C-terminal tail length guides insertion and assembly of membrane proteins
2020; Elsevier BV; Volume: 295; Issue: 46 Linguagem: Inglês
10.1074/jbc.ra120.012992
ISSN1083-351X
AutoresSha Sun, Malaiyalam Mariappan,
Tópico(s)RNA and protein synthesis mechanisms
ResumoA large number of newly synthesized membrane proteins in the endoplasmic reticulum (ER) are assembled into multiprotein complexes, but little is known about the mechanisms required for assembly membrane proteins. It has been suggested that membrane chaperones might exist, akin to the molecular chaperones that stabilize and direct the assembly of soluble protein complexes, but the mechanisms by which these proteins would bring together membrane protein components is unclear. Here, we have identified that the tail length of the C-terminal transmembrane domains (C-TMDs) determines efficient insertion and assembly of membrane proteins in the ER. We found that membrane proteins with C-TMD tails shorter than ∼60 amino acids are poorly inserted into the ER membrane, which suggests that translation is terminated before they are recognized by the Sec61 translocon for insertion. These C-TMDs with insufficient hydrophobicity are post-translationally recognized and retained by the Sec61 translocon complex, providing a time window for efficient assembly with TMDs from partner proteins. Retained TMDs that fail to assemble with their cognate TMDs are slowly translocated into the ER lumen and are recognized by the ER-associated degradation (ERAD) pathway for removal. In contrast, C-TMDs with sufficient hydrophobicity or tails longer than ∼80 residues are quickly released from the Sec61 translocon into the membrane or the ER lumen, resulting in inefficient assembly with partner TMDs. Thus, our data suggest that C-terminal tails harbor crucial signals for both the insertion and assembly of membrane proteins. A large number of newly synthesized membrane proteins in the endoplasmic reticulum (ER) are assembled into multiprotein complexes, but little is known about the mechanisms required for assembly membrane proteins. It has been suggested that membrane chaperones might exist, akin to the molecular chaperones that stabilize and direct the assembly of soluble protein complexes, but the mechanisms by which these proteins would bring together membrane protein components is unclear. Here, we have identified that the tail length of the C-terminal transmembrane domains (C-TMDs) determines efficient insertion and assembly of membrane proteins in the ER. We found that membrane proteins with C-TMD tails shorter than ∼60 amino acids are poorly inserted into the ER membrane, which suggests that translation is terminated before they are recognized by the Sec61 translocon for insertion. These C-TMDs with insufficient hydrophobicity are post-translationally recognized and retained by the Sec61 translocon complex, providing a time window for efficient assembly with TMDs from partner proteins. Retained TMDs that fail to assemble with their cognate TMDs are slowly translocated into the ER lumen and are recognized by the ER-associated degradation (ERAD) pathway for removal. In contrast, C-TMDs with sufficient hydrophobicity or tails longer than ∼80 residues are quickly released from the Sec61 translocon into the membrane or the ER lumen, resulting in inefficient assembly with partner TMDs. Thus, our data suggest that C-terminal tails harbor crucial signals for both the insertion and assembly of membrane proteins. Membrane proteins represent one-third of the proteins encoded by the human genome and carry out essential cellular processes including molecular transport, signaling, and cell–cell communication (1Krogh A. Larsson B. von Heijne G. Sonnhammer E.L. Predicting transmembrane protein topology with a hidden Markov model: application to complete genomes.J. Mol. Biol. 2001; 305 (11152613): 567-58010.1006/jmbi.2000.4315Crossref PubMed Scopus (8103) Google Scholar, 2Shao S. 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Here, there, and everywhere: the importance of ER membrane contact sites.Science. 2018; 361: eaan583510.1126/science.aan5835Crossref PubMed Scopus (197) Google Scholar), thus diluting their concentration as well as reducing their chance to find each other. Last, proteins have often the propensity to form homooligomers because they are translated from the same polysome and their local subunit concentration is high, thus reducing the chance to assemble with the partner protein. In recent years, much attention has been focused on understanding the assembly of soluble protein complexes (15Schwarz A. Beck M. The benefits of cotranslational assembly: a structural perspective.Trends Cell Biol. 2019; 29 (31427208): 791-80310.1016/j.tcb.2019.07.006Abstract Full Text Full Text PDF PubMed Scopus (20) Google Scholar). These