(A) 293 cells were subfractionated by differential centrifugation to get mitochondrial fraction (Heavy Membrane, HM) and cytosolic/Light Membrane(LM) fractions. Equal protein amounts (40 µg) of the whole cell extract (cell), cytosolic and LM fraction (cytosol), and HM fraction (Mitochondira) were blotted with anti-RNF185 pAb to detect endogenous RNF185. The mitochondrial protein VDAC and cytosolic protein β-actin served as positive and negative controls respectively for determining mitochondria localized proteins. (B) Mitochondria fraction was treated with or without 20µg/ml proteinase K for 20 min on ice, and samples were collected for western blot analysis to detect endogenous RNF185. Mfn1, OPA1 and Tim23 were used as markers for MOM, MIM and intermembrane space proteins respectively. (C) Intact mitochondria isolated from 293 cells expressing Flag-RNF185-Myc were treated with 20 µg/ml proteinase K for the indicated time points, and were subjected to western blot analysis with the indicated antibodies. Tom20, cytochrome c and Tim23 were used as markers for MOM, MIM and intermembrane space proteins respectively. (D)Topological structure model for Flag-RNF185-Myc at mitochondrial outer membrane. MOM, mitochondrial outer membrane; MIM, mitochondrial inner membrane.

(A) – (C) Over-expression of RNF185 promoted the formation of autophagolysosomes. GFP-LC3 and RFP-CD63 colocalized well after the expression of RNF185 (pCI-RNF185), as detected by confocal microscope at 24 h post transfection (A). Lysosome accumulation was assayed by LysoTracker Red staining. At 24 h post transfection of empty vector or GFP-RNF185 expressing construct, GFP positive HeLa cells were gated to check the staining of LysoTracker Red by analyzing histogram plot (B) and mean fluorescence intensity (MFI) (C). (D) HeLa cells were transiently transfected with RFP tagged RNF185 (RFP-RNF185) or RFP vector alone, and 24 h later cells were collected for staining by fluorescent probe DCFH-DA to detect the ROS level. The column graph depicts the mean fluorescence intensity of DCF-DA. (E) HeLa cells were transiently transfected with GFP tagged RNF185 (GFP-RNF185) or GFP vector alone, and at 24 h post transfection cells were subjected to Mitotracker Red staining. GFP positive cells (GFP+, upper panel) and cells with extremely high GFP intensity (GFP+++, lower panel) were gated to analyze the staining of MitoTracker Red. The column graphs depict the mean fluorescence intensity of MitoTracker Red. Numbers in gates represent percentages of gated cells. n = 3 for each group above. **, P<0.01; ***, P<0.001. White bar, 20 µm.

(A) Colocalization of RNF185 and BNIP1. Confocal microscopic analysis was taken at 24 h post transfection of GFP tagged BNIP1 and RFP tagged RNF185 in HeLa cells. (B) – (D) BNIP1 localized at mitochondria. Both exogenously expressed wild type BNIP1 (GFP-BNIP1-WT) and mutated BNIP1 with BH3 domain deletion (GFP-BNIP1-dBH3) colocalized well with mitochondria markers Mitotracker Red (B) and DsRed2-Mito (C). Endogenous BNIP1 overlapped well with Mitotracker Red (D). White bar, 20 µm. All the experimental procedures for confocal microscopic analysis were the same as in Figure 1.

Mitochondrial outer membrane E3 ubiquitin ligase RNF185 interacts with its substrate BNIP1, which also localizes at mitochondria. BNIP1 is polyubiquitinated by RNF185 through K63-based ubiquitin linkage, and recruits autophagy receptor p62, which can interact with both LC3 and ubiquitins. The accumulation of p62 and LC3 promotes the formation of autophagosome. Ub, ubiquitin; ATG12-ATG5-ATG16, the protein complex required for the expansion of the autophagosomal membrane.

(A) schematic presentation of GFP tagged full-length RNF185 and its mutants. RNF185-WT, wild type RNF185; RNF185-RM, RING domain mutated; RNF185-TM, two TM domains deleted; RNF185-TM1, the first TM domain mutated; RNF185-TM2, the second TM domain deleted. (B) RNF185's predicted TM domains mediated its localization to mitochondria. GFP tagged wild type or mutated RNF185 was transfected into HeLa cells and MitoTracker Red was used to stain mitochondria at 24 h post transfection. (C) GFP-RNF185 colocalized with DsRed2-Mito. Plasmid encoding GFP tagged wild type RNF185 (GFP-RNF185) and pDsRed2-Mito were co-transfected into HeLa cells and confocal microscopic analysis was taken at 24 h post transfection. (D) Endogenous RNF185 localized at mitochondria. HeLa cells were subjected to immunocytochemistry-based confocal microscopic analysis with Rabbit IgG or affinity chromatography purified polyclonal antibody (pAb) raised against RNF185. Alexa fluor 488-conjugated goat anti Rabbit IgG(H+L) (Green) served as the secondary antibody. Mitochondria were stained with MitoTracker Red (Red) and DNA was stained with Hoechst 33258 (Blue). White bar, 10 µm.

