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An intramembrane chaperone complex facilitates membrane protein biogenesis

Abstract

Integral membrane proteins are encoded by approximately 25% of all protein-coding genes1. In eukaryotes, the majority of membrane proteins are inserted, modified and folded at the endoplasmic reticulum (ER)2. Research over the past several decades has determined how membrane proteins are targeted to the ER and how individual transmembrane domains (TMDs) are inserted into the lipid bilayer3. By contrast, very little is known about how multi-spanning membrane proteins with several TMDs are assembled within the membrane. During the assembly of TMDs, interactions between polar or charged amino acids typically stabilize the final folded configuration4,5,6,7,8. TMDs with hydrophilic amino acids are likely to be chaperoned during the co-translational biogenesis of membrane proteins; however, ER-resident intramembrane chaperones are poorly defined. Here we identify the PAT complex, an abundant obligate heterodimer of the widely conserved ER-resident membrane proteins CCDC47 and Asterix. The PAT complex engages nascent TMDs that contain unshielded hydrophilic side chains within the lipid bilayer, and it disengages concomitant with substrate folding. Cells that lack either subunit of the PAT complex show reduced biogenesis of numerous multi-spanning membrane proteins. Thus, the PAT complex is an intramembrane chaperone that protects TMDs during assembly to minimize misfolding of multi-spanning membrane proteins and maintain cellular protein homeostasis.

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Fig. 1: A protein complex containing CCDC47 engages nascent membrane proteins.
Fig. 2: Asterix is the substrate-binding subunit of the PAT complex.
Fig. 3: The PAT complex facilitates biogenesis of multi-spanning membrane proteins.
Fig. 4: The PAT complex engages TMDs via exposed polar residues.

Data availability

All data supporting the findings of this study are available within the Article files. Uncropped images of all gels and autoradiographs in the figures are provided in Supplementary Fig. 1.

References

  1. 1.

    The UniProt Consortium. UniProt: the universal protein knowledgebase. Nucleic Acids Res. 46, D158–D169 (2018).

    Google Scholar 

  2. 2.

    von Heijne, G. The membrane protein universe: what’s out there and why bother? J. Intern. Med. 261, 543–557 (2007).

    Article  Google Scholar 

  3. 3.

    Shao, S. & Hegde, R. S. Membrane protein insertion at the endoplasmic reticulum. Annu. Rev. Cell Dev. Biol. 27, 25–56 (2011).

    CAS  Article  Google Scholar 

  4. 4.

    Lu, P. et al. Accurate computational design of multipass transmembrane proteins. Science 359, 1042–1046 (2018).

    ADS  CAS  Article  Google Scholar 

  5. 5.

    Harrington, S. E. & Ben-Tal, N. Structural determinants of transmembrane helical proteins. Structure 17, 1092–1103 (2009).

    CAS  Article  Google Scholar 

  6. 6.

    Zhou, F. X., Cocco, M. J., Russ, W. P., Brunger, A. T. & Engelman, D. M. Interhelical hydrogen bonding drives strong interactions in membrane proteins. Nat. Struct. Biol. 7, 154–160 (2000).

    CAS  Article  Google Scholar 

  7. 7.

    Venkatakrishnan, A. J. et al. Molecular signatures of G-protein-coupled receptors. Nature 494, 185–194 (2013).

    ADS  CAS  Article  Google Scholar 

  8. 8.

    Choma, C., Gratkowski, H., Lear, J. D. & DeGrado, W. F. Asparagine-mediated self-association of a model transmembrane helix. Nat. Struct. Biol. 7, 161–166 (2000).

    CAS  Article  Google Scholar 

  9. 9.

    Shurtleff, M. J. et al. The ER membrane protein complex interacts cotranslationally to enable biogenesis of multipass membrane proteins. eLife 7, e37018 (2018).

    Article  Google Scholar 

  10. 10.

    Meacock, S. L., Lecomte, F. J. L., Crawshaw, S. G. & High, S. Different transmembrane domains associate with distinct endoplasmic reticulum components during membrane integration of a polytopic protein. Mol. Biol. Cell 13, 4114–4129 (2002).

    CAS  Article  Google Scholar 

  11. 11.

    Park, P. S.-H. Rhodopsin oligomerization and aggregation. J. Membr. Biol. 252, 413–423 (2019).

    CAS  Article  Google Scholar 

  12. 12.

