Reticulophagy receptor FAM134C restrains BMP receptor signaling

Abstract

FAM134/RETREG family members are ER-phagy receptors that maintain cellular homeostasis by regulating endoplasmic reticulum turnover. However, possible non-ER-phagy functions of FAM134 proteins remain elusive. Here, we show that RETREG3/FAM134C functions as a selective autophagy receptor for the type I BMP receptor (BMPRIA/ALK3) and recruits BMPRIA into LC3-containing autophagosomes for subsequent degradation. FAM134C-induced degradation diminishes the availability of BMP receptors and thus the strength of BMP signaling. Inhibition of autophagy through chemical means or knockdown of key autophagy regulators, ATG5 or Beclin-1, prevents BMPR1A degradation. Additionally, disruption of the putative LC3-interacting region (LIR) motif in FAM134C completely abolishes its interaction with LC3, thereby impeding its ability to degrade BMPR1A. Moreover, FAM134C-deficient mice exhibit enhanced BMP responses in the intestines, which affects intestinal crypt regeneration. Our findings suggest that FAM134C acts as a specific receptor that controls BMP signaling through the autophagic degradation of the type I BMP receptor, independent of its canonical role in ER-phagy.

Keywords: Autophagy; Degradation; RETREG; Smad; TGF-β.

Figure 1. FAM134C promotes autophagic degradation of BMPR1A.

(A) Amino acids withdrawal attenuates BMP-induced Id1-luc reporter activity. A549 cells were transfected with Id1-luc reporter plasmids, after 24 h change medium to complete culture medium or nutrient depleted medium (without amino acids or FBS) with or without BMP2 (50 ng/ml) for 8 h. Relative luciferase activity was determined using a microplate-type luminometer with luciferase assay reagent. Statistical analysis by unpaired two-tailed Student’s t-test; *p < 0.05, **p < 0.01, ***p < 0.001; mean ± SD (n  =  3 independent experiments). After BMP stimulation, Medium vs -FBS p  =  8.39E-03, (B) mTOR inhibition attenuates BMP-induced Id1-luc reporter activity. A549 cells were transfected with Id1-luc reporter plasmids, after 24 h cells stimulated with Torin1, Rapamycin or MHY1486 and together with or without BMP2 for 8 h. Relative luciferase activity were determined using a microplate-type luminometer with luciferase assay reagent. Statistical analysis by unpaired two-tailed Student’s t-test; *p < 0.05, **p < 0.01, ***p < 0.001; mean ± SD (n  =  3 independent experiments). After BMP stimulation, DMSO vs Torin1 p  =  4.34E-04; DMSO vs Rapamycin p = 1.35E-04, (C) Amino acids withdrawal blocks BMP signaling. A549 cells were cultured with complete culture medium or amino acids withdrawal medium for 4 h, then treated with BMP2 (50 ng/ml) for 1 or 24 h. Expression of p-Smad1/5/8, Smad1, Id1, p-mTOR, mTOR, p-S6K, S6K, BMPR1A, and GAPDH were measured by Western blotting. (D) mTOR inhibition blocks BMP signaling. A549 cells were stimulated with Torin1, Rapamycin or MHY1485 and together with or without BMP2 for 1 or 24 h. Expression of p-Smad1/5/8, Smad1, p-mTOR, mTOR, p-S6K, S6K, BMPR1A, and GAPDH were measured by Western blot. (E) FAM134C degrades BMPR1A by autophagy. HEK293T cells were co-transfected with vectors encoding FAM134C-FLAG or Smurf1-FLAG and BMPR1A-HA, 24 h after transfection, cells were treated with MG132 (20 μM), CQ (10 μM), or 3-MA (200 μM) for 4 h, and expression of FAM134C, Smurf1 and BMPR1A were measured by Western blotting. (F) FAM134C promotes BMPR1A degradation and inhibits Smad1/5/8 phosphorylation. U2OS cells were stably expressing FAM134C-FLAG or GFP-FLAG as a control, pretreated with CQ (10 μM) or Rapamycin (100 nM) for 4 h, and then treated with BMP2 (50 ng/ml) for 1 h. Expression of BMPR1A, p-Smad1/5/8, Smad1, FAM134C, LC3, and GAPDH were measured by Western blotting. (G) FAM134C knockout stabilizes BMPR1A. U2OS or U2OS FAM134C knockout cells were treated with Rapamycin (100 nM) and BMP2 (50 ng/ml) for 1 or 24 h. Expression of BMPR1A, p-Smad1/5/8, Smad1, FAM134C, LC3, and GAPDH were measured by Western blotting. (H) ATG5 knockdown blocks FAM134C-dependent degradation of BMPR1A. ATG5 stable knockdown U2OS cells were transfected with FAM134C-FLAG or the FLAG vector. Twenty-four hours after transfection, cells were treated with BMP2 (50 ng/ml) for 0.5 or 1 h. Expression of BMPR1A, p-Smad1/5/8, Smad1, FAM134C, and GAPDH were measured by Western blotting. (I) FAM134C is colocalized with BMPR1A in lysosomes. U2OS cells were co-transfected with GFP-FAM134C and mCherry-BMPR1A. After treatment with CQ (10 μM) 4 h, cells were analyzed by immunofluorescence for FAM134C and BMPR1A, and lysosomes (labeled by an anti-LAMP1 antibody) using Zeiss LSM880. DAPI (blue fluorescence) was used to stain nuclei (scale bar, 10 μm). Percentage of FAM134C colocalized with BMPR1A in the lysosome was quantified by ImageJ. Statistical analysis by unpaired two-tailed Student’s t-test; *p < 0.05, **p < 0.01, ***p < 0.001; mean ± SD (n = 3 independent experiments; each with five technical replicates). Vector vs FAM134C p = 1.00E-02. Source data are available online for this figure.

