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• W2979161746 abstract "Newly synthesized integral membrane proteins must traverse the aqueous cytosolic environment before arrival at their membrane destination and are prone to aggregation, misfolding, and mislocalization during this process. The biogenesis of integral membrane proteins therefore poses acute challenges to protein homeostasis within a cell and requires the action of effective molecular chaperones. Chaperones that mediate membrane protein targeting not only need to protect the nascent transmembrane domains from improper exposure in the cytosol, but also need to accurately select client proteins and actively guide their clients to the appropriate target membrane. The mechanisms by which cellular chaperones work together to coordinate this complex process are only beginning to be delineated. Here, we summarize recent advances in studies of the tail-anchored membrane protein targeting pathway, which revealed a network of chaperones, cochaperones, and targeting factors that together drive and regulate this essential process. This pathway is emerging as an excellent model system to decipher the mechanism by which molecular chaperones overcome the multiple challenges during post-translational membrane protein biogenesis and to gain insights into the functional organization of multicomponent chaperone networks. Newly synthesized integral membrane proteins must traverse the aqueous cytosolic environment before arrival at their membrane destination and are prone to aggregation, misfolding, and mislocalization during this process. The biogenesis of integral membrane proteins therefore poses acute challenges to protein homeostasis within a cell and requires the action of effective molecular chaperones. Chaperones that mediate membrane protein targeting not only need to protect the nascent transmembrane domains from improper exposure in the cytosol, but also need to accurately select client proteins and actively guide their clients to the appropriate target membrane. The mechanisms by which cellular chaperones work together to coordinate this complex process are only beginning to be delineated. Here, we summarize recent advances in studies of the tail-anchored membrane protein targeting pathway, which revealed a network of chaperones, cochaperones, and targeting factors that together drive and regulate this essential process. This pathway is emerging as an excellent model system to decipher the mechanism by which molecular chaperones overcome the multiple challenges during post-translational membrane protein biogenesis and to gain insights into the functional organization of multicomponent chaperone networks. Generation and maintenance of a functional proteome requires the proper folding, assembly, and localization of all of the cellular proteins. Integral membrane proteins comprise over 30% of the proteins encoded by the genome and mediate numerous essential cellular processes, including molecular transport, energy generation, signaling, and cell-to-cell communication. Compared with soluble proteins, the biogenesis of integral membrane proteins poses particularly acute challenges to protein homeostasis in the cell. Before arrival at the appropriate membrane destination, newly synthesized membrane proteins must traverse the cytosol and, in some cases, multiple other aqueous cellular compartments where improper exposure of their transmembrane domains (TMDs) 2The abbreviations used are: TMDtransmembrane domainERendoplasmic reticulumGETguided entry of tail-anchored proteinSRPsignal recognition particleEMCER membrane protein complexTAtail-anchored membrane proteinTPRtetratricopeptide repeatCaMcalmodulinUBLubiquitin-likePDBProtein