    Estabrooks, S. & Brodsky, J. L. Regulation of CFTR biogenesis by the proteostatic network and pharmacological modulators. Int. J. Mol. Sci. 21, 452 (2020).

    Article  Google Scholar 

  13. 13.

    Keenan, R. J., Freymann, D. M., Stroud, R. M. & Walter, P. The signal recognition particle. Annu. Rev. Biochem. 70, 755–775 (2001).

    CAS  Article  Google Scholar 

  14. 14.

    Rapoport, T. A. Protein translocation across the eukaryotic endoplasmic reticulum and bacterial plasma membranes. Nature 450, 663–669 (2007).

    ADS  CAS  Article  Google Scholar 

  15. 15.

    Chitwood, P. J., Juszkiewicz, S., Guna, A., Shao, S. & Hegde, R. S. EMC is required to initiate accurate membrane protein topogenesis. Cell 175, 1507–1519.e16 (2018).

    CAS  Article  Google Scholar 

  16. 16.

    Chitwood, P. J. & Hegde, R. S. The role of EMC during membrane protein biogenesis. Trends Cell Biol. 29, 371–384 (2019).

    CAS  Article  Google Scholar 

  17. 17.

    Ismail, N., Crawshaw, S. G. & High, S. Active and passive displacement of transmembrane domains both occur during opsin biogenesis at the Sec61 translocon. J. Cell Sci. 119, 2826–2836 (2006).

    CAS  Article  Google Scholar 

  18. 18.

    Lin, Z. et al. TTC5 mediates autoregulation of tubulin via mRNA degradation. Science 367, 100–104 (2020).

    ADS  CAS  Article  Google Scholar 

  19. 19.

    Itakura, E. et al. Ubiquilins chaperone and triage mitochondrial membrane proteins for degradation. Mol. Cell 63, 21–33 (2016).

    CAS  Article  Google Scholar 

  20. 20.

    Talbot, B. E., Vandorpe, D. H., Stotter, B. R., Alper, S. L. & Schlondorff, J. S. Transmembrane insertases and N-glycosylation critically determine synthesis, trafficking, and activity of the nonselective cation channel TRPC6. J. Biol. Chem. 294, 12655–12669 (2019).

    CAS  Article  Google Scholar 

  21. 21.

    Guna, A., Volkmar, N., Christianson, J. C. & Hegde, R. S. The ER membrane protein complex is a transmembrane domain insertase. Science 359, 470–473 (2018).

    ADS  CAS  Article  Google Scholar 

  22. 22.

    Görlich, D. & Rapoport, T. A. Protein translocation into proteoliposomes reconstituted from purified components of the endoplasmic reticulum membrane. Cell 75, 615–630 (1993).

    Article  Google Scholar 

  23. 23.

    Döring, K. et al. Profiling Ssb-nascent chain interactions reveals principles of Hsp70-assisted folding. Cell 170, 298–311.e20 (2017).

    Article  Google Scholar 

  24. 24.

    Stein, K. C., Kriel, A. & Frydman, J. Nascent polypeptide domain topology and elongation rate direct the cotranslational hierarchy of Hsp70 and TRiC/CCT. Mol. Cell 75, 1117–1130.e5 (2019).

    CAS  Article  Google Scholar 

  25. 25.

    Mayer, M. P. & Bukau, B. Hsp70 chaperones: cellular functions and molecular mechanism. Cell. Mol. Life Sci. 62, 670–684 (2005).

    CAS  Article  Google Scholar 

  26. 26.

    Sato, B. K., Schulz, D., Do, P. H. & Hampton, R. Y. Misfolded membrane proteins are specifically recognized by the transmembrane domain of the Hrd1p ubiquitin ligase. Mol. Cell 34, 212–222 (2009).

    CAS  Article  Google Scholar 

  27. 27.

    Sato, K., Sato, M. & Nakano, A. Rer1p, a retrieval receptor for ER membrane proteins, recognizes transmembrane domains in multiple modes. Mol. Biol. Cell 14, 3605–3616 (2003).

    CAS  Article  Google Scholar 

  28. 28.

    Natarajan, N., Foresti, O., Wendrich, K., Stein, A. & Carvalho, P. Quality control of protein complex assembly by a transmembrane recognition factor. Mol. Cell 77, 108–119.e9 (2020).

    CAS  Article  Google Scholar 

  29. 29.

    Itzhak, D. N., Tyanova, S., Cox, J. & Borner, G. H. Global, quantitative and dynamic mapping of protein subcellular localization. eLife 5, e16950 (2016).

