From Europe PMC via Jisc Publications RouterHistory: epub 2021-07-01, ppub 2021-07-01Publication status: PublishedFunder: Ball State University (Ball State); Grant(s): Provost Startup AwardFunder: Wellcome Trust; Grant(s): 204957/Z/16/ZFunder: NIGMS NIH HHS; Grant(s): R15 GM116032The heterotrimeric Sec61 complex is a major site for the biogenesis of transmembrane proteins (TMPs), accepting nascent TMP precursors that are targeted to the endoplasmic reticulum (ER) by the signal recognition particle (SRP). Unlike most single-spanning membrane proteins, the integration of type III TMPs is completely resistant to small molecule inhibitors of the Sec61 translocon. Using siRNA-mediated depletion of specific ER components, in combination with the potent Sec61 inhibitor ipomoeassin F (Ipom-F), we show that type III TMPs utilise a distinct pathway for membrane integration at the ER. Hence, following SRP-mediated delivery to the ER, type III TMPs can uniquely access the membrane insertase activity of the ER membrane complex (EMC) via a mechanism that is facilitated by the Sec61 translocon. This alternative EMC-mediated insertion pathway allows type III TMPs to bypass the Ipom-F-mediated blockade of membrane integration that is seen with obligate Sec61 clients

Andrews, Lauren E

Duah, Kwabena B

High, Stephen

O'Keefe, Sarah

Shi, Wei Q

Zong, Guanghui

English

From Springer Nature via Jisc Publications RouterHistory: received 2020-11-18, accepted 2021-06-17, registration 2021-06-18, pub-electronic 2021-07-01, online 2021-07-01, collection 2021-12Publication status: PublishedFunder: Wellcome Trust (Wellcome); doi: https://doi.org/10.13039/100010269; Grant(s): 204957/Z/16/ZFunder: Ball State University (Ball State); doi: https://doi.org/10.13039/100008326; Grant(s): Provost Startup AwardAbstract: The heterotrimeric Sec61 complex is a major site for the biogenesis of transmembrane proteins (TMPs), accepting nascent TMP precursors that are targeted to the endoplasmic reticulum (ER) by the signal recognition particle (SRP). Unlike most single-spanning membrane proteins, the integration of type III TMPs is completely resistant to small molecule inhibitors of the Sec61 translocon. Using siRNA-mediated depletion of specific ER components, in combination with the potent Sec61 inhibitor ipomoeassin F (Ipom-F), we show that type III TMPs utilise a distinct pathway for membrane integration at the ER. Hence, following SRP-mediated delivery to the ER, type III TMPs can uniquely access the membrane insertase activity of the ER membrane complex (EMC) via a mechanism that is facilitated by the Sec61 translocon. This alternative EMC-mediated insertion pathway allows type III TMPs to bypass the Ipom-F-mediated blockade of membrane integration that is seen with obligate Sec61 clients

O’Keefe, Sarah

Duah, Kwabena B.

Andrews, Lauren E.

Shi, Wei Q.