Data Bank. will lead to rapid and irreversible aggregation. In addition, the degeneracy of TMD-lipid interactions poses challenges to the fidelity of their insertion at the appropriate biological membrane, especially in eukaryotic cells that contain multiple membrane-enclosed organelles. The proper localization and folding of membrane proteins therefore relies critically on molecular chaperones, which not only protect nascent membrane proteins from off-pathway interactions but also actively guide them to the correct biological membrane. The mechanism by which the cellular chaperone network overcomes these challenges during membrane protein biogenesis remains an outstanding question. transmembrane domain endoplasmic reticulum guided entry of tail-anchored protein signal recognition particle ER membrane protein complex tail-anchored membrane protein tetratricopeptide repeat calmodulin ubiquitin-like Protein Data Bank. In the past decade, an increasing number of factors have been described that represent components of multiple, distinct protein-targeting pathways that deliver nascent membrane proteins to diverse organelles such as the endoplasmic reticulum (ER), mitochondria, and peroxisomes (1Becker T. Song J. Pfanner N. Versatility of preprotein transfer from the cytosol to mitochondria.Trends Cell Biol. 2019; 29 (31030976): 534-54810.1016/j.tcb.2019.03.007Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar2Chio U.S. Cho H. Shan S.O. Mechanisms of tail-anchored membrane protein targeting and insertion.Annu. Rev. Cell Dev. Biol. 2017; 33 (28992441): 417-43810.1146/annurev-cellbio-100616-060839Crossref PubMed Scopus (61) Google Scholar, 3Cichocki B.A. Krumpe K. Vitali D.G. Rapaport D. Pex19 is involved in importing dually targeted tail-anchored proteins to both mitochondria and peroxisomes.Traffic. 2018; 19 (30033679): 770-78510.1111/tra.12604Crossref PubMed Scopus (32) Google Scholar, 4Chen Y. Pieuchot L. Loh R.A. Yang J. Kari T.M. Wong J.Y. Jedd G. Hydrophobic handoff for direct delivery of peroxisome tail-anchored proteins.Nat. Commun. 2014; 5 (25517356): 579010.1038/ncomms6790Crossref PubMed Scopus (49) Google Scholar, 5Yagita Y. Hiromasa T. Fujiki Y. Tail-anchored PEX26 targets peroxisomes via a PEX19-dependent and TRC40-independent class I pathway.J. Cell Biol. 2013; 200 (23460677): 651-66610.1083/jcb.201211077Crossref PubMed Scopus (69) Google Scholar6Borgese N. Coy-Vergara J. Colombo S.F. Schwappach B. The ways of tails: the GET pathway and more.Protein J. 2019; 38 (31203484): 289-30510.1007/s10930-019-09845-4Crossref PubMed Scopus (45) Google Scholar). One of these pathways, the guided entry of tail-anchored protein (GET) pathway, has been studied in exquisite mechanistic detail. This review will summarize recent advances in our understanding of the GET pathway, with a focus on a hierarchical chaperone network found in this pathway. These findings suggest sophisticated solutions to the challenges of membrane protein biogenesis as well as new questions about the role and mechanisms of molecular chaperones during this process. Diverse pathways mediate the targeting of nascent membrane proteins to the ER, via which proteins enter the endomembrane system in eukaryotic cells. Despite being overly simplistic, it has been useful to conceptualize the multitude of targeting mechanisms in terms of the needs of membrane proteins with distinct TMD locations. For example, most membrane proteins harboring a TMD near the N terminus are recognized by the universally conserved signal recognition particle (SRP) as soon as their first TMD emerges from the exit tunnel of the translating ribosome. Ribosome profiling work in yeast further suggested that SRP can engage ribosomes even earlier, before the targeting signals on the nascent polypeptide are translated (7Chartron J.W. Hunt K.C. Frydman J. Cotranslational signal-independent SRP preloading during membrane targeting.Nature. 