic pulse-chase analysis of CD3-δ turnover showing stabilization of CD3-δ in the presence of Rfp2[C¹³A]. HEK293 cells transfected with indicated constructs were labeled with [³⁵S] cell labeling mix and chased in complete medium for the indicated times. CD3-δ turnover was monitored by autoradiography (top panel). (B) Rfp2[C¹³A] does not affect Ub-R-YFP stability. Autoradiography after metabolic pulse-chase analysis of Ub-R shows no stabilization of substrate in the presence of Rfp2[C¹³A] (top panel). Bottom panels, Western blot of whole cell lysate using 5% of the input used for immunoprecipitation showing amount of GST-tagged product in each lane. Numbers indicate protein size in kDa. Asterisk indicates an unspecific band. (C) Knockdown of Rfp2 expression stabilizes CD3-δ. HEK293 cells were transfected with CD3-δ-YFP together with two different Rfp2 siRNA oligos or scrambled control oligos, respectively, and CD3-δ turnover was analyzed by metabolic labeling (left panels). Right panel, Western blot on 5% input controls showing effective silencing of Rfp2.

Rfp2 coimmunoprecipates with several ER-resident proteins. (A) MS analysis of Rfp2 protein complexes. Rfp2 immunoprecipitations were performed as described in Material and Methods, separated on SDS-PAGE gels, and stained with Coomassie. In parallel, a set of immunoprecipitations using irrelevant antibodies were used as controls. Entire lanes were cut into 10-kDa pieces and were analyzed by LC-MS/MS. Peptides unique for Rfp2 immunoprecipitates were regarded as putative Rfp2 interacting proteins. Boxed text lists sequences of peptides from ER-resident proteins exclusively found in Rfp2 immunoprecipitates. IPI accession numbers for the corresponding proteins are included. Numbers indicate protein size in kDa. (B) Rfp2 interacts with VCP. HEK293 were transfected with ECFP-VCP together with indicated GST constructs. Lysates were prepared in NP40 buffer and incubated with glutathione Sepharose beads. After extensive washing, proteins bound to the resin were extracted and subjected to Western blot analysis with antibodies against the indicated proteins. The GFP antibody recognizes the ECFP tag fused to VCP. A VCP band can be observed in the lane containing transfected wild-type Rfp2 protein but not in lanes containing indicated Rfp2 deletion mutants (top panel). Middle panel, Western blot with GST antibodies showing amount of GST-tagged product in each lane. Schematic figures represent the Rfp2 variants used in the transfections. Lower panel, Western blot of whole cell lysate using 5% of the input used for glutathione precipitation showing equal amounts of transfected ECFP-VCP. (C) VCP interacts with endogenous Rfp2 protein. ECFP-VCP overexpressing cells were subjected to immunoprecipitaion with Rfp2 antibodies, and VCP was detected with Western blotting using GFP antibodies. Extracts (5% of input) were immunoblotted with GFP and Rfp2 antibodies to confirm expression of VCP and Rfp2, respectively.