    Article  Google Scholar 

  30. 30.

    Yamamoto, S. et al. Contribution of calumin to embryogenesis through participation in the endoplasmic reticulum-associated degradation activity. Dev. Biol. 393, 33–43 (2014).

    CAS  Article  Google Scholar 

  31. 31.

    Morimoto, M. et al. Bi-allelic CCDC47 variants cause a disorder characterized by woolly hair, liver dysfunction, dysmorphic features, and global developmental delay. Am. J. Hum. Genet. 103, 794–807 (2018).

    CAS  Article  Google Scholar 

  32. 32.

    Wang, T. et al. Identification and characterization of essential genes in the human genome. Science 350, 1096–1101 (2015).

    ADS  CAS  Article  Google Scholar 

  33. 33.

    Kota, J. & Ljungdahl, P. O. Specialized membrane-localized chaperones prevent aggregation of polytopic proteins in the ER. J. Cell Biol. 168, 79–88 (2005).

    CAS  Article  Google Scholar 

  34. 34.

    Gu, S. et al. Brain α7 nicotinic acetylcholine receptor assembly requires NACHO. Neuron 89, 948–955 (2016).

    CAS  Article  Google Scholar 

  35. 35.

    Brechet, A. et al. AMPA-receptor specific biogenesis complexes control synaptic transmission and intellectual ability. Nat. Commun. 8, 15910 (2017).

    ADS  CAS  Article  Google Scholar 

  36. 36.

    Fons, R. D., Bogert, B. A. & Hegde, R. S. Substrate-specific function of the translocon-associated protein complex during translocation across the ER membrane. J. Cell Biol. 160, 529–539 (2003).

    CAS  Article  Google Scholar 

  37. 37.

    Walter, P. & Blobel, G. Preparation of microsomal membranes for cotranslational protein translocation. Methods Enzymol. 96, 84–93 (1983).

    CAS  Article  Google Scholar 

  38. 38.

    Sharma, A., Mariappan, M., Appathurai, S. & Hegde, R. S. In vitro dissection of protein translocation into the mammalian endoplasmic reticulum. Methods Mol. Biol. 619, 339–363 (2010).

    CAS  Article  Google Scholar 

  39. 39.

    Feng, Q. & Shao, S. In vitro reconstitution of translational arrest pathways. Methods 137, 20–36 (2018).

    CAS  Article  Google Scholar 

  40. 40.

    Shao, S., von der Malsburg, K. & Hegde, R. S. Listerin-dependent nascent protein ubiquitination relies on ribosome subunit dissociation. Mol. Cell 50, 637–648 (2013).

    CAS  Article  Google Scholar 

Download references

Acknowledgements

We thank S.-Y. Peak-Chew, F. Begum and M. Skehel for mass spectrometry analysis; S. Juszkiewicz, J. O’Donnell and E. Zavodszky for discussions and advice; and N. Peters and H. Damstra for initial characterization of photo-crosslinking methods. This work was supported by the UK Medical Research Council (MC_UP_A022_1007 to R.S.H.) and a studentship (to P.J.C.) from the MRC International PhD Programme.

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P.J.C. and R.S.H. conceived the project, analysed the results and wrote the manuscript. P.J.C. performed all of the experimental work with advice from R.S.H.

Corresponding author

Correspondence to Ramanujan S. Hegde.

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Extended data figures and tables

Extended Data Fig. 1 Characterization of Rho TM1+2 insertion.