2016; 536 (27487213): 224-22810.1038/nature19309Crossref PubMed Scopus (99) Google Scholar). Via interaction with the SRP receptor, SRP delivers translating ribosomes to the Sec61p translocase at the ER membrane (or the SecYEG translocase at the bacterial plasma membrane), often before an additional 60–100 residues of the nascent protein is synthesized (Fig. 1, left path) (8Walter P. Johnson A.E. Signal sequence recognition and protein targeting to the endoplasmic reticulum membrane.Annu. Rev. Cell Biol. 1994; 10 (7888184): 87-11910.1146/annurev.cb.10.110194.000511Crossref PubMed Scopus (719) Google Scholar9Akopian D. Dalal K. Shen K. Duong F. Shan S.O. SecYEG activates GTPases to drive the completion of cotranslational protein targeting.J. Cell Biol. 2013; 200 (23401005): 397-40510.1083/jcb.201208045Crossref PubMed Scopus (40) Google Scholar, 10Voorhees R.M. Hegde R.S. Toward a structural understanding of co-translational protein translocation.Curr. Opin. Cell Biol. 2016; 41 (27155805): 91-9910.1016/j.ceb.2016.04.009Crossref PubMed Scopus (67) Google Scholar, 11Costa E.A. Subramanian K. Nunnari J. Weissman J.S. Defining the physiological role of SRP in protein-targeting efficiency and specificity.Science. 2018; 359 (29348368): 689-69210.1126/science.aar3607Crossref PubMed Scopus (116) Google Scholar12Jan C.H. Williams C.C. Weissman J.S. Principles of ER cotranslational translocation revealed by proximity-specific ribosome profiling.Science. 2014; 346 (25378630): 125752110.1126/science.1257521Crossref PubMed Scopus (245) Google Scholar). The strictly co-translational nature of the SRP pathway ensures that the nascent TMDs are effectively shielded by proteinaceous environments in either the SRP or the Sec61p (or SecYEG) complex, thus minimizing exposure to the aqueous cytosolic environment during their biogenesis. Much less is known about the targeting of membrane proteins harboring internal TMDs (Fig. 1, middle path). A genetic screen identified three genetically linked SND (for SRP-independent targeting) proteins, Snd1 in the cytosol, and Snd2 and Snd3 at the ER membrane, whose loss led to mislocalization of this class of proteins (13Aviram N. Ast T. Costa E.A. Arakel E.C. Chuartzman S.G. Jan C.H. Hassdenteufel S. Dudek J. Jung M. Schorr S. Zimmermann R. Schwappach B. Weissman J.S. Schuldiner M. The SND proteins constitute an alternative targeting route to the endoplasmic reticulum.Nature. 2016; 540 (27905431): 134-13810.1038/nature20169Crossref PubMed Scopus (128) Google Scholar). More recently, the human orthologue of yeast Snd2 has been described (14Hassdenteufel S. Sicking M. Schorr S. Aviram N. Fecher-Trost C. Schuldiner M. Jung M. Zimmermann R. Lang S. hSnd2 protein represents an alternative targeting factor to the endoplasmic reticulum in human cells.FEBS Lett. 2017; 591 (28862756): 3211-322410.1002/1873-3468.12831Crossref PubMed Scopus (47) Google Scholar). Nevertheless, localized ribosome-profiling data suggested that SRP is responsible for the cotranslational ER localization of most membrane proteins containing internal TMDs (Fig. 1, dashed arrow a (11Costa E.A. Subramanian K. Nunnari J. Weissman J.S. Defining the physiological role of SRP in protein-targeting efficiency and specificity.Science. 2018; 359 (29348368): 689-69210.1126/science.aar3607Crossref PubMed Scopus (116) Google Scholar)). In addition, the SND genes are synthetically lethal with the GET genes (13Aviram N. Ast T. Costa E.A. Arakel E.C. Chuartzman S.G. Jan C.H. Hassdenteufel S. Dudek J. Jung M. Schorr S. Zimmermann R. Schwappach B. Weissman J.S. Schuldiner M. The SND proteins constitute an alternative targeting route to the endoplasmic reticulum.Nature. 