ChesterRep

Supplementary information for  An alternative pathway for membrane protein biogenesis at the endoplasmic reticulum  Sarah O’Keefe, Guanghui Zong, Kwabena B. Duah, Lauren E. Andrews, Wei Q. Shi and Stephen High                    Page 2 of 32   Supplementary Figure 1. The specificity of an Ipom-F induced translocational block is consistent and reproducible in ER-derived microsomes and SP cells, Related to Figures 1-6. The tail-anchored protein precursors, (Ai) C-terminally OPG2-tagged cytochrome b5 (Cytb5OPG2) and (Aii) C-terminally OPG2-tagged Sec61 beta subunit (Sec61βOPG2); secretory proteins, (Bi) bovine preprolactin (PPL) and (Bii) yeast prepro-alpha-factor (ppαF); single-pass type I transmembrane protein (TMP), (C) T-cell surface glycoprotein CD3 delta chain (CD3δ) and single-pass type II TMP (D) asialoglycoprotein receptor 1 (ASGR1) were synthesised in rabbit reticulocyte lysate supplemented with ER-derived canine pancreatic microsomes in the presence and Page 3 of 32  absence of 1 µM Ipom-F. (E) The effect of 1 µM Ipom-F on insertion into both ER-derived microsomes and SP cells was determined using a model substrate from each class of single-pass TMP; the type I TMP, vascular cell adhesion protein 1 (VCAM1), the type II TMP, isoform 2 (residues 17-232) of the short form of HLA class II histocompatibility antigen gamma chain (Ii) and the type III TMP OPG2Vpu (as Fig. 1di). Phosphorimages of membrane-associated products resolved by SDS-PAGE together with representative substrate outlines are shown. N-glycosylation or signal sequence cleavage were used as a read-out for the qualitative efficiency of translocation/insertion. N-glycosylated (X-Gly) versus non-N-glycosylated (0Gly) species were identified by treatment with endoglycosidase H (Endo H, lane 2). Sc, signal cleaved form, nc, non-signal cleaved form.                Page 4 of 32  Supplementary Figure 2    Subunit Structurally integral? Protein Type Mw (kDa)  Human Dog Sequence identity EMC1 Core subunit Type I TMP 112 Q8N766 F1PN78 95.5% EMC2 Core subunit Cytosolic 35 Q15006 E2R0P8 100% EMC3 Core subunit Multi-pass TMP 30 QP012 E3RMQ3 99.5% EMC4 X Multi-pass TMP 20 Q5J8M3 E2R5T1 97.8% EMC5 Core subunit Multi-pass TMP 15 Q8N4V1 N/A N/A EMC6 Core subunit Multi-pass TMP 12 Q8N766 E2R7Q9 99.1% EMC7 X Type I TMP 26 Q9NPA0 J6P692 95.5% EMC8 X Cytosolic 24 O4302 E2QUA5 98.6% EMC9 X Cytosolic 23 Q9Y3B6 F6UPE2 92.7% EMC10 X Type I TMP 27 Q5UCC4 E2R112 92.0% Page 5 of 32  Supplementary Figure 2. The human EMC is a 9-subunit multimeric complex, Related to Fig. 2.  (A) A schematic of the structure of the human EMC, depicting its tripartite organisation across the ER membrane (a basket-shaped cytosolic region, an ordered membrane-spanning core and an L-shaped ER lumenal region)1,2,3 and the topology of each of its subunits; three soluble/cytosolic (EMC2, EMC8, EMC9), three single-pass type I TMPs (EMC1, EMC7, EMC10), three multi-pass TMPs (EMC3, EMC5, EMC6) and one topologically ambiguous subunit whose N-terminus is cytosolic3,4 but may be comprised of one1,2,3, two5 or three2,3 transmembrane domains (EMC4). Of these 10 subunits, EMC8 and EMC9 are functional paralogues, thus the EMC complex may either be comprised of EMC1-8 or EMC1-7,9 together with EMC101,2,3. In addition, EMC6 is topologically dependent on the presence of EMC5, whereby the poorly hydrophobic first transmembrane domain of EMC6 is only capable of inserting into the membrane upon assembly with EMC51. EMC subunits with variable or an unconfirmed number of TMDs are indicated by (*). (B) Based on subunit depletion studies, and consistent with its ER membrane organisation, EMC1, EMC2, EMC3, EMC5, EMC6 are structurally integral to the assembly of the wider EMC complex1,6. Subunits EMC1-10, whether structurally integral or not, are highly conserved between humans and dogs, with the exception of EMC5 which is not present at all in dogs. (C) The structurally-integral subunit human EMC5 (H. sapiens, Q8N4V1-1) is conserved in yeast (S. cerevisiae, P40540, 22% identity, 54% similarity) and shares high homology with its sheep (O. aries, W5PPL4, 79% identity, 79% similarity) and mouse (M. musculus, Q8K273, 95% identity, 99% similarity) equivalents.         