Secretory kinase Fam20C tunes endoplasmic reticulum redox state via phosphorylation of Ero1α

Abstract

Family with sequence similarity 20C (Fam20C), the physiological Golgi casein kinase, phosphorylates numerous secreted proteins that are involved in a wide variety of biological processes. However, the role of Fam20C in regulating proteins in the endoplasmic reticulum (ER) lumen is largely unknown. Here, we report that Fam20C interacts with various luminal proteins and that its depletion results in a more reduced ER lumen. We further show that ER oxidoreductin 1α (Ero1α), the pivotal sulfhydryl oxidase that catalyzes disulfide formation in the ER, is phosphorylated by Fam20C in the Golgi apparatus and retrograde-transported to the ER mediated by ERp44. The phosphorylation of Ser145 greatly enhances Ero1α oxidase activity and is critical for maintaining ER redox homeostasis and promoting oxidative protein folding. Notably, phosphorylation of Ero1α is induced under hypoxia, reductive stress, and secretion-demanding conditions such as mammalian lactation. Collectively, our findings open a door to uncover how oxidative protein folding is regulated by phosphorylation in the secretory pathway.

Keywords: ER redox; Ero1α; Fam20C; oxidative protein folding; phosphorylation.

Figure 1. FAM20C KO triggers a reduced ER

1. Coomassie Blue Staining of Flag immunoprecipitates from HeLa cells expressing Fam20C‐Flag for MS analysis.

2. Venn diagram of the 349 proteins interacting with Fam20C identified in the ER and Golgi apparatus based on DAVID GO term analysis.

3. KEGG pathway mapping of the most enriched biological processes in the Fam20C interactome in the ER and Golgi. The graph represents the top six statistically significant enriched gene clusters ordered by FDR (false discovery rate), with the number of genes in each cluster indicated beside the bars.

4. (Left) Fluorescence intensities of superfolded‐roGFP‐iE_ER in wild‐type (WT) and two clones (C3 and C5) of FAM20C KO HeLa cells at 525 nm were measured with excitation at 390 and 465 nm. The fluorescence ratio at 390/465 nm excitation was calculated. Data are shown as mean ± SEM from five (WT and C3) or three (C5) independent experiments performed in six technical replicates. **P < 0.01, ***P < 0.001 (one‐way ANOVA, the post hoc Tukey's HSD test). (Right) Protein immunoblotting of Concanavalin A (ConA) precipitates from the culture medium of WT and FAM20C KO HeLa cells. Ponceau staining is shown as a loading control.

5. (Left) Fluorescence intensities of superfolded‐roGFP‐iE_ER in FAM20C KO HeLa cells expressing Fam20C WT or its inactive mutant D478A (DA) were measured as in (D). Data are shown as mean ± SEM from four independent experiments performed in six technical replicates. **P < 0.01 (one‐way ANOVA, the post hoc Tukey's HSD test). (Right) Protein immunoblotting of FAM20C KO HeLa cells expressing Fam20C WT or DA.

6. Schematic representation of the pivotal pathway constituted by Ero1α and PDI for oxidative protein folding in the mammalian ER.

7. Co‐immunoprecipitation of endogenous Ero1α and Flag‐tagged Fam20C in HeLa cells.

Source data are available online for this figure.

Figure EV1. Generation of FAM20C KO HeLa cells using CRISPR/Cas9 genome editing

1. Schematic representation of the base pairing between the guide RNA (sgRNA) and the targeting locus of exon 1 in the human FAM20C gene. The sequences of the mutated alleles in FAM20C clone 3 (C3) and clone 5 (C5) and representative chromatograms depicting the insertions/deletions (INDELs) are shown. The INDELs were predicted to cause frameshift mutations producing inactive copies of the protein. SP, signal peptide.

2. Protein immunoblotting of Concanavalin A‐Sepharose (ConA) precipitates from the culture medium of WT and FAM20C KO HeLa cells. Ponceau staining was used as a loading control.

3. FAM20C KO shows little effect on unfolded protein response signaling. Protein immunoblotting of cell extracts from WT and FAM20C KO HeLa cells treated with or without 5 μM Tg for 6 h.

Figure EV2. Redox response ability of the superfolded‐roGFP‐iE_ER in HeLa cells

1. Schematic representation of superfolded‐roGFP‐iE_ER exhibiting different fluorescence characteristics under oxidizing (blue) or reducing (green) condition.