Figure 2. FAM134C targets membrane-bound BMPR1A for degradation.

(A) Scheme diagram of the RUSH system. The RUSH system consists of two expression plasmids: KDEL-SA, an ER hook (KDEL) fused to streptavidin (SA); BMPR1A-SBP-GFP, BMPR1A linked to green fluorescent protein (GFP) through a streptavidin-binding peptide (SBP). (B) BMPR1A is localized on the plasma membrane. Immunofluorescence images show the trafficking of BMPR1A from the ER in the absence of biotin to the plasma membrane in the presence of biotin (50 μM, 1 h). Cells were analyzed using Zeiss LSM880. BMPR1A is marked with BMPR1A-RUSH (green fluorescence). ER tracker (red fluorescence) was used to stain the ER. DAPI (blue fluorescence) was used to stain nuclei (scale bar, 10 μm). (C) FAM134C degrades BMPR1A. HEK293T cells were co-transfected with empty vector (negative control) or FAM134C-FLAG and BMPR1A-RUSH. Twenty-four hours after transfection, cells were treated with or without Biotin 50 μM for 1 h, and expression of FAM134C and BMPR1A were detected by Western blotting. (D) FAM134C does not degrade newly synthesized BMPR1A. BONCAT assay in A549 cells stably expressing empty vector (negative control) and FAM134C-FLAG. The newly synthesized protein was precipitated by Streptavidin agarose beads. Endogenous BMPR1A expression was detected by Western blotting. (E) FAM134C overexpression causes BMPR1A degradation. U2OS cells stably expressing FAM134C-FLAG or empty vector control were treated with cycloheximide (CHX, 10 μM) for the indicated times. Expression of BMPR1A, FAM134C, and GAPDH were measured by Western blotting. (F) BMPR1A degradation is blocked in FAM134C knockout cells. FAM134C knockout cells or parental U2OS cells were treated with CHX (10 μM) for the indicated times. Expression of BMPR1A, FAM134C, and GAPDH were measured by Western blotting. (G) FAM134C reduces the levels of BMPR1A on the plasma membrane and endosomes, but not the ER. HCQ blocks BMPR1A degradation in the lysosome. Organelles were collected and analyzed for BMPR1A by Western blotting in FAM134C-expressing A549 cells. Isolation of the plasma membrane, endosome, ER, and lysosome is described in the Materials and Methods section. Expression of BMPR1A, FAM134C, E-cadherin (membrane marker), EEA1 (endosome marker), PDAI3 (ER marker), and LAMP1 (lysosome marker) were measured by Western blotting. (H) FAM134C degrades internalized BMPR1A. U2OS cells stably expressing FAM134C-FLAG or empty vector control were treated with Chlorpromazine (Chl, 50 nM), Nystatin (NY, 50 nM), Thapsigargin (TG, 500 μM), or ethyl-isopropyl amiloride (EIPA, 10 μM) for 15 h, sucrose (0.2 M) and Brefeldin A (BFA,10 μg/ml) for 2 h. Levels of BMPR1A and FAM134C-FLAG were measured by Western blotting. (I) Live cell image shows that FAM134C targets membrane-bound BMPR1A to lysosomes for degradation. FAM134C-KO U2OS cells stably express FAM134C-GFP and BMPR1A-mCherry. Lysotracker (blue fluorescence) was used to stain the lysosome. Cells were analyzed under Zeiss LSM880 (scale bar, 10 μm). Source data are available online for this figure.