Page 6 of 32   Supplementary Figure 3. The OPG2-tag does not affect Ipom-F sensitivity of TMPs and permits efficient recovery of TMP substrates by immunoprecipitation independent of OPG2-tag location, Related to Figures 1-2 Page 7 of 32  and 4-6. (A) An N-terminal fragment of bovine rhodopsin containing two sites for N-glycosylation (OPG2-tag) was incorporated either at the N-terminus (residues 1-26 rhodopsin) or C-terminus (residues 2-18 rhodopsin) of model TMPs. (B) Phosphorimages of the membrane-associated radiolabelled products synthesised in ER-derived canine pancreatic microsomes. Precursor polypeptides include OPG2-tagged (Bi) asialoglycoprotein receptor 1 (ASGR1OPG2), (Bii) small cell adhesion glycoprotein containing an artificial N-glycan site (SMAGPOPG2), (Biii) glycophorin C (GypCOPG2), (Biv) tumour necrosis factor receptor superfamily member 17 containing an artificial N-glycan site (BCMAOPG2), (Bv) synaptotagmin 1 (Syt1OPG2), (Bvi) a truncated version of Syt1OPG2 (Syt1-127-OPG2) and (Bvii) a non-tagged version of Syt-127-OPG2 (Syt1-127). (C) Each of the OPG2-tagged precursor polypeptides used in this study were synthesised with semi-permeabilised cells, recovered via the OPG2-tag by immunoprecipitation (IP1; lanes 1 and 2) and analysed by SDS-PAGE and phosphorimaging. Any remaining proteins contained in the supernatant were then isolated by immunoprecipitating a second time (IP2; lanes 3 and 4). Inputs are 10% of the total material. The relative efficiencies of TMP recovery were calculated as a ratio of the total signal intensity present in IP1 compared to that in present in IP1+IP2. Quantifications are given as means ±SEM for three independent immunoprecipitations (n=3). (!) indicates a slightly reduced calculation of recovery due to the presence of radiolabelled material unrelated to the protein of interest (Syt1-127-OPG2, lane 3).             Page 8 of 32   Supplementary Figure 4. Predicted hydrophobicity of transmembrane domains from human type III TMPs, Related to Figures 1-7. Predicted transmembrane domain (TMD) hydrophobicity values (∆Gapp)5, the number of charged and/or polar amino acid residues in the transmembrane domain and lumenal/cytoplasmic (Nexo/Ccyt) domain lengths were calculated by inputting the protein sequences of 38 human, 1 rat (Syt1) and 1 viral (HIV-Vpu) type III TMPs (Uniprot annotated) into the ‘full protein scan’ mode using http://dgpred.cbr.su.se/. Type III TMPs analysed in this study are in yellow. All other Uniprot annotated type III TMPs that enter the secretory pathway (i.e. are localized in the ER, Golgi apparatus or plasma membrane) are in black. For a full list of type III TMPs analysed, and those discounted from analysis, see Supplementary Data 1. Charged amino acid residues, D, E, H, K, R; polar amino acid residues, N, Q, S, T, Y.               