studies propose two prevailing models. First, the large protein complexes such as proteasome often have dedicated molecular chaperones that can shield the nascent proteins from inappropriate interactions and facilitate assembly with partner proteins (16Le Tallec B. Barrault M.B. Courbeyrette R. Guérois R. Marsolier-Kergoat M.C. Peyroche A. 20S proteasome assembly is orchestrated by two distinct pairs of chaperones in yeast and in mammals.Mol. Cell. 2007; 27 (17707236): 660-67410.1016/j.molcel.2007.06.025Abstract Full Text Full Text PDF PubMed Scopus (136) Google Scholar). Second, the newly synthesized proteins can be co-translationally assembled into heterooligomeric complexes in eukaryotes (17Shiber A. Döring K. Friedrich U. Klann K. Merker D. Zedan M. Tippmann F. Kramer G. Bukau B. Cotranslational assembly of protein complexes in eukaryotes revealed by ribosome profiling.Nature. 2018; 561 (30158700): 268-27210.1038/s41586-018-0462-yCrossref PubMed Scopus (109) Google Scholar). In contrast to soluble protein complexes, less is known about the assembly of membrane protein complexes in the ER membrane, where the majority of the membrane proteins are synthesized in cells. Pioneering early studies have used the T-cell receptor (TCR) complex as a model membrane protein complex to investigate this problem (18Manolios N. Bonifacino J.S. Klausner R.D. Transmembrane helical interactions and the assembly of the T cell receptor complex.Science. 1990; 249 (2142801): 274-27710.1126/science.2142801Crossref PubMed Scopus (189) Google Scholar). The opposite charge residues in TMDs of membrane proteins drive assembly by forming ionic bonds between subunits (18Manolios N. Bonifacino J.S. Klausner R.D. Transmembrane helical interactions and the assembly of the T cell receptor complex.Science. 1990; 249 (2142801): 274-27710.1126/science.2142801Crossref PubMed Scopus (189) Google Scholar, 19Bonifacino J.S. Cosson P. Klausner R.D. 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However, it is unclear how these two different subunits find each other in the extensive ER membrane network after their synthesis and form a specific ionic pair. Moreover, this is even less understood for assembly of polytopic membrane protein complexes, but it is often proposed that membrane chaperones may mediate the assembly process. However, experimental evidence of membrane chaperones mediating the assembly of membrane protein complexes is lacking. To reveal the biochemical features that are necessary for both assembly and degradation of membrane protein complexes, we investigated the assembly of the tail-anchored membrane protein insertion complex composed of WRB and CAML proteins (22Vilardi F. Lorenz H. Dobberstein B. WRB is the receptor for TRC40/Asna1-mediated insertion of tail-anchored proteins into the ER membrane.J. Cell Sci. 2011; 124 (21444755): 1301-130710.1242/jcs.084277Crossref PubMed Scopus (72) Google Scholar, 23Vilardi F. Stephan M. Clancy A. Janshoff A. 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The WRB subunit of the Get3 receptor is required for the correct integration of its partner CAML into the ER.Sci. Rep. 2019; 9 (31417168): 1188710.1038/s41598-019-48363-2Crossref PubMed Scopus (6) Google Scholar). In the absence of WRB, CAML exhibits two different topologies: one with both TMD2 and TMD3 translocated into the ER lumen and the other with only TMD2 translocated into the ER lumen (25Inglis A.J. Page K.R. Guna A. Voorhees R.M. Differential modes of orphan subunit recognition for the WRB/CAML complex.Cell Rep. 2020; 30 (32187542): 3691-3698.e510.1016/j.celrep.2020.02.084Abstract Full Text Full Text PDF PubMed Scopus (6) Google Scholar, 26Carvalho H.J.F. Del Bondio A. Maltecca F. Colombo S.F. Borgese N. The WRB subunit of the Get3 receptor is required for the correct integration of its partner CAML into the ER.Sci. Rep. 2019; 9 (31417168): 1188710.1038/s41598-019-48363-2Crossref PubMed Scopus (6) Google Scholar). The molecular mechanism by which newly synthesized WRB and CAML are brought together for efficient assembly is unclear. In this study, we identified that the Sec61 translocon can post-translationally retain membrane proteins. This retention mechanism depends on less hydrophobic C-TMDs with shorter tails. The Sec61 translocon-retained TMD provides a time window for efficient assembly with TMDs from partner membrane proteins. If they missed assembly with their partner TMDs, C-TMDs are slowly translocated into the ER lumen and are recognized for degradation by ERAD. Thus, our studies suggest that the C-terminal TMD and its flanking cytosolic tails are important biochemical features for the assembly of membrane protein complexes. To determine the biochemical features that are required for the assembly of multimembrane protein complexes, we sought for