a, Diagram of Rho TM1+2 constructs used throughout this study. Variations on this construct include different N-terminal epitope tags, the presence or absence of a glycosylation site near the N terminus, the presence, absence, or position of a cysteine within TMD1, various mutations within TMD1, and the presence or absence of TMD2. All of the constructs were tested either by protease protection or glycosylation to verify that no appreciable differences were observed in their insertion efficiencies. Although the exact amino acid numbering varies depending on the N-terminal tag, the numbering system corresponding to the Flag-tagged version is used throughout. Thus, the 146mer refers to a truncation at the 146th codon in the numbering scheme indicated in Fig. 1a and in this diagram, even in constructs containing a different tag. b, Representative example of insertion assays on two different tagged versions of Rho TM1+2. The TwinStrep tagged version (Strep) lacking a glycosylation site was compared to an HA-tagged version containing a glycosylation site. Identical constructs containing either the wild-type Rho TM1 sequence or a point mutant (F53C) were tested in parallel to confirm no insertion defects result from insertion of a cysteine in TM1 (used for BMH-mediated crosslinking in later experiments). In this experiment, [35S]methionine labelled ribosome nascent chain complexes (RNCs) of 181 amino acids were produced by in vitro translation using rabbit reticulocyte lysate (RRL) in the presence of ER-derived RMs after which the microsomes were isolated and resuspended. Aliquots of the reactions were left untreated or digested with PK and analysed directly by SDS–PAGE and visualized by autoradiography (left). Green arrowheads represent the fully inserted and PK-protected population and red arrowheads denote the non-inserted and proteolytically cleaved products. The cleaved product contains the region of polypeptide protected by the ribosomal tunnel and the attached tRNA. Aliquots of the PK-digested sample were treated with EDTA and RNase to release the polypeptide from the ribosome and tRNA and immunoprecipitated (IP) via the N-terminal tag. Only the fully inserted products are recovered by IP (green arrowheads). c, Comparison of the topology of truncated RNCs and terminated Rho TM1+2. In this experiment, the Flag-tagged Rho TM1+2 containing a glycosylation site with (term.) or without (trunc.) a stop codon was translated in the presence of RMs, after which the RMs were isolated by centrifugation. Aliquots of the isolated RMs were analysed directly (−PK), after PK digestion (+PK), or after PK digestion in the presence of detergent (+PK/det). Where indicated, the +PK and +PK/det samples were released from the attached tRNA and immunoprecipitated via the N-terminal tag. Note comparable glycosylation near the N terminus and complete protection from PK for both the truncated and terminated products. d, Diagram representing the interpretation of the experiments in b and c. The relatively short cytosolic loop between TMD1 and TMD2 is not accessible to PK digestion either as an RNC or a terminated product. For gel source data, see Supplementary Fig. 1.

Extended Data Fig. 2 Additional characterization of crosslinking to PAT10.

a, Diagram of constructs either lacking or containing a cysteine in place of Gly48 or Phe53 within TMD1 of a Rho TM1+2 cassette. b, 35S-labelled Flag-tagged Rho TM1+2 RNCs of varying lengths (as described in Fig. 1a) containing a Cys at position 53 were generated by in vitro translation in the presence of RMs. Membranes were isolated by centrifugation through a sucrose cushion and resuspended in physiological salt buffer (PSB). An equivalent amount of each translation reaction was taken before (−BMH) and after (+BMH) the addition of BMH for analysis by SDS–PAGE. The tRNA-linked nascent chains and free nascent chains are indicated. The free nascent chains arise from partial hydrolysis of the tRNA during electrophoresis under moderately basic conditions. Glycosylation is first observed at the 96mer length, which is 38 amino acids downstream of the end of TMD1. This matches the length of the ribosome tunnel, and indicates that membrane insertion and glycosylation occurs only after TMD1 is fully exposed outside the ribosome. Crosslinks to Sec61α are most prominent for the 106mer. Crosslinks to PAT10 are most prominent from the 126mer onwards, after the Sec61α crosslinks diminish. All of the crosslinked adducts are seen to the tRNA-attached nascent chain, verifying that they are co-translational. The Sec61α crosslink and others are not as visible when total translation products are analysed, which is why we typically immunoprecipitate the sample via the nascent chain (for example, in Fig. 1a). This reduces the background, allowing otherwise obscured crosslinks to be visualized clearly. Furthermore, we usually digest the samples with RNase after the experiment but before SDS–PAGE to remove the tRNA, thereby avoiding the heterogeneity that results from partial tRNA hydrolysis during sample handling and SDS–PAGE. All of the indicated crosslinking adducts observed were completely dependent on the presence of BMH. c, The indicated ‘Input’ crosslinking sample from Fig. 2b (reproduced here on the left) was subjected to immunoprecipitation using anti-Flag or anti-Sec61β antibodies under denaturing conditions. The IP samples were either left untreated or digested with PNGase F to remove N-linked glycans. Equivalent amounts were loaded in each lane. Sec61β is not an appreciable crosslinking partner of these RNCs, and to the extent a crosslink is observed, it migrates slightly faster than the PAT10 crosslink. d, The indicated Rho TM1+2 variants were translated in vitro in the presence of RMs and treated with BMH as indicated. In this experiment, the crosslinking was performed directly on total translation reactions, not after isolation of the microsome fraction. Instead, translation reactions were diluted fivefold with buffer to dilute the reduced glutathione and minimize quenching of BMH. An aliquot of each reaction was analysed directly by SDS–PAGE. Cross-linking efficiency is reduced compared to other experiments because membranes were not isolated by centrifugation through a sucrose cushion to remove reduced glutathione from translation extract. One aliquot of the BMH-treated translation reactions were treated with RNase A and EDTA, denatured, and IPed via the N-terminal Flag tag (IP). e, As in d, except Rho TM1+2 variants were generated in the presence of RMs derived from either canine pancreas (cRMs) or HEK293 cells (hRMs). In this experiment, the microsomes were isolated by centrifugation through a sucrose cushion before BMH crosslinking (note the higher crosslinking efficiency). Although the PAT10 crosslink is seen in both cRMs and hRMs, crosslinking efficiency of the inserted (glycosylated) product is appreciably lower in cRMs, which is one reason why we used HEK293-derived RMs for most of the experiments in this study. For gel source data, see Supplementary Fig. 1.