Figure 3. FAM134C interacts with BMPR1A under physiological conditions.

(A) FAM134C interacts with BMPR1A at endogenous levels. Endogenous FAM134C was immunoprecipitated by FAM134C antibody in HEK293T, C2C12, A549, U2OS, and FAM134C Knockout U2OS cells, and BMPR1A was detected by Western blotting. (B) FAM134C specifically interacts with BMPR1A, but not BMPR2 or Smad5. HEK293T cells were co-transfected with vectors encoding FAM134C-FLAG and Smad5-HA, BMPR1A-HA, or BMPR2-HA, then FAM134C was immunoprecipitated by anti-FLAG antibody, and the protein Smad5, BMPR1A, or BMPR2 was detected by HA antibody. (C) FAM134C binds to the intercellular domain of BMPR1A. HEK293T cells were transfected with vectors encoding FAM134C-FLAG and BMPR1A-HA, BMPR1A(ICD)-HA, or BMPR1A(ECD)-HA, then FAM134C was immunoprecipitated by anti-FLAG antibody and BMPR1A or BMPR1A mutation was detected by HA antibody. (D) FAM134C interacts with BMPR1A in vitro. MYC-FAM134C, Smad5-HA, and BMPR1A-HA were purified using the TNT in vitro transcription/translation System. Purified Smad5-HA or BMPR1A-HA proteins were incubated with MYC-FAM134C, and then immunoprecipitation was performed using anti-HA antibodies. The IP products were detected by Western blotting with the indicated antibodies. (E) FAM134C interacts with BMPR1A in cells. In BiFC assays, A549 or U2OS cells were co-transfected with YC-tagged FAM134C and YN-tagged BMPR1A or YN-tagged vector as a negative control. Cells were analyzed using Zeiss LSM880. The green YFP fluorescence signal indicates the interaction part. DAPI (blue fluorescence) was used to stain nuclei (scale bar, 10 μm). (F) Endogenous FAM134C is colocalized with BMPR1A. GFP-tagged FAM134C and mCherry-tagged BMPR1A were expressed in FAM134C-KO U2OS cells (top row), or mCherry-tagged BMPR1A was expressed in A549 cells. These cells were visualized by immunofluorescence under Zeiss LSM880. DAPI (blue fluorescence) was used to stain nuclei (scale bar, 10 μm). (G, H) FAM134C is colocalized with BMPR1A in autophagolysosomes under starvation. FAM134C-KO U2OS cells were stably expressed FAM134C-GFP and BMPR1A-mCherry. Cells were starved for 1 h and then analyzed using Zeiss LSM880. Panel (G), Lysotracker (blue fluorescence) was used to stain lysosomes (scale bar, 10 μm), and the percentage of FAM134C and BMPR1A colocalized with lysosomes was quantified by ImageJ. Panel (H), endogenous LC3 (blue fluorescence) were stained by immunofluorescence (scale bar, 10 μm), and the percentage of FAM134C and BMPR1A colocalized with LC3 were quantified by ImageJ. Statistical analysis by unpaired two-tailed Student’s t-test; *p < 0.05, **p < 0.01, ***p < 0.001; mean ± SD. (n = 3 independent experiments; each with three technical replicates). 0 h vs 2 h p = 4.36E-08(G); 0 h vs 2 h p = 2.88E-07(H). Source data are available online for this figure.

Figure 4. FAM134C targets BMPR1A into autophagosomes through LC3.