Page 9 of 32   Supplementary Figure 5. Knockdowns of subunits of the Sec61, EMC and/or SRP receptor complexes variably affect the levels of ER protein translocation components, Related to Figures 2-6. (Ai) Representative structures of known membrane-bound receptors involved in protein translocation. The SRP-(signal recognition particle) receptor (co-translational pathway) is a heterodimer comprised of the membrane-integrated beta-subunit (SRβ) which anchors the peripherally associated cytosolic alpha-subunit (SRα) to the Page 10 of 32  ER membrane. SRα binds to SRP and transfers SRP-targeted ribosome nascent chains to the Sec61 complex7. hSnd2 (co/post-translational pathway) is the human orthologue of a component involved in the distinct SRP-independent (SND) targeting pathway present in yeast8. hSnd2 plays a role in the ER targeting of certain proteins, although this pathway is yet to be fully defined in humans9. Here, we show hSnd2 as a TMP containing three transmembrane domains5,10, although a four transmembrane domain model has also been hypothesised9. The TRC40 receptor (post-translational pathway), comprised of the WRB and CAML-(calcium signal-modulating cyclophilin ligand) membrane proteins, may either insert TRC40-targeted tail-anchor proteins into the ER membrane directly11 or re-route certain proteins to the Sec61 complex12. (Aii) A schematic of SRP-(signal recognition particle) which is comprised of a highly base paired SRP RNA (7SL) and six protein subunits (SRP9, SRP14, SRP19, SRP54, SRP68 and SRP72) that are assembled into a functionally independent Alu domain (responsible for translational retardation) and the S domain (signal sequence engagement and SRP receptor binding). Each subunit of the S domain is essential for SRP function whereas those of the Alu domain are dispensable for protein translocation/insertion7. (B) The effect on SRα, hSnd2, CAML and SRP54 of transfecting HeLa cells with non-targeting (NT; lanes 1, 5, 8 and 10), Sec61α-targeting (lane 2), EMC2-targeting (lane 3), EMC5-targeting (lane 4), Sec61α+EMC2-targeting (lane 6), Sec61α+EMC5-targeting (lane 7), SRα-targeting (lane 9), SRα+EMC5-targeting (lane 11) and SRα+Sec61α-targeting (lane 12) siRNAs were determined after semi-permeabilisation by immunoblotting. (C) The effects of each siRNA-mediated knockdown were calculated as a proportion of the signal intensity obtained with the NT control (set as 100%). Quantifications are given as means ±SEM for two or three separate siRNA treatments (n=2 or n=3 as indicated in figure) with statistical significance of siRNA-mediated knockdowns (two-way ANOVA) determined using Sidak’s multiple comparisons test. Statistical significance is given as n.s., non-significant; *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001.                Supplementary Figure 6  Uncropped and unedited blot/gel images                   Page 12 of 32   Fig. 1d: Phosphorimages of +/- Ipom-F insertion assay in microsomes                  Page 13 of 32  Fig. 2c: Efficiency of single knockdowns; NT, Se61α-kd, EMC2-kd, EMC5-kd  SP Cells                                  Page 14 of 32  Microsomes                              Page 15 of 32      Fig. 3c: Phosphorimages of NT, Se61α-kd, EMC2-kd, EMC5-kd (+/- Ipom-F) insertion assay                            Page 16 of 32  Fig. 4b: Phosphorimages of NT, Se61α-kd, EMC2-kd, EMC5-kd (+/- Ipom-F) insertion assay                                   Page 17 of 32  Fig. 5a: Efficiency of double knockdowns; NT, Se61α+EMC2-kd, Se61α+EMC5                                    Page 18 of 32        Fig. 5c: Phosphorimages of NT, Se61α+EMC2-kd, Se61α+EMC5-kd (+/- Ipom-F) insertion assay                             Page 19 of 32  Fig. 5e: Phosphorimages of NT, Se61α+EMC2-kd, Se61α+EMC5-kd (+/- Ipom-F) insertion assay                     Page 20 of 32  Fig. 6b: Efficiency of single and combined SRα knockdowns; NT, SRα-kd, SRα+EMC5-kd, SRα+Sec61α-kd                                   Page 21 of 32  Fig. 6d: Phosphorimages of NT, SRα-kd, SRα+EMC5-kd, SRα+Sec61α-kd insertion assay                                   Page 22 of 32  Supplementary Fig. 1: Phosphorimages of +/- Ipom-F insertion assay in microsomes and SP cells  