Broadly conserved roles of TMEM131 family proteins in intracellular collagen assembly and secretory cargo trafficking

Abstract

Collagen is the most abundant protein in animals. Its dysregulation contributes to aging and many human disorders, including pathological tissue fibrosis in major organs. How premature collagen proteins in the endoplasmic reticulum (ER) assemble and route for secretion remains molecularly undefined. From an RNA interference screen, we identified an uncharacterized Caenorhabditis elegans gene tmem-131, deficiency of which impairs collagen production and activates ER stress response. We find that amino termini of human TMEM131 contain bacterial PapD chaperone-like domains, which recruit premature collagen monomers for proper assembly and secretion. Carboxy termini of TMEM131 interact with TRAPPC8, a component of the TRAPP tethering complex, to drive collagen cargo trafficking from ER to the Golgi. We provide evidence that previously undescribed roles of TMEM131 in collagen recruitment and secretion are evolutionarily conserved in C. elegans, Drosophila, and humans.

Fig. 1. Genome-wide RNAi screen identifies tmem-131 regulating ER stress response in C. elegans.

(A) Schematic of RNAi screen for regulators of asp-17p::GFP. For each 96-well plate, 10 L4-stage animals carrying asp-17p::GFP were placed in each well, triplicated for each gene RNAi treatment, and observed under stereoscopes when reaching adult stages. (B) Exemplar fluorescence and bright-field images for rrf-3; asp-17p::GFP with control, ostb-1, and tmem-131 RNAi. (C) Table listing ER proteostasis genes whose RNAi also suppresses rrf-3; asp-17p::GFP (n ≥ 20 for each group). (D) Schematic of Ce-tmem-131 LOF alleles and RT-PCR P1 and P2 primers for tmem-131 mRNA measurement. (E) Quantification of mRNA fold changes in tmem-131 and hsp-4::GFP expression levels in wild-type and tm6119 mutants. ***P < 0.001 (n ≥ 3 biological replicates). (F) Exemplar fluorescence and bright-field images showing rescue of hsp-4p::GFP induction in tmem-131(tm6119) mutants with transgenic expression of tmem-131p::tmem-131::gfp marked by pharyngeal myo-2p::mCherry. (G to I) Exemplar fluorescence and bright-field images for the UPR reporter hsp-4p::GFP with tmem-131 RNAi in wild-type (Wt) (G) and xbp-1 mutants (H) and (I) in tmem-131(tm6119) mutants with control, xbp-1, or ire-1 RNAi. Over 50 animals were observed, with three shown to indicate representative reporter expression. Scale bars, 100 μm.

Fig. 2. Ce-TMEM131 is essential for secretion of GFP-labeled collagen COL-19 and COL-101.

(A) Exemplar epifluorescence image of tmem-131p::GFP transcriptional reporter. Arrows indicate major tissues of reporter expression. (B) Exemplar confocal images of tmem-131p::tmem-131::GFP and ER transgenic reporter tmem-131p::ERSig::mCherry::KDEL showing colocalization (indicated by arrows) of both reporters with vesicular puncta patterns in the perinuclear areas of epithelial cells. (C) Exemplar epifluorescence image of col-19::GFP (i.e., col-19p::col-19::GFP) and tm6119; col-19::GFP. (D and E) Exemplar confocal fluorescence images with indicated phenotypic penetrance of col-19::GFP in wild-type (D) and tmem-131 mutant (E). (F) Quantification of COL-19::GFP fluorescence intensity (n ≥ 4 for each group) and endogenous col-19 mRNA levels in wild-type and tmem-131(tm6119) mutants. a.u., arbitrary units. (G and H) Exemplar confocal fluorescence images with the indicated phenotypic penetrance of col-19::GFP with xbp-1 RNAi in wild type (G) or tmem-131 mutants (H). ***P < 0.001 (n ≥ 3 biological replicates). n.s., no significant differences. (I) Exemplar SDS-PAGE and Western blot analysis of col-101::GFP and tm6119; col-101::GFP proteins from total animal lysates. IB, immunoblotting. (J and K) Exemplar confocal images with indicated phenotypic penetrance of col-101::GFP in wild-type (J) and tmem-131 mutants (K). Scale bars, 20 μm.

Fig. 3. PapD-L chaperone domains are essential for TMEM131 functions.

(A) Schematic of C. elegans TMEM131 domain organization with PapD-L domain in green, ER localization in blue, and cytosol localization in red as predicted by the TOPCONS program (http://topcons.net/). The biological hydrophobicity scale calculates the free energy (ΔG) of membrane insertion for a window of 21 amino acids (aa) centered on each position in the TMEM131 sequence. (B) Schematic of tmem-131 gene structure with the PapD-L deletion generated by CRISPR-Cas9 and RT-PCR P1 and P2 primers for tmem-131 mRNA measurement. (C and D) Exemplar fluorescence images for hsp-4p::GFP (C) and col-19p::col-19::GFP with the indicated phenotypic penetrance (D) in wild-type and PapD-L deletion mutants. Scale bars, 20 μm. (E) Quantification of the COL-19::GFP fluorescence intensity in wild-type, tmem-131(tm6119), and tmem-131(ΔPapD-L) mutants. ***P < 0.001 (n ≥ 3 biological replicates). (F) qRT-PCR measurements of endogenous tmem-131, col-19, and hsp-4p::gfp mRNA levels in wild-type and ΔPapD mutants. ***P < 0.001 (n ≥ 3 biological replicates). (G) Exemplar Western blot analysis of COL-19::GFP in different fractions from wild-type, tmem-131(tm6119), and tmem-131(ΔPapD-L) mutants.