(A) FAM134C binds to LC3. In BiFC assays, A549 cells were co-transfected with YC-FAM134C and YN-LC3 or YN-tagged vector as a negative control. Cells were analyzed using Zeiss LSM880. Green YFP fluorescence signal indicates the location of subcellular interaction. DAPI (blue fluorescence) was used to stain nuclei (scale bar, 10 μm). (B) FAM134C interacts with BMPR1A in the LC3-containing autophagosome. U2OS cells were co-transfected with LC3-mCherry with YC-FAM134C and YN-BMPR1A, starved for 2 h, and then analyzed using Zeiss LSM880. Green YFP fluorescence signal indicates the FAM134C-BMPR1A interaction. DAPI (blue fluorescence) was used to stain nuclei (scale bar, 10 μm), and the percentage of FAM134C and BMPR1A colocalized with LC3 were quantified by ImageJ. Statistical analysis by unpaired two-tailed Student’s t-test; *p < 0.05, **p < 0.01, ***p < 0.001; mean ± SD (n = 3 independent experiments; each with three technical replicates). 0 h vs 2 h p = 1.23E-07. (C) LC3 is associated with BMPR1A in the presence of FAM134C. U2OS cells were stably expressed with FAM134C-FLAG, treated with CQ (10 μM), and immunoprecipitated by anti-FLAG antibody. LC3-bound BMPR1A or FAM134C were analyzed by Western blotting using the indicated antibodies. (D) FAM134C LIR mutation fails to interact with LC3 and degrade BMPR1A. Top, schematic diagram of domain structures and mutation design of FAM134C. Bottom, HEK293T cells were co-transfected with vectors encoding BMPR1A-HA and FAM134C-FLAG, FAM134C-M-FLAG, or FAM134C-D-FLAG, then FAM134C was immunoprecipitated by anti-FLAG antibody and BMPR1A or LC3 was detected by Western blot. (E) FAM134C, but not its LIR mutants, are colocalized with LC3 after starvation. U2OS cells were co-transfected with GFP-LC3 and FAM134-FLAG, FAM134C-M-FLAG, or FAM134C-D-FLAG. Upon starvation buffer for 1 h, cells were analyzed by fluorescence microscopy using Zeiss LSM880. DAP (blue fluorescence) was used to stain nuclei (scale bar, 10 μm). Bottom, the percentage of FAM134C, FAM134C-M-FLAG, or FAM134C-D-FLAG colocalized with LC3 was quantified by ImageJ. Statistical analysis by unpaired two-tailed Student’s t-test; *p < 0.05, **p < 0.01, ***p < 0.001; mean ± SD (n = 3 independent experiments; each with five technical replicates). FAM134C vs FAM134C-M p = 1.19E-06, FAM134C vs FAM134C-D p = 2.07E-07. (F) FAM134C LIR mutants fail to degrade BMPR1A. FAM134C Knockout U2OS cells were transfected with FAM134C-FLAG, FAM134C-M-FLAG, or FAM134C-D-FLAG. Twenty-four hours after transfection, cells were treated with BMP2 (50 ng/ml) for 0.5 h. Levels of BMPR1A, p-Smad1/5/8, Smad1, FAM134C, and GAPDH were measured by Western blotting. Source data are available online for this figure.

Figure 5. FAM134C blocks BMP signaling.