(A) Morphology for HeLa cells that were transiently transfected with GFP tagged RNF185 (GFP-RNF185) or GFP vector alone. Fluorescent microscopic analysis was taken at 24 h and 48 h post transfection. (B) Over-expression of RNF185 led to changes in LC3I to LC3II ratio. At 24 h post transfection of empty vector (pCI-neo) and RNF185-expressing vector (pCI-R185), 293T cells were collected for western blot with the indicated antobodies. For positive controls, 293T cells were treated with 10 µM rapamycin for 24 h or maintained in Hank's Buffered Salt Solution (HBSS) for 4 h to induce autophagy. As the vehicle for rapamycin, DMSO served as negative control. (C) Knocking down of RNF185 decreased the base level of LC3II. At 36h post transfection of siRNA oligos (siR-341 and siR-440), 293T cells were collected to detect LC3 level by western blot. Bands were detected with a longer exposure time to display the difference on LC3II level. (D) Over-expression of RNF185 induced the formation of autophagosome, as detected by the distribution of GFP-LC3 from ubiquitous localization to punctate form. Confocal microscopic analyses were taken at 24 h post co-transfection of the indicated constructs. (E) Quantitative analysis of GFP-LC3 vesicular distribution. Experiments were done as in Fig. S6. Data represent mean±SD of 3 independent experiments in which 100–200 RFP+ cells per condition were analyzed. ***, P<0.001. White bar, 20 µm.

(A) RNF185 pulled down BNIP1 and ATG5. 293T cells were co-transfected with expression constructs for 2HA tagged candidate gene and 3Flag tagged RNF185 or empty vector as control. At 24 h post transfection, cell lysates were immunoprecipitated (IP) with anti-Flag conjugated beads and immunoblotted (IB) with anti-Flag and anti-HA antibodies. Bcl-w represented one of the candidate genes that could not interact with RNF185. (B) BNIP1 or ATG5 co-immunoprecipitated with RNF185. 3Flag tagged BNIP1 or ATG5 or empty vector and 2HA tagged RNF185 were co-transfected into 293T cells and followed by the same experimental procedures as in (A). HRP-conjugated goat anti mouse/rabbit IgG Fc fragment antibodies were used as secondary antibodies for IB to avoid antibody light chain in (B) to (E). (C) Exogenously expressed RNF185 associated with endogenous BNIP1. 293T cells were transfected with 3Flag tagged RNF185 or empty vector, and cell lysates were subjected to IP and IB analysis with the indicated antibodies as in (A). (D) The binding of RNF185 to BNIP1 was dependent on its TM domains. RM, RING domain mutated; TM, transmembrane domains deleted. (E) RNF185 interacted with the coiled-coil domain of BNIP1. dCC, coiled-coil domain deleted; dBH3, BH3 domain deleted; dTM, transmembrane domain deleted. The arrows indicate non-specific bands. The bands appeared in whole lysates but disappeared after IP are labeled by boxes with solid lines and dotted lines respectively. IP and IB were performed by the same experimental procedures as in (A). β-tubulin was detected as the loading control for all the western blots.

(A) – (B) The self-ubiquitination of RNF185 was dependent on its RING domain and TM domains. Empty vector, or plasmid encoding Flag tagged wild type RNF185 or mutant forms of RNF185 was transfected individually (A) or cotransfected with Myc tagged ubiquitin (B) into 293T cells. Proteins were prepared 24 h after transfection and subjected to IP, followed by IB analysis. Ub, ubiquitin. (C) – (D) RNF185 polyubiquitinated BNIP1 through K63-based ubiquitin linkage. BNIP1 was polyubiquitinated by RNF185 (C). 3Flag tagged BNIP1 and 2HA tagged RNF185 were cotransfected with Myc tagged ubiquitin or its variants (K29R, K48R, K63R) into 293T cells, and the polyubiquitination of BNIP1 was detected by IP and IB analysis (D). (E) BNIP1 associated with p62. 293T cells were cotransfected with the expression constructs for Myc-Ub, 3Flag-BNIP1 and RNF185-2HA or empty vectors as controls. Cell lysates were subjected to IP and IB analysis with the antibodies indicated in the figures. (F) Colocalization of endogenous BNIP1 and endogenous p62 in the cytosol. HeLa cells were stained with anti-BNIP1 rabbit polyclonal antibody and anti-p62 mouse monoclonal antibody. Alexa Flour 488-conjugated goat anti-rabbit IgG(H+L) antibody (Green) and TRITC-conjugated goat anti-mouse IgG antibody (Red) were used as secondary antibodies for detection of endogenous BNIP1 and p62 respectively. White bar, 10 µm.