Fig. 4. Evolutionarily conserved role of TMEM131 for collagen secretion in human cells.

(A) Cladogram of phylogenetic tree for the TMEM131 protein family from major metazoan species (adapted from www.wormbase.org). (B) Domain architectures of TMEM131 family proteins. (C) Schematic of Ce-PapD-L Y2H screens identifying the human COL1A2 C-terminal domain as an evolutionarily conserved binder of PapD-L. (D) Yeast colony growth in Y2H assays after retransformation of prey and bait vectors verifying the interaction between human COL1A2 C termini with PapD-L domains from C. elegans, Drosophila, and humans (photo credit: Zhe Zhang, UCSF). (E) Quantitative immunofluorescence measurements of extracellular collagen I fibers (green) in control and TMEM131 shRNA–depleted cells; nuclei (blue). ***P < 0.001 (N > 10 cells for each condition; n ≥ 3 biological replicates). (F) Exemplar confocal fluorescence images of U2OS cells with type I collagen staining after lentiviral expression of control shRNA and TMEM131 shRNAs (#2 and #5). Scale bars, 20 μm.

Fig. 5. Evolutionarily conserved role of TMEM131 for collagen secretion in Drosophila.

(A) Exemplar confocal images of Drosophila fat body cells showing normal COL4A1 collagen secretion with control transgenic RNAi. (B) Exemplar confocal images of Drosophila fat body cells showing defective collagen secretion after fat body cell–specific transgenic RNAi of Drosophila tmem131. Both low-magnification (scale bar, 100 μm) and high-magnification (scale bar, 200 μm) views indicating collagen accumulation in fat body cells because of defective secretion of collagen to the hemolymph (insect blood) are shown.

Fig. 6. Roles of TRAPPC8 in binding to TMEM131 and collagen secretion.

(A) Schematic showing identification of TRAPPC8 as a TMEM131 interactor from Y2H screens. The C terminus of human TMEM131 as a bait yielded a prey cDNA clone encoding the amino acids 1002 to 1063 C-terminal region of TRAPPC8. (B) Yeast growth colonies verifying interaction of TMEM131 Ct and TRAPPC8 Ct (photo credit: Zhe Zhang, UCSF). (C) Coimmunoprecipitation and Western blot showing biochemical interaction of GFP-labeled TRAPPC8 fragment and mCherry-labeled TMEM131 Ct in human embryonic kidney (HEK) 293 cells. Cells were cotransfected with expression vectors, lysed for immunoprecipitation by mCherry-TRAP, and blotted by antibodies against GFP and mCherry. (D) Yeast growth colonies testing interaction of various TMEM131 Ct mutants with TRAPPC8 Ct or a mutant carrying WRD->AAA substitution, which attenuated interaction (photo credit: Zhe Zhang, UCSF). (E) Exemplar fluorescence images showing strongly decreased COL-19::GFP in C. elegans treated with RNAi against tmem-131 or trpp-8. Scale bar, 50 μm.

Fig. 7. TMEM131 promotes collagen secretion through cytoplasmic C-terminal interaction with TRAPPC8.

(A) Exemplar fluorescence (left) and differential interference contrast merged (right) images showing activation of hsp-4p::GFP by CRISPR-mediated specific deletion of C. elegans tmem-131 cytoplasmic C terminus. (B) Exemplar fluorescence images showing the abolished COL-19::GFP signal in the cuticle of mutants (right) with deletion of tmem-131 cytoplasmic C terminus compared with wild type (left). Scale bar, 50 μm. (C) Schematic showing a series of deletions in human TMEM131 used for Y2H assays to identify regions responsible for binding to TRAPPC8. (D) Yeast colonies from Y2H assays showing interaction of TRAPPC8 with various TMEM131 Ct mutants (photo credit: Zhe Zhang, UCSF). (E) Yeast growth colonies from Y2H assays showing interaction of TRAPPC8 and a WRD->AAA mutant with TMEM131 Ct mutants with single alanine substitutions in the conserved C-terminal domain (photo credit: Zhe Zhang, UCSF). (F) Schematic model illustrating how TMEM131 regulates collagen secretion through its N-terminal PapD-L domain and C-terminal TRAPID. PapD-L binds to the C-propeptide domain of COL1A2 and facilitates assembly of procollagen trimers. TRAPID binds to TRAPPC8 and facilitates TRAPP III activation of Rab GTPase to promote the ER-to-Golgi transport of collagen cargo in COPII. For clarity, procollagen trimers in COPII and other essential factors for collagen secretion, including TANGO, HSP47, and SEC13/31, are not shown.