Extended Data Fig. 3 Analysis of PAT complex candidates.

a, Radiolabelled 146mer RNCs of Rho TM1+2 constructs containing or lacking a glycosylation site were generated by in vitro translation in the presence of RMs. Membranes were isolated by centrifugation through a sucrose cushion, subjected to BMH crosslinking where indicated, treated with RNase A, then analysed by SDS–PAGE. One aliquot of the BMH treated reactions were solubilized under native conditions and immunoprecipitated with either an antibody raised against the HA epitope tag (Cntrl) for a specificity control, or an antibody against signal peptide peptidase (SPP). Direct crosslinks to SPP are observed as two distinct adducts seen on very long exposures (compare to Fig. 1d, which is a shorter exposure), but native IPs do not enrich for a PAT10 engaged substrate. b, Radiolabelled 146mer RNCs of Flag-tagged Rho TM1+2 containing (+) or lacking (−) a glycosylation site (glyc.) were generated by in vitro translation with RMs, crosslinked with BMH, then immunoprecipitated under native conditions via the N-terminal Flag tag on the substrate (Nterm) or with an antibody recognizing the C terminus of calnexin (CNX). Only the glycosylated substrate is recovered with CNX, consistent with its binding via the glycan. c, Aliquots of the crosslinked and natively solubilized samples from b were run on a 5–25% sucrose gradient before analysis by SDS–PAGE and autoradiography. Red asterisks denote peak fractions containing the PAT complex as detected by the PAT10 crosslinking product. The PAT complex crosslinked to unglycosylated Rho TM1+2 migrates slightly smaller on the gradient than glycosylated Rho TM1+2, probably the result of CNX (around 90 kDa) no longer being associated with the nascent chain. d, The insertion and BMH-mediated crosslinking for 146mer RNCs of the parent Rho TM1+2 construct or versions lacking a cysteine (NoCys) or lacking a glycosylation site (Cys53No glyc.). The radiolabelled RNCs were produced by in vitro translation in the presence of RMs isolated from wild-type (WT) or two different CCDC47 knockout cell lines (∆1 and ∆2) generated from two different guide RNAs. Aliquots of the reaction before (−BMH) and after (+BMH) addition BMH were analysed by SDS–PAGE. No appreciable difference in insertion efficiency was observed in knockout microsomes for Rho TM1+2 as monitored by glycosylation efficiency. Red arrowheads indicate the PAT10 crosslink which is lost upon CCDC47 knockout. The faint crosslinked adduct observed in the knockout samples (black asterisks) migrates slightly faster on the gel and probably represents weak Rho TM1+2 crosslinks to Sec61β (Extended Data Fig. 2c). For gel source data, see Supplementary Fig. 1.

Extended Data Fig. 4 Conservation and topology of Asterix.