2016; 540 (27905431): 134-13810.1038/nature20169Crossref PubMed Scopus (128) Google Scholar). These observations suggest that the SND components provide a backup system for the SRP and GET pathways to deliver membrane proteins with relatively downstream TMDs. Analogous diversity is observed with translocases at the ER membrane. The insertion of some SRP-dependent membrane proteins and less hydrophobic TAs are dependent on the ER membrane protein complex (EMC) (Fig. 1) (15Chitwood P.J. Juszkiewicz S. Guna A. Shao S. Hegde R.S. EMC is required to initiate accurate membrane protein topogenesis.Cell. 2018; 175 (30415835): 1507-1519.e1610.1016/j.cell.2018.10.009Abstract Full Text Full Text PDF PubMed Scopus (111) Google Scholar, 16Guna A. Volkmar N. Christianson J.C. Hegde R.S. The ER membrane protein complex is a transmembrane domain insertase.Science. 2018; 359 (29242231): 470-47310.1126/science.aao3099Crossref PubMed Scopus (147) Google Scholar17Shurtleff M.J. Itzhak D.N. Hussmann J.A. Schirle Oakdale N.T. Costa E.A. Jonikas M. Weibezahn J. Popova K.D. Jan C.H. Sinitcyn P. Vembar S.S. Hernandez H. Cox J. Burlingame A.L. Brodsky J.L. et al.The ER membrane protein complex interacts cotranslationally to enable biogenesis of multipass membrane proteins.Elife. 2018; 7 (29809151): e3701810.7554/eLife.37018Crossref PubMed Scopus (100) Google Scholar). In addition, Snd2/Snd3 genetically and physically interacts with Sec72p (13Aviram N. Ast T. Costa E.A. Arakel E.C. Chuartzman S.G. Jan C.H. Hassdenteufel S. Dudek J. Jung M. Schorr S. Zimmermann R. Schwappach B. Weissman J.S. Schuldiner M. The SND proteins constitute an alternative targeting route to the endoplasmic reticulum.Nature. 2016; 540 (27905431): 134-13810.1038/nature20169Crossref PubMed Scopus (128) Google Scholar), a component of the post-translational Sec62/63/71/72 translocase conserved across eukaryotic organisms (18Linxweiler M. Schick B. Zimmermann R. Let's talk about Secs: Sec61, Sec62 and Sec63 in signal transduction, oncology and personalized medicine.Signal Transduct. Target Ther. 2017; 2 (29263911): 1700210.1038/sigtrans.2017.2Crossref PubMed Scopus (85) Google Scholar, 19Wu 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 (75) Google Scholar). The diversity and redundancy of targeting and translocation machineries are thought to provide a robust network that accommodates the targeting needs of diverse membrane proteins with different TMD location, topology, and charge distribution. At the other extreme is the class of tail-anchored membrane proteins (TAs), whose TMD is near the C terminus (Fig. 1, right path). TAs comprise up to 5% of the eukaryotic membrane proteome and mediate diverse cellular processes, including protein translocation across organellar membranes, vesicle fusion, apoptosis, and protein quality control (2Chio U.S. Cho H. Shan S.O. Mechanisms of tail-anchored membrane protein targeting and insertion.Annu. Rev. Cell Dev. Biol. 2017; 33 (28992441): 417-43810.1146/annurev-cellbio-100616-060839Crossref PubMed Scopus (61) Google Scholar, 20Chartron J.W. Clemons Jr, W.M. Suloway C.J.M. The complex process of GETting tail-anchored membrane proteins to the ER.Curr. Opin. Struct. Biol. 2012; 22 (22444563): 217-22410.1016/j.sbi.2012.03.001Crossref PubMed Scopus (58) Google Scholar, 21Hegde R.S. Keenan R.J. Tail-anchored membrane protein insertion into the endoplasmic reticulum.Nat. Rev. Mol. Cell Biol. 2011; 12 (22086371): 787-79810.1038/nrm3226Crossref PubMed Scopus (205) Google Scholar22Kutay U. Hartmann E. Rapoport T.A. A class of membrane proteins with