model membrane protein substrates that should fulfill three criteria. First, they must be relatively small and amenable to biochemical manipulations. Second, they should be quickly degraded when they failed to assemble. Third, degradation signals or degrons should be buried when they are assembled, but exposed when they fail to assemble. We reasoned that tail-anchored membrane protein insertase complex, WRB and CAML, may be ideal substrates to investigate this fundamental problem because both proteins are relatively small in their sizes (20 and 33 kDa, respectively) with each having three TMDs (Fig. 1A). We first tested whether these proteins are degraded when transiently expressed individually in HEK293 cells. The cycloheximide chase assay revealed that the expression of either WRB or CAML alone led to degradation in a proteasomal dependent manner because the turnover could be inhibited by the proteasomal inhibitor MG132 (Fig. 1, B and C). However, the co-expression of both WRB and CAML slowed their degradation compared with when they were expressed separately (Fig. 1D). We hypothesized that the unassembled membrane proteins must expose a degradation signal or degron for recognition by ERAD. To investigate this, we focused on WRB as a model substrate. We reasoned that the degron must lie in one of three TMDs of WRB because WRB lacks a prominent luminal domain. Also, the cytosolic tryptophan-rich domain of WRB is functional without the rest of the protein (22Vilardi F. Lorenz H. Dobberstein B. WRB is the receptor for TRC40/Asna1-mediated insertion of tail-anchored proteins into the ER membrane.J. Cell Sci. 2011; 124 (21444755): 1301-130710.1242/jcs.084277Crossref PubMed Scopus (72) Google Scholar). Therefore, we swapped one TMD at a time with a stable TMD from either Sec61β or Ost4p (Fig. 1E) based on the previously described protocol (27Wang F. Chan C. Weir N.R. Denic V. The Get1/2 transmembrane complex is an endoplasmic-reticulum membrane protein insertase.Nature. 2014; 512 (25043001): 441-44410.1038/nature13471Crossref PubMed Scopus (63) Google Scholar). Replacing the first TMD of WRB led to the degradation similar to the WT (Fig. 1F). The second TMD swap exhibited a slightly faster degradation. Remarkably, swapping the 3rd TMD resulted in the complete stabilization of WRB, suggesting that the 3rd TMD contains a degron (Fig. 1F). The close inspection of the amino acid sequence of the C-TMD revealed that it has a single positively charged lysine residue (Fig. 1G). To test whether the lysine residue in the C-TMD is required for the recognition by ERAD, we mutated lysine to the hydrophobic leucine residue and analyzed the residue by protein turnover assay. In agreement with our view, the degradation of WRB (K164L) was completely inhibited compared with the WT (Fig. 1H), confirming that the lysine residue in the C-TMD is crucial for degradation. To our surprise, replacing the lysine residue with either a charged residue or a hydrophilic residue also caused destabilization (Fig. 1H). These results suggested that the lysine residue in the C-TMD of WRB is not the direct signal for degradation, but rather indicated that the overall hydrophobicity of the C-TMD plays a role in recognition by ERAD factors for degradation. Because the C-TMD of WRB has a relatively low apparent free energy (ΔGapp = +2.28) (28Hessa T. Kim H. Bihlmaier K. Lundin C. Boekel J. Andersson H. Nilsson I. White S.H. von Heijne G. Recognition of transmembrane helices by the endoplasmic reticulum translocon.Nature. 2005; 433 (15674282): 377-38110.1038/nature03216Crossref PubMed Scopus (753) Google Scholar, 29Hessa T. Meindl-Beinker N.M. Bernsel A. Kim H. Sato Y. Lerch-Bader M. Nilsson I. White S.H. von Heijne G. Molecular code for transmembrane-helix recognition by the Sec61 translocon.Nature. 2007; 450 (18075582): 1026-103010.1038/nature06387Crossref PubMed Scopus (494) Google Scholar), we asked whether the C-TMD is properly inserted into the ER membrane. A negative value of free energy indicates that the sequence can be efficiently recognized as a TMD by the Sec61 translocon and integrated into the lipid bilayer. Conversely, a positive value of free energy indicates that the sequence is less efficiently inserted unless it is helped by interactions with neighboring TMDs (30Ojemalm K. Halling K.K. Nilsson I. von Heijne G. Orientational preferences of neighboring helices can drive ER insertion of a marginally hydrophobic transmembrane helix.Mol. Cell. 2012; 45 (22281052): 529-54010.1016/j.molcel.2011.12.024Abstract Full Text Full Text PDF PubMed Scopus (41) Google Scholar). To test if the C-TMD of WRB is translocated into the ER lumen, we appended a glycosylation site (NFT) at the C terminus of WRB (Fig. S1A). Interestingly, about 37% of the C-TMD of WRB was translocated into the ER lumen (Fig. 2A). The glycosylated band of WRB was verified by the treatment with endoglycosidase H (Endo H). The pulse-chase experiment revealed that both the glycosylated (translocated) and nonglycosylated (inserted) forms of WRB were recognized by ERAD for degradation (Fig. 2B). We next asked whether the C-TMD translocation into the ER lumen is a common feature for unassembled membrane proteins. To address this, we looked for other membrane protein complexes. The Sec61 translocon is composed of three subunits, α, β, and γ. We reasoned that the C-TMD of the α subunit, which has a low apparent free energy of +2.95 (Fig. 2C), might translocate into the ER lumen if it failed to assemble with other two subunits. To test this, we added an N-glycosylation motif to the C terminus of Sec61α and monitored its translocation into the lumen. Consistent with our prediction, a small fraction of Sec61α was translocated into the ER lumen as shown by an Endo H-sensitive glycosylated band (Fig. 2C). We next tested the translocation of the C-TMD of TRAPγ, which forms a heterotrimeric complex with α and β subunits (31Lang S. Pfeffer S. Lee P.H. Cavalie A. Helms V. Forster F. Zimmermann R. An update on Sec61 channel functions, mechanisms, and related diseases.Front. Physiol. 2017; 8 (29163222): 88710.3389/fphys.2017.00887Crossref PubMed Scopus (45) Google Scholar). The C-TMD of TRAPγ exhibits a low apparent free energy (ΔGapp = +2.06) due to the presence of several hydrophilic serine residues (Fig. 2D). About 50% of TRAPγ was translocated into the ER lumen as shown by the glycosylation assay (Fig. 2D). We further examined whether the translocation of the C-TMD of a polytopic membrane protein might be an indicator of defects in the assembly of TMDs within the same protein. To test this, we used peripheral myelin protein 22 (PMP22) as a model substrate because many patients affected by Charcot-Marie-Tooth disease possess mutations in TMDs of PMP22. We reasoned that introducing a patient mutation into the C-TMD of PMP22 (32Navon R. Seifried B. Gal-On N.S. Sadeh M. A new point mutation affecting the fourth transmembrane domain of PMP22 results in severe de novo Charcot-Marie-Tooth disease.Hum. Genet. 1996; 97 (8655153): 685-68710.1007/BF02281883Crossref PubMed Scopus (43) Google Scholar) may lead to its translocation into the ER lumen due to defects in the assembly with neighboring TMDs. Although C-TMD of WT PMP22 was not translocated into the ER, the C-TMD of PMP22 carrying the positively charged arginine residue was translocated and thereby glycosylated in the ER lumen (Fig. 2E). Collectively, these findings suggest that translocation of the C-TMD is a general phenomenon for unassembled membrane proteins. We investigated whether the translocation of C-TMD into the ER lumen is caused by its low hydrophobicity. To test this, we constructed variants of C-TMDs with either increasing hydrophobicity by introducing hydrophobic leucine residues or decreasing hydrophobicity by introducing hydrophilic residues (Fig. 3A). We analyzed the translocation of C-TMDs of WRB variants by the glycosylation assay combined with immunoblotting. C-TMDs of WRBs with insufficient hydrophobicity were translocated into the ER lumen as evidenced by glycosylated bands (Fig. 3B). The efficiency of the C-TMD translocation generally correlated with its hydrophobicity. For instance, about 90% of very hydrophilic C-TMD of WRB (DDRRK) translocated into the ER lumen. Strikingly, increasing the hydrophobicity of C-TMD did not prevent its translocation into the ER lumen, arguing that strong hydrophobicity of a C-TMD is not a determinant for insertion into the membrane (Fig. 3B). This result is further corroborated with the data derived from metabolically labeled cells expressing WRB variants as well as the data from an in vitro experiment where WRB variants were translated in the presence of rough microsomes (Fig. 3C, Fig. S2). We asked why the C-TMD with strong hydrophobicity does not obey the biological hydrophobicity (29Hessa T. Meindl-Beinker N.M. Bernsel A. Kim H. Sato Y. Lerch-Bader M. Nilsson I. White S.H. von Heijne G. Molecular code for transmembrane-helix recognition by the Sec61 translocon.Nature. 2007; 450 (18075582): 1026-103010.1038/nature06387Crossref PubMed Scopus (494) Google Scholar), as it is not efficiently inserted into the membrane. We reasoned that when the translation is terminated, most of the sequence of C-TMD and its tail might still be within the exit tunnel of the ribosome, which can accommodate ∼40 amino acids (33Voss N.R. Gerstein M. Steitz T.A. Moore P.B. The geometry of the ribosomal polypeptide exit tunnel.J. Mol. Biol. 2006; 360 (16784753): 893-90610.1016/j.jmb.2006.05.023Crossref PubMed Scopus (225) Google Scholar) (Fig. S1A). This might force the translocon to post-translationally recognize and insert the C-TMD into the lipid bilayer. We hypothesized this post-translational recognition of C-TMD by the Sec61 translocon might be slow and inefficient because the previous structural studies have shown that the lateral gate of the translocon is closed in the absence ribosome (34Wu X. Cabanos C. Rapoport T.A. Structure of the post-translational protein translocation machinery of the ER membrane.Nature. 