2. Localization of superfolded‐roGFP‐iE_ER in HeLa cells analyzed by immunofluorescence. Scale bar = 20 μm.

3. Fluorescence excitation spectrum of superfolded‐roGFP‐iE_ER in HeLa cells untreated or treated with 1 mM diamide or 1 mM DTT.

4. The ratios of fluorescence intensities at 525 nm with excitation at 390 and 465 nm were calculated from (C).

Figure EV3. Fam20C is the kinase catalyzing Ero1α phosphorylation

1. Coomassie Blue Staining (Left) and protein immunoblotting (Right) of HA immunoprecipitates from HeLa cells expressing Ero1α‐HA. h.c.: heavy chain, l.c.: light chain.

2. Non‐phosphopeptide (NP‐Pep) and phosphopeptide (P‐Pep) used for generating non‐phospho‐antibody (NP‐Ab) and phospho‐antibody (P‐Ab). 5 ng NP‐Pep and P‐Pep were spotted on membranes separately, followed by dot blot analysis with NP‐Ab and P‐Ab, respectively.

3. Protein immunoblotting of ConA precipitates from the conditioned medium of HeLa cells transfected with Ero1α‐myc and/or shRNA targeting FAM20C. The arrow indicates an unspecific background band.

4. Co‐immunoprecipitation of Ero1α and Fam20C in HeLa cells expressing Fam20C‐Flag WT/DA and/or Ero1α‐myc WT/S145A.

Figure 2. Ero1α can be phosphorylated at Ser145

1. A, B

   Representative MS/MS fragmentation spectra of tryptic phosphorylated (A) and non‐phosphorylated (B) peptides (Ero1α Leu137–Lys150) depicting Ero1α Ser145 phosphorylation enriched from Ero1α‐overexpressed HeLa cells.

2. C

   Schematic representation of human Ero1α active and inactive forms depicting signal peptide (SP), flexible loop (lilac), and cofactor FAD. The cysteines are shown as circles of white, yellow (outer active site), orange (inner active site), or green (regulatory cysteines) with amino acid numbering, disulfides as lines of blue (structural), black (active site), or green (regulatory), and Ser145 as a red square.

3. D

   Amino acid sequence alignments of Ero1α homologues in several species by BLAST. Residue positions are indicated by numbers counted from the N‐terminus. Human Ero1α Ser145 and its counterparts are shown in red.

4. E

   Detection of phosphorylated Ero1α (p‐Ero1α) in HeLa cells overexpressing HA‐tagged Ero1α WT or S145A (Left), or in ConA precipitates from the conditioned medium (Right). h.c.: heavy chain.

5. F

   Protein immunoblotting of ConA precipitates treated with λ‐phosphatase (λ‐PP) from the conditioned medium of HeLa cells overexpressing Ero1α.

Source data are available online for this figure.

Figure 3. Ero1α phosphorylation is catalyzed by Fam20C

1. A

   Time‐dependent incorporation of phosphate group into Ero1α catalyzed by recombinant Fam20C WT or DA mutant. Reaction products were analyzed by Coomassie Blue staining and p‐Ero1α immunoblotting.

2. B

   Detection of endogenous p‐Ero1α in HeLa and HepG2 cells overexpressing Fam20C.

3. C

   Protein immunoblotting of ConA precipitates from the conditioned medium of WT or FAM20C KO HeLa cells transfected with or without Ero1α‐myc.

4. D

   Protein immunoblotting of cell extracts (Left) and ConA precipitates from the conditioned medium (Right) of HeLa cells co‐expressing Ero1α‐myc and/or Fam20C‐Flag WT or DA.

5. E, F

   Immunofluorescence analysis of p‐Ero1α in HepG2 cells co‐expressing Ero1α‐HA and Fam20C‐Flag WT or DA. Scale bars = 10 μm.

Source data are available online for this figure.

Figure 4. Ero1α is phosphorylated by Fam20C in the Golgi and retained in the ER by ERp44

1. Protein immunoblotting of cell extracts and ConA precipitates from the conditioned medium of HeLa cells transfected with Ero1α (1) or Fam20C (2) alone, or co‐transfected with Ero1α and Fam20C (4), or co‐cultured cells expressing Ero1α and Fam20C separately (3).

2. Immunofluorescence analysis showing the subcellular localization of p‐Ero1α in HepG2 cells co‐expressing Ero1α and Fam20C. PDI and GM130 were used as ER and Golgi markers, respectively. Scale bars = 10 μm.