a C-terminal anchor.Trends Cell Biol. 1993; 3 (14731773): 72-7510.1016/0962-8924(93)90066-AAbstract Full Text PDF PubMed Scopus (268) Google Scholar). As the C-terminal TMD is obscured by the ribosome during translation, it was predicted early on that TAs undergo obligatorily post-translational mechanisms of targeting (22Kutay U. Hartmann E. Rapoport T.A. A class of membrane proteins with a C-terminal anchor.Trends Cell Biol. 1993; 3 (14731773): 72-7510.1016/0962-8924(93)90066-AAbstract Full Text PDF PubMed Scopus (268) Google Scholar). The past decade has witnessed the discovery of several pathways that mediate the targeted delivery and insertion of TAs, including the GET-, SND-, and EMC-dependent pathways (Fig. 1) (2Chio U.S. Cho H. Shan S.O. Mechanisms of tail-anchored membrane protein targeting and insertion.Annu. Rev. Cell Dev. Biol. 2017; 33 (28992441): 417-43810.1146/annurev-cellbio-100616-060839Crossref PubMed Scopus (61) Google Scholar, 6Borgese N. Coy-Vergara J. Colombo S.F. Schwappach B. The ways of tails: the GET pathway and more.Protein J. 2019; 38 (31203484): 289-30510.1007/s10930-019-09845-4Crossref PubMed Scopus (45) Google Scholar, 13Aviram N. Ast T. Costa E.A. Arakel E.C. Chuartzman S.G. Jan C.H. Hassdenteufel S. Dudek J. Jung M. Schorr S. Zimmermann R. Schwappach B. Weissman J.S. Schuldiner M. The SND proteins constitute an alternative targeting route to the endoplasmic reticulum.Nature. 2016; 540 (27905431): 134-13810.1038/nature20169Crossref PubMed Scopus (128) Google Scholar, 16Guna A. Volkmar N. Christianson J.C. Hegde R.S. The ER membrane protein complex is a transmembrane domain insertase.Science. 2018; 359 (29242231): 470-47310.1126/science.aao3099Crossref PubMed Scopus (147) Google Scholar, 20Chartron J.W. Clemons Jr, W.M. Suloway C.J.M. The complex process of GETting tail-anchored membrane proteins to the ER.Curr. Opin. Struct. Biol. 2012; 22 (22444563): 217-22410.1016/j.sbi.2012.03.001Crossref PubMed Scopus (58) Google Scholar, 21Hegde R.S. Keenan R.J. Tail-anchored membrane protein insertion into the endoplasmic reticulum.Nat. Rev. Mol. Cell Biol. 2011; 12 (22086371): 787-79810.1038/nrm3226Crossref PubMed Scopus (205) Google Scholar, 23Cho H. Shan S.O. Substrate relay in an Hsp70-cochaperone cascade safeguards tail-anchored membrane protein targeting.EMBO J. 2018; 37 (29973361): e9926410.15252/embj.201899264Crossref PubMed Scopus (30) Google Scholar). The GET pathway, which targets relatively hydrophobic TAs to the ER, is especially well-studied. This pathway is also remarkably conserved among eukaryotic cells; all of the components in the yeast GET pathway have orthologues or functional homologs in mammalian cells. The readers are referred to Refs. 2Chio U.S. Cho H. Shan S.O. Mechanisms of tail-anchored membrane protein targeting and insertion.Annu. Rev. Cell Dev. Biol. 2017; 33 (28992441): 417-43810.1146/annurev-cellbio-100616-060839Crossref PubMed Scopus (61) Google Scholar, 6Borgese N. Coy-Vergara J. Colombo S.F. Schwappach B. The ways of tails: the GET pathway and more.Protein J. 2019; 38 (31203484): 289-30510.1007/s10930-019-09845-4Crossref PubMed Scopus (45) Google Scholar, 20Chartron J.W. Clemons Jr, W.M. Suloway C.J.M. The complex process of GETting tail-anchored membrane proteins to the ER.Curr. Opin. Struct. Biol. 2012; 22 (22444563): 217-22410.1016/j.sbi.2012.03.001Crossref PubMed Scopus (58) Google Scholar, and 21Hegde R.S. Keenan R.J. Tail-anchored membrane protein insertion into the endoplasmic reticulum.Nat. Rev. Mol. Cell Biol. 2011; 12 (22086371): 787-79810.1038/nrm3226Crossref PubMed Scopus (205) Google Scholar for comprehensive reviews of the GET pathway and the targeting of tail-anchored proteins in general. Here, I will focus