(A) FAM134C inhibits BMP-induced Id1 and Id2 mRNA transcription. U2OS cells stably expressing FAM134C-FLAG or empty vector control) were treated by BMP (50 ng/ml) for 12 h, Id1 and Id2 mRNA were measured by qPCR. Statistical analysis by unpaired two-tailed Student’s t-test; *p < 0.05, **p < 0.01, ***p < 0.001; mean ± SD (n  =  3 independent experiments). In Id1 mRNA expression, treated with BMP, Vector vs FAM134C-Flag p = 2.20E-02. In Id2 mRNA expression, treated with BMP, Vector vs FAM134C-Flag p = 1.76E-04. (B) Knockout of FAM134C increases BMP-induced Id1 and Id2 mRNA transcription. WT or FAM134C-KO U2OS cells were treated by BMP (50 ng/ml) for 12 h, Id1 and Id2 mRNA were measured by qPCR. Statistical analysis by unpaired two-tailed Student’s t-test; *p < 0.05, **p < 0.01, ***p < 0.001; mean ± SD (n  =  3 independent experiments). In Id1 mRNA expression, treatment with BMP, KO control vs FAM134C-KO-1 p = 5.01E-03, KO control vs FAM134C-KO-2 p = 4.71E-03. In Id2 mRNA expression, treatment with BMP, KO control vs FAM134C-KO-1 p = 1.62E-04, KO control vs FAM134C-KO-2 p = 1.12E-04. (C) BMPR1A is increased in the small intestine of FAM134C−/− mice. Immunofluorescence staining of BMPR1A, p-Smad1/5/8, MUC2, and ChgA were done in the proximal jejunum of WT and KO mice at week 8. DAPI (blue fluorescence) was used to stain nuclei (scale bar, 50 μm). Images are representative of n = 6 mice per genotype. (D) FAM134C deficiency increases BMPR1A expression in mouse intestines. WB analysis of BMPR1A, FAM134C, and GAPDH in the colon of WT and FAM134C knockout mice (n = 5 mice per genotype). (E) Hematoxylin-eosin staining and immunohistochemical staining of Sox9 in the small intestines of WT and FAM134C knockout mice. Proximal jejunum sections were made at week 8 (scale bar, 100 μm). Images are representative of six mice per genotype. (F, G) FAM134C deficiency increases the level of BMPR1A in the mouse intestines during fasting. (F) Immunofluorescence staining of BMPR1A and p62, and hematoxylin-eosin staining were done in the small intestines of WT and FAM134C knockout mice with or without fasting. Proximal jejunum sections were harvested at week 8. DAPI (blue fluorescence) was used to stain nuclei (scale bar, 100 μm). (G) Analysis of BMPR1A expression in the small intestines of WT and FAM134C knockout mice with or without fasting (n = 5 mice per genotype) was done by Western Blotting. (H–J) Loss of FAM134C hampers crypt regeneration after exposure to ionizing radiation. Proximal jejunum sections were collected at different time points in WT and FAM134C knockout mice after 12 Gy abdominal X-ray radiation (n = 5 mice per genotype). (H) Haematoxylin-eosin staining, representative images with quantification of crypt number. (I) Representative immunohistochemical staining of Ki67 and its quantification of Ki67+ cells. (J) Representative immunohistochemical staining of Sox9 with quantification of Sox9+ cells. Statistical analysis by unpaired two-tailed Student’s t-test; *p < 0.05, **p < 0.01, ***p < 0.001; mean± SD. of n = 5 mice per genotype (scale bar, 50 μm). Crypts number in day 4 WT vs KO p = 6.72E-04; KI67⁺ cell/crypt in day 4 WT vs KO p = 3.31E-06; Sox9⁺ cell/crypt in day0 WT vs KO p = 4.00E-02, Sox9⁺ cell/crypt in day4 WT vs KO p = 3.53E-03. Source data are available online for this figure.

Figure 6. Block BMP signaling help budding organoid in FAM134c−/− mice.

(A) FAM134C deficiency attenuates organoid formation. Organoids were cultured in the medium containing 100 ng/ml of Noggin. Morphology of intestine organoids from WT and FAM134C knockout mice after 1 and 4 days are shown. Right, quantification of organoid budding percentage at 4 days (scale bar, 200 μm). Statistical analysis by unpaired two-tailed Student’s t-test; *p < 0.05, **p < 0.01, ***p < 0.001; mean ± SD (n = 5 mice per genotype; each with five technical replicates). WT vs KO p = 2.93E-26. (B) FAM134C deficiency causes the disappearance of Lgr5 and increased BMPR1A. Immunofluorescence was performed using organoid sections at day 4 from WT and FAM134C knockout mice (scale bar, 50 μm). DAPI (blue fluorescence) was used to stain nuclei. Images are representative of n = 3 mice per genotype. (C) Quantitative RT-PCR analysis of Id1 and Id2 in intestinal organoids at day 4. Statistical analysis by unpaired two-tailed Student’s t-test; *p < 0.05, **p < 0.01, ***p < 0.001; mean ± SD (n  =  3 independent experiments). Id1 expression WT vs KO p = 4.22E-02; Id2 expression WT vs KO p = 5.89E-03. (D) Quantitative RT-PCR analysis of Lgr5 and Olfm4 in intestinal organoids at day 4. Statistical analysis by unpaired two-tailed Student’s t-test; *p < 0.05, **p < 0.01, ***p < 0.001; mean ± SD (n  =  3 independent experiments). Lgr5 expression WT vs KO p = 6.46E-05; Id2 expression WT vs KO p = 6.95E-04. (E, F) Noggin counteracts FAM134C deficiency during organoid formation. Panel E, morphology of intestine organoids from WT and FAM134C knockout mice after treatment with noggin at the indicated concentration after 1 and 4 days (scale bar, 200 μm). (F) Quantification of organoid budding percentage at 4 days in the intestine organoids. Statistical analysis by unpaired two-tailed Student’s t-test; *p < 0.05, **p < 0.01, ***p < 0.001; mean ± SD (n = 3 mice per genotype; each with ten technical replicates). In 50 ng/ml WT vs KO p = 1.54E-45; In 100 ng/ml WT vs KO p = 4.10E-61; In 500 ng/ml WT vs KO p = 2.11E-46. (G, H) Quantitative RT-PCR analysis of Lgr5 (G) and Id1 (H) in the intestine organoids of WT and FAM134C knockout mice after treatment with Noggin at the indicated concentration on day 4. Statistical analysis by unpaired two-tailed Student’s t-test; *p < 0.05, **p < 0.01, ***p < 0.001; mean ± SD (n  =  3 independent experiments). For (G), In 50 ng/ml WT vs KO p = 6.03E-04; In 100 ng/ml WT vs KO p = 9.97E-05; In 500 ng/ml WT vs KO p = 4.46E-03; In 1000 ng/ml WT vs KO p = 4.19E-04. For (H), In 0 ng/ml WT vs KO p = 3.45E-04; In 50 g/ml WT vs KO p = 1.04E-03; In 100 ng/ml WT vs KO p = 1.64E-04. (I) A working model for the effect of FAM134C in BMP signaling. In cells, FAM134C interacts with BMPR1A and promotes its degradation through the autophagy-lysosome pathway. Source data are available online for this figure.