3. Protein immunoblotting of cell extracts and ConA precipitates from the conditioned medium of HeLa cells overexpressing Ero1α‐HA or Ero1α‐HA‐KDEL.

4. Co‐immunoprecipitation of ERp44 and Ero1α/p‐Ero1α (Left) and protein immunoblotting of ConA precipitates from the conditioned medium (Right) in HeLa cells expressing Ero1α‐myc alone or with HA‐ERp44.

5. Protein immunoblotting of cell extracts and ConA precipitates from the conditioned medium of HeLa cells transfected with Ero1α‐HA and scramble siRNA or siRNA targeting ERP44.

Source data are available online for this figure.

Figure EV4. The ER retention of p‐Ero1α is mediated by ERp44

1. Subcellular fractionation of HeLa cells. The postnuclear supernatant (PNS) of HeLa cells expressing Ero1α‐HA and Fam20C‐Flag was separated on a 30% Percoll gradient and the fractions were collected and subjected to protein immunoblotting. Calnexin, ER marker; GM130, Golgi marker.

2. Co‐immunoprecipitation of Ero1α and endogenous ERp44 in HeLa cells expressing Ero1α‐HA WT/S145A/S145E.

3. Protein immunoblotting of cell extracts and ConA precipitates from the conditioned medium of HeLa cells overexpressing Ero1α‐myc WT/S145A/S145E and increasing amounts of HA‐ERp44.

Figure 5. Phosphorylation of Ero1α increases its sulfhydryl oxidase activity in vitro

1. A

   Ribbon representation of the active form of human Ero1α structure (PDB: 3AHQ). Outer active site (yellow ball), inner active site (orange ball), cofactor FAD (orange stick), and Ser145 (red ball) are indicated. The outer active site‐containing loop is shown in lilac and the missing region by the dashed line.

2. B

   Schematic representation of the electron transfer pathway between the active sites of Ero1α and its physiological substrate PDI/GSH or small molecule reducing agent DTT.

3. C–F

   Oxygen consumption catalyzed by Ero1α phosphorylation mimics (C, D) or Fam20C‐treated Ero1α (E, F), on a background of Ero1α WT or hyperactive C104A/C131A, was monitored in the presence of DTT (C, E) or PDI and GSH (D, F). The relative oxidase activity of Ero1α was calculated from the slope of the linear phase of oxygen decrease. Data are shown as mean ± SEM from three independent experiments. **P < 0.01, ***P < 0.001 (two‐tailed, Student's t‐test).

Figure EV5. Biophysical and biochemical characterization of Ero1α phosphorylation mimics

1. Oxygen consumption catalyzed by Ero1α WT, inactive Ero1α C94A/C99A, and corresponding phosphorylation mimics was monitored in the presence of PDI and GSH.

2. The redox states of recombinant Ero1α WT, hyperactive Ero1α C104A/C131A, and corresponding phosphorylation mimics were determined under reducing and non‐reducing conditions. The active form (OX1), inactive form (OX2), and fully reduced form (Red) are indicated.

3. Recombinant oxidized Ero1α C99A/C104A/C166A and Ero1α C99A/C104A/C166A/S145E were mixed with reduced PDI, and aliquots were taken at indicated times for analysis by non‐reducing SDS–PAGE and Ero1α blotting.

4. Far UV circular dichroism spectrum of recombinant Ero1α WT and S145E.

5. Intrinsic fluorescence spectrum of recombinant Ero1α WT and S145E.

6. Ribbon representation of non‐phosphorylated Ero1α (Left) and Ser145‐phosphorylated Ero1α (Right) based on the structure of active human Ero1α (PDB: 3AHQ). The outer active site‐containing loop is shown in lilac, and the missing region in dashed line. Outer active site (yellow ball), inner active site (orange ball), cofactor FAD (orange stick), and the side chains of Ser145 and p‐Ser145 (stick) are indicated. The distance between O atom of Ser145 side chain and N atom of Thr148 main chain is shown.

Figure EV6. Generation of ERO1A KO HeLa cells using CRISPR/Cas9 genome editing

1. Schematic representation of the base pairing between the sgRNA and the targeting locus of exon 1 in the human ERO1A gene. The sequences of the mutated alleles in ERO1A clone 10 (C10) and clone 12 (C12) and representative chromatograms depicting the INDELs are shown. The INDELs were predicted to cause frameshift mutations producing inactive copies or terminating translation of the protein. SP, signal peptide.