a, Alignment of Asterix homologues for five divergent species with a bar chart representing conservation scores of each amino acid. Indicated are three hydrophobic domains (HD1 to HD3), each consisting of approximately 15 amino acids, that are candidate TMDs. b, Representation of human Asterix amino acid sequence and the relative lengths of hydrophilic (grey bar) and hydrophobic (blue) regions. c, Two matched human Asterix constructs containing either an N- or C-terminal Flag tag were generated by in vitro translation in the presence of RMs. One aliquot of the reaction was set aside for analysis by SDS–PAGE of total translation products (IVT). The remainder of the reaction was treated with proteinase K (+PK) without or with detergent (det). These protease-digested samples were either analysed directly or after immunoprecipitation via the Flag tag. Red arrowheads indicate fragments protected from PK in the absence, but not in the presence, of detergent. The PK-protected fragment from the C-terminally tagged Asterix was recovered by IP, suggesting that the C terminus is located within the ER lumen and the N terminus is located in the cytosol. The relative size difference between the N-and C-terminally tagged constructs observed after PK digestion can be attributed to digestion or protection of the Flag tag. Below the gel is a cartoon depiction of one possible topology based on the results and the protease protected fragments that remain after digestion with PK. The other possible topology is a single-spanning orientation with HD2 and HD3 in the lumen. d, Schematic of human Asterix with a C-terminal Flag tag in its predicted 3-TMD topology based on the protease digestion results in c. To test this prediction, a cysteine-free version of Asterix (No Cys) was modified with single cysteines at the position indicated by the red-asterisks. If the topology prediction is correct, only the N-terminal domain (NTD) cysteine and the loop 2 cysteine should be accessible to sulfhydryl modifying reagents added to the cytosolic side of them membrane. If the protein spans the membrane only once with the N terminus facing the cytosol, then the loop 2 cysteine should not be modified. As shown in a, wild-type Asterix naturally has four cysteines, only one of which should be exposed to the cytosol because it is in the NTD. e, Asterix knockout HEK-293 cells were transiently transfected with the indicated Asterix–Flag constructs, semi-permeabilized in 0.01% digitonin, washed to remove digitonin, and treated with PEG-maleimide (average molecular weight 5 kDa) in order to modify any cytosolically exposed cysteine residues. Wild-type Asterix contains four native cysteine residues, one in the N terminus preceding TMD1 and three others within the putative TMD regions. Modification was observed only for the NTD cysteine and the cysteine in loop 2, supporting a 3-TMD topology as depicted in d. The single cysteine present in the cytosolic domain of Sec61β was used as a positive control demonstrating equal modification efficiency in all samples, and the no-Cys construct verifies sulfhydryl-dependent modification. Protection of the cysteines in TMD1 and TMD2 from modification verifies that membrane integrity was maintained in the experiment. For gel source data, see Supplementary Fig. 1.

Extended Data Fig. 5 Characterization of site-specific photo-crosslinking.

a, Schematic of the strategy for site-specific incorporation of the photo-crosslinking amino acid BPA during in vitro translation (IVT). BPA, a synthetic amber-suppressor tRNA, and recombinant BPA tRNA synthetase are added to an IVT reaction. The nascent protein that is produced incorporates BPA at an amber codon. UV irradiation results in activation of BPA and crosslinking to adjacent proteins. b, The photo-crosslinking strategy was tested using a well-validated translocation intermediate: the 86mer of the secretory protein prolactin. The amber codon was installed at position 9, within the hydrophobic core of the signal sequence. At this length, the majority of the nascent chain is precursor (pre), with a small amount that is signal-cleaved (s.c.). The primary crosslinks to SRP54 and components of the translocation site (Sec61α, TRAM, and TRAPα) were verified by immunoprecipitation. c, Site-specific photo-crosslinking of a 141mer RNC containing the UV-activated photo-crosslinking amino acid BPA at the indicated amber positions (Amb). The diagram above the autoradiographs shows a schematic of the construct with the appropriate amino acid numbering. Amino acids in red show the strongest crosslinks to Asterix, pink show reduced crosslinks, and grey no detectable crosslinks. Total translation products recovered by IP via an HA tag on the nascent chain are shown adjacent to parallel IPs of selected samples using the indicated antibodies. Although not all IPs are shown, each position was tested for crosslinking to Asterix and CCDC47. RNCs that failed to engage SRP crosslink to UBQLN2, a quality control factor that binds exposed TMDs. This crosslink diminishes markedly when increased RMs are used in the reaction (lanes 9–16, compared to lanes 5–8), presumably because the RMs contribute SRP, which is otherwise limiting in the reaction. A subset of RNCs fail to release from SRP and crosslink to SRP54. The crosslink indicated by the hashtag (#) is likely to be a mixture of similarly migrating crosslinks. Because this crosslink diminishes substantially with increased RMs (similar to the UBQLN2 crosslink), it is likely to be SGTA, another TMD-binding factor in the cytosol of this size. A small proportion of this crosslink could be the similarly sized Sec61α or TRAM. Of the membrane-inserted RNCs, the main crosslink is to Asterix, seen prominently for residues 52 to 63. At this length, the TMD has moved away from Sec61α, so crosslinks to this factor are minimal. No crosslinking to CCDC47 were ever observed. By testing five sequential positions in the centre of the TMD, all sides of the helix have been sampled.