Figure EV1. FAM134C degrades BMPR1A by autophagy.

(A, B) Quantitative RT-PCR analysis of FAM134C and BMPR1A mRNAs in U2OS cells stably expressing FAM134C-FLAG or carrying an empty vector control. mean ± SD (n  =  3 independent experiments). (C) Expression of FAM134C and GAPDH were measured in wildtype (WT) or FAM134C-KO U2OS cells by Western blotting. (D) Quantitative RT-PCR analysis of BMPR1A mRNA in WT or FAM134C-KO U2OS cells. mean ± SD (n  =  3 independent experiments). (E) HEK293T cells were co-transfected with vectors encoding FAM134C-FLAG and ALK1-HA, ALK2-HA, ALK3-HA, ALK6-HA, or BMPR2-HA, then FAM134C was detected by anti-FLAG antibody and the protein ALK1, ALK2, ALK3, ALK6, or BMPR2 was detected by HA antibody. (F) Quantitative RT-PCR analysis of ATG5 mRNA in U2OS with stable knockdown of ATG5 or control. mean ± SD (n  =  3 independent experiments). (G) Quantitative RT-PCR analysis of BECN1 mRNA in U2OS with stable knockdown of BECN1 or control. mean ± SD (n  =  3 independent experiments). (H) BECN1 downregulation blocks the degradation of BMPR1A by FAM134C. U2OS cells with BECN1 stable knockdown were transfected with FAM134C-FLAG or the FLAG vector. Twenty-four hours after transfection, cells were treated with BMP2 (50 ng/ml) for 0.5 or 1 h. Levels of BMPR1A, p-Smad1/5/8, Smad1, FAM134C, and GAPDH were measured by Western blotting. Source data are available online for this figure.

Figure EV2. FAM134C targets membrane-bound BMPR1A for degradation.

(A) Schema of the BONCAT translation assay. (B, C) FAM134C does not degrade newly synthesized BMPR1A. HEK293T cells were co-transfected with FAM134C-FLAG and BMPR1A-HA and/or empty vector control for 24 h. In (C), transfected cells were further treated with cycloheximide (10 μM) or HCQ (50 nM) for 8 h. The newly synthesized protein was collected by Streptavidin agarose beads. BMPR1A expression was detected by Western blotting. (D) FAM134C overexpression causes BMPR1A degradation. HEK293T cells were co-transfected with FAM134C-FLAG and BMPR1A-HA, and treated with cycloheximide (CHX, 10 mM) for the indicated times. Expression of BMPR1A, FAM134C, and GAPDH were measured by Western blotting. (E) FAM134C reduces the levels of BMPR1A on the plasma membrane and endosomes, but not the ER. HCQ blocks BMPR1A degradation in the lysosome. Organelles were collected and analyzed for BMPR1A by Western blotting in HEK293T cells expressing FAM134C-FLAG and BMPR1A-HA. Isolation of the plasma membrane, endosome, ER, and lysosome is described in the Materials and Methods section. Expression of BMPR1A, FAM134C, E-cadherin (membrane marker), EEA1 (endosome marker), PDIA3 (ER marker), and LAMP1 (lysosome marker) were measured by Western blotting. (F) FAM134C degrades internalized BMPR1A. HEK293T cells were co-transfected with FAM134C-FLAG and BMPR1A-HA, and treated with Chlorpromazine (Chl, 50 nM), Nystatin (NY, 50 nM), Thapsigargin (TG, 500 mM), or ethyl-isopropyl amiloride (EIPA, 10 mM) for 15 h, sucrose (0.2 M) and Brefeldin A (BFA, 10 mg/ml) for 2 h. Levels of BMPR1A and FAM134C-FLAG were measured by Western Blotting. (G) Inhibitors used in (F) above. Source data are available online for this figure.