2. Protein immunoblotting of cell extracts from WT and ERO1A KO HeLa cells.

Figure 6. Phosphorylation of Ero1α Ser145 plays a critical role in ER redox homeostasis

1. (Left) The fluorescence intensities of superfolded‐roGFP‐iE_ER in WT and two clones (C10 and C12) of ERO1A KO HeLa cells were measured as in Fig 1D. (Right) Aliquots of cells in the left panel were analyzed by immunoblotting.

2. (Left) The fluorescence intensities of superfolded‐roGFP‐iE_ER in ERO1A KO HeLa cells expressing Ero1α‐myc WT or S145E were measured as in Fig 1D. (Right) Aliquots of cells in the left panel were analyzed by immunoblotting.

3. (Left) The fluorescence intensities of superfolded‐roGFP‐iE_ER in ERO1A KO HeLa cells expressing Ero1α‐myc WT or S145A and/or Fam20C‐Flag were measured as in Fig 1D. (Right) Aliquots of cells in the left panel were analyzed by immunoblotting.

Data information: All of the data are shown as mean ± SEM from four independent experiments performed in six technical replicates. **P < 0.01, ***P < 0.001 (one‐way ANOVA, the post hoc Tukey's HSD test).Source data are available online for this figure.

Figure 7. Phosphorylation of Ero1α promotes oxidative protein folding in cells

1. (Left) HeLa transfectants expressing myc‐tagged JcM with Ero1α‐HA C99A/C104A, WT, or S145E were pulsed with DTT, washed, and chased at indicated time points by non‐reducing myc blotting. The mobility of reduced JcM monomers (Red), oxidized monomers (Oxi), homodimers (Dim), and high‐molecular‐weight (HMW) species is indicated. (Right) Aliquots from cell lysates in the left panel were resolved in reducing conditions and analyzed by immunoblotting.

2. The fraction of reduced JcM (Red/[Red + Oxi]) in (A) was quantified by densitometry. Data are shown as mean ± SEM from five independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001, between Ero1α WT and S145E (two‐tailed Student's t‐test).

3. (Left) JcM re‐oxidation in WT and ERO1A KO HeLa cells overexpressing Fam20C‐Flag WT or DA was monitored as in (A). (Right) Aliquots from cell lysates in the left were resolved in reducing conditions and analyzed by immunoblotting.

4. The fraction of reduced JcM in (C) was quantified by densitometry. Data are shown as mean ± SEM from four independent experiments. *P < 0.05, **P < 0.01, between Fam20C‐Flag WT and DA in WT HeLa cells (two‐tailed Student's t‐test).

Source data are available online for this figure.

Figure 8. p‐Ero1α is induced during mammalian lactation, hypoxia, and reductive stress

1. mRNA levels of several ER folding catalysts and Fam20 family members in the mouse mammary gland. The mammary glands of four virgin and four lactating (Lact) mice were isolated, and their mRNA levels were determined by RT–qPCR and normalized to the mean value of the virgin group. Data are represented as mean ± SEM performed in six technical replicates.

2. Protein immunoblotting of extracts from the whole mammary glands of virgin and lactating mice.

3. Quantification of relative p‐Ero1α/Ero1α ratio in (B). Data are shown as mean ± SEM of four groups. *P < 0.05 (two‐tailed, Student's t‐test).

4. Protein immunoblotting of HeLa cells expressing Fam20C‐Flag following exposure to hypoxia (0.1% oxygen) for the indicated times.

5. Protein immunoblotting of HeLa cells expressing Fam20C‐Flag treated with 5 μM thapsigargin (Tg), 5 μg/ml tunicamycin (Tm), 5 μg/ml Brefeldin A (BFA), or 200 μM DTT for 6 h. The arrow indicates an unglycosylated form of Fam20C.

6. Protein immunoblotting of HeLa cells transfected with or without Fam20C‐Flag treated with 200 μM DTT for the indicated times.

7. A working model for Fam20C tuning ER redox homeostasis and oxidative protein folding. Ero1α and PDI catalyze protein disulfide formation in the ER lumen. During mammalian lactation, hypoxia, and reductive stress, non‐phosphorylated Ero1α with basal activity is transported to the Golgi apparatus and phosphorylated by Fam20C at Ser145. Phosphorylated Ero1α is sequestrated by ERp44, a chaperone primarily localized in the ER‐Golgi intermediate compartment (ERGIC), and relocated to the ER mediated by KDEL receptor (KDELR). Phosphorylated Ero1α displays higher oxidase activity to promote disulfide bond formation and maintain ER redox homeostasis.

Source data are available online for this figure.