on the works that uncovered and characterized a multicomponent chaperone system required for the biogenesis of this essential class of membrane proteins. Components of the GET pathway were initially identified through biochemical reconstitutions and genetic interaction analyses of the secretory pathway in yeast. Work in rabbit reticulocyte lysate identified a 40-kDa ATPase, TRC40, which cross-links efficiently to the C-terminal TMD of model TAs and allows insertion of the bound TA into ER microsomes (24Favaloro V. Vilardi F. Schlecht R. Mayer M.P. Dobberstein B. Asna1/TRC40-mediated membrane insertion of tail-anchored proteins.J. Cell Sci. 2010; 123 (20375064): 1522-153010.1242/jcs.055970Crossref PubMed Scopus (48) Google Scholar, 25Stefanovic S. Hegde R.S. Identification of a targeting factor for post-translational membrane protein insertion into the ER.Cell. 2007; 128 (17382883): 1147-115910.1016/j.cell.2007.01.036Abstract Full Text Full Text PDF PubMed Scopus (312) Google Scholar). The yeast homologue of TRC40, Get3, was epistatically linked to two ER-localized membrane proteins, Get1 and Get2 (26Schuldiner M. Collins S.R. Thompson N.J. Denic V. Bhamidipati A. Punna T. Ihmels J. Andrews B. Boone C. Greenblatt J.F. Weissman J.S. Krogan N.J. Exploration of the function and organization of the yeast early secretory pathway through an epistatic miniarray profile.Cell. 2005; 123 (16269340): 507-51910.1016/j.cell.2005.08.031Abstract Full Text Full Text PDF PubMed Scopus (688) Google Scholar), which were subsequently shown to act as both a receptor complex for TA-loaded Get3 and a translocase that mediates TA insertion into the ER membrane (Fig. 2, step 7) (27Schuldiner M. Metz J. Schmid V. Denic V. Rakwalska M. Schmitt H.D. Schwappach B. Weissman J.S. The GET complex mediates insertion of tail-anchored proteins into the ER membrane.Cell. 2008; 134 (18724936): 634-64510.1016/j.cell.2008.06.025Abstract Full Text Full Text PDF PubMed Scopus (366) Google Scholar, 28Wang 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 (81) Google Scholar). As the central targeting factor in the GET pathway, the structure, dynamics, and activity of Get3 have been extensively studied, providing a high-resolution mechanistic model for how this targeting factor couples its ATPase cycle to the ER targeting of TAs (see “Get3: An ATP-driven protean clamp”). Nevertheless, it soon became clear that TA capture by Get3 (or TRC40) is a facilitated process in the crowded cytosolic environment. Nascent TA released from the ribosome was poorly captured by partially purified TRC40 (29Mariappan M. Li X. Stefanovic S. Sharma A. Mateja A. Keenan R.J. Hegde R.S. A ribosome-associating factor chaperones tail-anchored membrane proteins.Nature. 2010; 466 (20676083): 1120-112410.1038/nature09296Crossref PubMed Scopus (205) Google Scholar). Using biochemical reconstitutions, Wang et al. showed that the products of two additional genes epistatically linked to Get3, Get4 and Get5, form a scaffold complex that bridges between Get3 and an upstream cochaperone, Sgt2, and facilitates TA transfer from Sgt2 to Get3 (Fig. 2, steps 5–6) (30Wang F. Brown E.C. Mak G. Zhuang J. Denic V. A chaperone cascade sorts proteins for posttranslational membrane insertion into the endoplasmic reticulum.Mol. Cell. 2010; 40 (20850366): 159-17110.1016/j.molcel.2010.08.038Abstract Full Text Full Text PDF PubMed Scopus (142) Google Scholar). In mammalian cytosol, the C-terminal part of the BAG6 complex (composed of BAG6, TRC35, and UBL4A) was shown to be structurally and functionally homologous to Get4/5 and facilitates TA loading onto TRC40 from SGTA, the mammalian Sgt2 homologue (29Mariappan M. Li X. Stefanovic S. Sharma A. Mateja A. Keenan R.J. Hegde R.S. A ribosome-associating factor chaperones tail-anchored membrane proteins.Nature. 