Figure EV3. FAM134C interacts with BMPR1A.

(A) ATG5 or BECN1 downregulation had no effect on the interaction between FAM134C and BMPR1A. ATG5 or BECN1 were stably knocked down in U2OS cells, and then FAM134C was immunoprecipitated by anti-FAM134C and anti-BMPR1A antibodies was detected by Western blotting. (B) BMPR1A interacts with FAM134C and to a lesser extent FAM134B, but not FAM134A. HEK293T cells were transfected with vectors encoding BMPR1A-HA and FAM134A-FLAG, FAM134B-FLAG, or FAM134C-FLAG, and then FAM134A/B/C were immunoprecipitated by anti-FLAG antibody and FAM134-bound BMPR1A was detected by HA antibody. (C) FAM134C is colocalized with BMPR1A. U2OS cells were co-transfected with RTN4-mCherry, LAMP1-mCherry, Clathrin-mCherry, or Rab7a-mCherry together with YC-tagged FAM134C and YN-tagged BMPR1A. Cells were treated with HCQ for 2 h and then analyzed using Zeiss LSM880. The green YFP fluorescence signal indicates the FAM134C-BMPR1A interaction. DAPI (blue fluorescence) was used to stain nuclei (scale bar, 5 μm). The percentage of the FAM134C-BMPR1A interaction colocalized with an organelle was quantified by ImageJ. Bar and error bars show the mean ± SD (n = 3 independent experiments; each with three technical replicates). Source data are available online for this figure.

Figure EV4. FAM134C targets BMPR1A into autophagosomes through LC3.

Live cell image shows that FAM134C targets membrane-bound BMPR1A to lysosomes for degradation. FAM134C-KO U2OS cells stably express FAM134C-GFP and BMPR1A-mCherry. Lysotracker (blue fluorescence) was used to stain the lysosome. Cells were analyzed under Zeiss LSM880 (scale bar, 1 μm). Source data are available online for this figure.

Figure EV5. Lack of FAM134C increases BMP signaling in mouse colons.

(A) FAM134C inhibits BMP responses. U2OS cells stably expressing FAM134C-FLAG or with FAM134C knockout or parental cells were treated with BMP (50 ng/ml) for 1 and 24 h. Levels of Id1, p-Smad1/5/8, Smad1, FAM134C, BMPR1A, and GAPDH were measured by Western blotting. (B) Hematoxylin-eosin staining and immunohistochemical staining of BMPR1A, p-Smad1/5/8 and Sox9 in colons of WT and FAM134C knockout mice (scale bar, 50 mm). Colon sections were collected at week 8. Images are representative of n = 6 mice per genotype. (C) Immunohistochemical staining of MUC2 and ChgA in colons of WT and FAM134C knockout mice. Colon sections were collected at week 8 (scale bar, 100 mm). Images are representative of n = 6 mice per genotype. (D) Quantitative RT-PCR analysis of the indicated genes in the colons of WT and FAM134C knockout mice. Statistical analysis by unpaired two-tailed Student’s t-test; *p < 0.05, **p < 0.01, ***p < 0.001; mean ± SD (n = 3 mice per genotype; each with two technical replicates). Id1 expression, WT vs KO p = 2.36E-02; Id2 expression, WT vs KO p = 2.87E-02. (E) Immunofluorescence staining of BMPR1A and p62, and Hematoxylin-eosin staining in small intestines of WT and FAM134C knockout mice with or without fasting. Proximal jejunum sections were made at week 8. DAPI (blue fluorescence) was used to stain nuclei (scale bar, 100 mm). Source data are available online for this figure.