Microsomes                                   Page 23 of 32         SP Cells                            Page 24 of 32  Supplementary Fig. 3b: Phosphorimages of +/- Ipom-F insertion assay in microsomes                                    Page 25 of 32  Supplementary Fig. 3c: Phosphorimages to determine the immunoprecipitation efficiency OPG2-tagged TMPs                                    Page 26 of 32  Supplementary Fig. 5b: NT, Sec61α-kd, EMC2-kd, EMC5-kd, Sec61α+EMC2-kd, Sec61α+EMC5-kd, SRα-kd, SRα+EMC5-kd, SRα+Sec61α-kd Western blots  Single knockdowns of Sec61α, EMC2 and EMC5 (lanes 1-4, left panels)                 Page 27 of 32  Double knockdowns of Sec61α+EMC2 and Sec61α+EMC5 (lanes 5-7, left panels)                                   Page 28 of 32  Knockdowns of SRα (lanes 8-9, left panels)                    Page 29 of 32  Knockdowns of SRα+EMC5, SRα+Sec61α (lanes 10-12, left panels)    Page 30 of 32  Knockdowns of Sec61α, EMC2, EMC5, Sec61α+EMC5  (lanes 1-4, 7, right panels)          Supplementary Figure 6  Uncropped and unedited blot/gel images                   Page 12 of 32   Fig. 1d: Phosphorimages of +/- Ipom-F insertion assay in microsomes                  Page 13 of 32  Fig. 2c: Efficiency of single knockdowns; NT, Se61α-kd, EMC2-kd, EMC5-kd  SP Cells                                  Page 14 of 32  Microsomes                              Page 15 of 32      Fig. 3c: Phosphorimages of NT, Se61α-kd, EMC2-kd, EMC5-kd (+/- Ipom-F) insertion assay                            Page 16 of 32  Fig. 4b: Phosphorimages of NT, Se61α-kd, EMC2-kd, EMC5-kd (+/- Ipom-F) insertion assay                                   Page 17 of 32  Fig. 5a: Efficiency of double knockdowns; NT, Se61α+EMC2-kd, Se61α+EMC5                                    Page 18 of 32        Fig. 5c: Phosphorimages of NT, Se61α+EMC2-kd, Se61α+EMC5-kd (+/- Ipom-F) insertion assay                             Page 19 of 32  Fig. 5e: Phosphorimages of NT, Se61α+EMC2-kd, Se61α+EMC5-kd (+/- Ipom-F) insertion assay                     Page 20 of 32  Fig. 6b: Efficiency of single and combined SRα knockdowns; NT, SRα-kd, SRα+EMC5-kd, SRα+Sec61α-kd                                   Page 21 of 32  Fig. 6d: Phosphorimages of NT, SRα-kd, SRα+EMC5-kd, SRα+Sec61α-kd insertion assay                                   Page 22 of 32  Supplementary Fig. 1: Phosphorimages of +/- Ipom-F insertion assay in microsomes and SP cells  Microsomes                                   Page 23 of 32         SP Cells                            Page 24 of 32  Supplementary Fig. 3b: Phosphorimages of +/- Ipom-F insertion assay in microsomes                                    Page 25 of 32  Supplementary Fig. 3c: Phosphorimages to determine the immunoprecipitation efficiency OPG2-tagged TMPs                                    Page 26 of 32  Supplementary Fig. 5b: NT, Sec61α-kd, EMC2-kd, EMC5-kd, Sec61α+EMC2-kd, Sec61α+EMC5-kd, SRα-kd, SRα+EMC5-kd, SRα+Sec61α-kd Western blots  Single knockdowns of Sec61α, EMC2 and EMC5 (lanes 1-4, left panels)                 Page 27 of 32  Double knockdowns of Sec61α+EMC2 and Sec61α+EMC5 (lanes 5-7, left panels)                                   Page 28 of 32  Knockdowns of SRα (lanes 8-9, left panels)                    Page 29 of 32  Knockdowns of SRα+EMC5, SRα+Sec61α (lanes 10-12, left panels)    Page 30 of 32  Knockdowns of Sec61α, EMC2, EMC5, Sec61α+EMC5  (lanes 1-4, 7, right panels) 

An alternative pathway for membrane protein biogenesis at the endoplasmic reticulum

An alternative pathway for membrane protein biogenesis at the endoplasmic reticulum.

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An alternative pathway for membrane protein biogenesis at the endoplasmic reticulum.

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