Extended Data Fig. 6 Effect of Asterix depletion on multi-spanning membrane proteins.

a, The raw data for three of the histograms of the GFP:RFP ratio (or RFP:GFP ratio in the case of GFP-2A-RFP-SQS) shown in Fig. 3. The mode of the control histogram (dotted black line in Fig. 3) was used to determine the statistical mode of GFP:RFP (or RFP:GFP) ratio. This mode was used as a gate to colour the dot plots shown below the histograms such that all cells above the gate were coloured red and those below the gate were coloured grey. The percent of cells above the gate for each plot is indicated. b, Flow-cytometry analysis of the indicated GPCR reporters using the dual-colour assay system exactly as in Fig. 3. Cell lines containing the reporter stably integrated at a single FRT site located downstream of a doxycycline-inducible reporter were used for these assays. This allows assay of cells using a single transfection (which proved to be less toxic than sequential transfections with both the siRNA and the reporter), and provided control of the length of time of reporter expression. Each reporter cell line was treated with scrambled (Scr) or Asterix-targeting siRNAs, then at the time of effective knockdown (verified in separate experiments using immunoblotting), the reporter was induced for approximately 6–8 h. Induction only after knockdown allows us to monitor the reporter that was produced in the absence of Asterix rather than a heterogeneous mixture of reporter expressed during the knockdown. The histograms of the GFP:RFP ratio in the scrambled- versus Asterix-siRNA cells are shown in grey and blue, respectively, in the upper plot for each construct. The two dot plots below the histogram are the corresponding raw data plotted as described in a. Each reporter shows a distribution of lower GFP:RFP ratio, with some reporters being more impacted than others. This is not seen with the tail-anchored protein SQS using the same assay format.

Extended Data Fig. 7 Effect of CCDC47 depletion on membrane protein biogenesis.

a,l The diagrams depict dual-colour fluorescent reporters for protein stability as an indirect measure of successful biogenesis. The membrane protein of interest is tagged with one fluorescent protein (FP), which is separated from a second FP by the viral 2A peptide sequence. When the 2A sequence is translated, peptide bond formation is skipped without perturbing elongation by the ribosome. Thus, translation results in two separate proteins made in a 1:1 stoichiometry that are separated at the 2A sequence. If biogenesis of the membrane protein is impaired, it will be degraded along with its tagged FP, resulting in an altered ratio of the two FPs. Thus, treatment conditions that impair biogenesis of the membrane protein will be reflected as a relative change in the ratio of FPs. The three reporters encoding angiotensin type-2 receptor II (AGTR2), squalene synthase (SQS) and Asialglycoprotein receptor (ASGR) were transiently transfected into wild-type (WT), CCDC47 knockout (∆CCDC47) or Asterix knockout (∆Asterix) HEK293 cells and analysed by dual-colour flow cytometry. Histograms represent the distribution of FP ratio in wild-type (grey), ∆CCDC47 (red) and ∆Asterix (blue) cells. A biogenesis defect is only seen for the multi-spanning membrane protein AGTR2, but not for the tail-anchored protein SQS or the signal-anchored single pass protein ASGR. b, Assays similar to those in Fig. 3, but for cell lines treated with scrambled versus CCDC47 siRNAs as indicated. We find that the phenotypes for Asterix and CCDC47 knockdowns are very similar for all reporters (three are shown here), with CCDC47 consistently being somewhat more modest. The reason for this seems to be that CCDC47 knockdown is slower and less efficient than Asterix knockdown. Similar phenotypes are seen for AGTR2 and SS-T4L-AGTR2, a version that contains an N-terminal signal sequence and T4 lysozyme preceding TMD1. In earlier studies, we found that initiating translocation with a signal sequence completely bypasses the requirement for EMC-mediated TMD1 insertion. The fact that SS-T4L-AGTR2 remains sensitive to PAT complex depletion (as judged by either Asterix or CCDC47 knockdowns) argues that the PAT complex acts independently of EMC.

Extended Data Fig. 8 Expression of ER biogenesis factors in ∆CCDC47 and ∆EMC6 cells.