2019; 566 (30644436): 136-13910.1038/s41586-018-0856-xCrossref PubMed Scopus (48) Google Scholar, 35Voorhees R.M. Hegde R.S. Structure of the Sec61 channel opened by a signal sequence.Science. 2016; 351 (26721998): 88-9110.1126/science.aad4992Crossref PubMed Scopus (104) Google Scholar). To test this idea, we increased the length of the cytosolic C-tail by appending a large Venus tag that comprises 238 amino acids (Fig. S1C). In support of our hypothesis, the C-TMD translocation of WRB-Venus variants with strong hydrophobicity was significantly prevented as shown by both immunoblotting and metabolic labeling results (Fig. 3, D and E). Consistent with the biological hydrophobicity scale (29Hessa T. Meindl-Beinker N.M. Bernsel A. Kim H. Sato Y. Lerch-Bader M. Nilsson I. White S.H. von Heijne G. Molecular code for transmembrane-helix recognition by the Sec61 translocon.Nature. 2007; 450 (18075582): 1026-103010.1038/nature06387Crossref PubMed Scopus (494) Google Scholar), C-terminal TMDs with insufficient hydrophobicity were translocated into the ER lumen. Of note, WRB-Venus with insufficient hydrophobicity exhibited more efficient translocation into the ER lumen compared with WRB constructs with small tails (compare Fig. 3, C and E). These results suggest that the long cytosolic C-terminal tail is important for both efficient insertion of strong hydrophobic C-TMD and translocation of less hydrophobic C-TMD into the ER lumen. We next wanted to determine the minimum C-terminal cytosolic tail length required for either insertion of strong hydrophobic C-TMDs or translocation of insufficient hydrophobic C-TMDs. To this end, we prepared WRB (K164D) constructs with varying C-terminal length and tested them for insertion or translocation by metabolic labeling and immunoprecipitation. We found ∼100 residues at the C terminus rendered efficient translocation of C-TMD with insufficient hydrophobicity (Fig. 4F). In contrast, about 60 residues at the C terminus were sufficient to prevent the translocation of C-TMDs with strong hydrophobicity (Fig. 3F). This result is consistent with the model that the presence of the ribosome at the translocon is crucial for efficient insertion of C-TMDs with strong hydrophobicity, presumably by trigging the opening of the lateral gate of the translocon (Fig. 3G). Collectively, these findings suggest that hydrophobicity alone is not sufficient for the insertion of a TMD and that the C-terminal tail length also influences the insertion of a TMD into the ER membrane. We hypothesized that the translocation of C-TMD of WRB into the ER lumen may be slow to provide a time window for assembly with the partner protein CAML. To test this hypothesis, we monitored the dynamics of C-TMD translocation by performing pulse-chase experiments. The cells expressing WRB constructs with short C-terminal tails were briefly labeled and chased for 3 h. Also, we treated cells with a p97 ATPase inhibitor during the chase period to prevent retrotranslocation and degradation of WRB, thus allowing us to quantify the translocated C-TMD without losing the signal from degradation. A proportion of the C-TMD of WT WRB containing a positively charged residue translocated into the ER lumen even during labeling (0 h) as shown by glycosylation (Fig. 4A). Intriguingly, about 25% of the C-TMD of WRB was continuously post-translationally translocated into the ER lumen as reflected by increased glycosylation signals during the chase period (Fig. 4A). In contrast, the nonglycosylated inserted form was significantly reduced during the chase period. This reduction is likely caused by both continuous translocation into the ER lumen and degradation even in the presence of the p97 ATPase inhibitor. The inhibitor did not completely block the degradation as this was evidenced by the continuous loss of total signal during the chase period (Fig. 4A). The C-TMD containing a negative charge residue (K164D) also yielded a similar result (Fig. 4B). In sharp contrast, the translocation of C-TMD of WRB (K164L) with sufficient hydrophobicity occurred only during labeling, but did not translocate post-translationally into the ER lumen during the chase period (Fig. 4C). We then investigated whether the length of the C-tail influences the translocation dynamics of C-TMD into the ER lumen. In contrast to the short C-ter
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