ER rough microsomes were isolated from wild-type, ∆CCDC47 and ∆EMC6 HEK293 cells and normalized to an absorbance of 75 at 280 nm. Serial dilutions of each sample were analysed by SDS–PAGE and immunoblotting for the indicated antigens. BiP levels are increased in both knockout cell lines, consistent with an activated UPR caused by altered ER homeostasis. For gel source data, see Supplementary Fig. 1.

Extended Data Fig. 9 TMD1 insertion does not require the PAT complex or TMD2.

a, Rho TM1+2 RNCs of varying nascent chain lengths (indicated at top of gels) were translated in vitro in the presence of RMs prepared from ∆CCDC47 or ∆Asterix HEK293 cells as indicated. Membranes were isolated by centrifugation through a sucrose cushion and treated with the chemical cross linking reagent BMH. The samples were denatured in 1% SDS and immunoprecipitated via the N-terminal Flag tag of the substrate. Notice that the glycosylation of substrate is very similar in efficiency and timing as that seen in RMs prepared from wild-type HEK293 cells (see Fig. 1a for comparison). Furthermore, the appearance and disappearance of the SRP54 and Sec61α cross linking adducts are not appreciably altered from the results seen in RMs prepared from wild type cells. Thus, the early steps of rhodopsin biogenesis are not impaired appreciably in the absence of Asterix or CCDC47. As expected, the crosslink to Asterix/PAT10 is not seen (verified by anti-Asterix immunoprecipitation; not shown). Crosslinking products seen at the approximate size of the Asterix crosslink are therefore other protein(s). b, The Rho TM1 construct in which TMD2 is replaced with a hydrophilic linker sequence (see diagram in Fig. 4a) was analysed for crosslinking as in Fig. 1a. The absence of TMD2 does not affect the crosslinking between TMD1 and Asterix. By contrast, mutation of the most polar residue in TMD1 (N52) markedly reduces Asterix crosslinking and reduces TMD1 proximity to Sec61α (Fig. 4a).

Extended Data Fig. 10 Analysis of Asterix interaction with TMD1 by photo-crosslinking.

a, Experimental strategy for comparing Asterix interaction with a membrane protein intermediate versus full-length product. In this experiment, the photo-crosslinking amino acid BPA (yellow star) is incorporated into position 52 within TMD1 of β1AR by in vitro translation. The intermediate is represented by the TM1-3 product containing the first three TMDs of β1AR. The full-length (FL) β1AR contains all seven TMDs followed by a long flexible linker. TM1-3 is stalled 35 amino acids downstream of TMD3 (with TMD4 inside the ribosomal tunnel), allowing TMD3 to be outside the ribosome. β1AR FL is stalled 152 amino acids downstream of TMD7, providing a sufficiently long tether for all seven TMDs to have emerged, inserted into the membrane, and assembled together. The translation products are then irradiated with UV light to activate the BPA and any crosslinking to Asterix is subsequently detected by denaturing IP via Asterix. b, Results from a photo-crosslinking experiment as depicted in a. The microsomes from the IVT reaction were isolated, resuspended, irradiated with UV light (or left untreated), and denatured. The samples were then divided in two and immunoprecipitated via the nascent chain or via Asterix. Sixfold more of the Asterix IPs were loaded on the gel relative to the nascent chain IPs. As expected, the BPA in TMD1 crosslinks to Asterix in the TM1–3 intermediate. The crosslinked band in the 6× Asterix IP is the same intensity as the glycosylated band in the 1× nascent chain IP. Although elongation to the full-length product was somewhat inefficient, clear glycosylated and non-glycosylated products are observed in the nascent chain IPs. No band is seen in the 6× Asterix IP sample that is of comparable intensity to the glycosylated band in the 1× nascent chain IP. This argues that the proximity of TMD1 to Asterix has diminished substantially in the full-length nascent chain relative to the TM1-3 intermediate. Of note, a heterogeneous set of crosslinks (marked by red stars) are seen at a lower part of the gel in the 6× Asterix IP. These correspond to the sizes expected for Asterix crosslinks (that is, shifted by about 10 kDa) to the major incomplete translation products (marked by blue stars). These crosslinks provide an internal control and further supports the conclusion that incomplete products engage Asterix, while a complete 7-TMD product does not.

Supplementary information

Supplementary Figure 1

This file contains all uncropped autoradiographs and gels from this study.

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Chitwood, P.J., Hegde, R.S. An intramembrane chaperone complex facilitates membrane protein biogenesis. Nature 584, 630–634 (2020). https://doi.org/10.1038/s41586-020-2624-y

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