2010; 466 (20676083): 1120-112410.1038/nature09296Crossref PubMed Scopus (205) Google Scholar, 31Mock J.Y. Chartron J.W. Zaslaver M. Xu Y. Ye Y. Clemons Jr., W.M. Bag6 complex contains a minimal tail-anchor-targeting module and a mock BAG domain.Proc. Natl. Acad. Sci. U.S.A. 2015; 112 (25535373): 106-11110.1073/pnas.1402745112Crossref PubMed Scopus (63) Google Scholar, 32Shao S. Rodrigo-Brenni M.C. Kivlen M.H. Hegde R.S. Mechanistic basis for a molecular triage reaction.Science. 2017; 355 (28104892): 298-30210.1126/science.aah6130Crossref PubMed Scopus (77) Google Scholar). Thus, the substrate loading mechanism via the Sgt2-to-Get3 transfer is conserved among eukaryotic cells. Despite these advances, how newly synthesized TAs are captured by Sgt2 remained a longstanding puzzle. Purified Sgt2 is ineffective in capturing TAs in the soluble form, and attempts to directly load TA onto Sgt2 led to extensively aggregated complexes (23Cho H. Shan S.O. Substrate relay in an Hsp70-cochaperone cascade safeguards tail-anchored membrane protein targeting.EMBO J. 2018; 37 (29973361): e9926410.15252/embj.201899264Crossref PubMed Scopus (30) Google Scholar). For many years, generation of soluble, functional Sgt2·TA or SGTA·TA complexes has relied on cell lysates that contain endogenous chaperone (30Wang F. Brown E.C. Mak G. Zhuang J. Denic V. A chaperone cascade sorts proteins for posttranslational membrane insertion into the endoplasmic reticulum.Mol. Cell. 2010; 40 (20850366): 159-17110.1016/j.molcel.2010.08.038Abstract Full Text Full Text PDF PubMed Scopus (142) Google Scholar, 33Rao M. Okreglak V. Chio U.S. Cho H. Walter P. Shan S.O. Multiple selection filters ensure accurate tail-anchored membrane protein targeting.Elife. 2016; 5 (27925580): e2130110.7554/eLife.21301Crossref PubMed Scopus (52) Google Scholar) or superphysiological Sgt2/SGTA concentrations (32Shao S. Rodrigo-Brenni M.C. Kivlen M.H. Hegde R.S. Mechanistic basis for a molecular triage reaction.Science. 2017; 355 (28104892): 298-30210.1126/science.aah6130Crossref PubMed Scopus (77) Google Scholar). Importantly, Sgt2 contains a conserved tetratricopeptide repeat (TPR) domain that associates with multiple heat shock proteins, including Hsp70, Hsp90, and Hsp104 (30Wang F. Brown E.C. Mak G. Zhuang J. Denic V. A chaperone cascade sorts proteins for posttranslational membrane insertion into the endoplasmic reticulum.Mol. Cell. 2010; 40 (20850366): 159-17110.1016/j.molcel.2010.08.038Abstract Full Text Full Text PDF PubMed Scopus (142) Google Scholar, 34Krysztofinska E.M. Evans N.J. Thapaliya A. Murray J.W. Morgan R.M.L. Martinez-Lumbreras S. Isaacson R.L. Structure and interactions of the TPR domain of Sgt2 with yeast chaperones and Ybr137wp.Front. Mol. Biosci. 2017; 4 (29075633): 6810.3389/fmolb.2017.00068Crossref PubMed Scopus (14) Google Scholar, 35Chartron J.W. Gonzalez G.M. Clemons Jr., W.M. A structural model of the Sgt2 protein and its interactions with chaperones and the Get4/Get5 complex.J. Biol. Chem. 2011; 286 (21832041): 34325-3433410.1074/jbc.M111.277798Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar). This observation led to the hypothesis that heat shock proteins are further required to facilitate TA loading on Sgt2 (35Chartron J.W. Gonzalez G.M. Clemons Jr., W.M. A structural model of the Sgt2 protein and its interactions with chaperones and the Get4/Get5 complex.J. Biol. Chem. 2011; 286 (21832041): 34325-3433410.1074/jbc.M111.277798Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar). Experimental evidence for this model emerged recently through the work of Cho et al. (23Cho H. Shan S.O. Substrate relay in an Hsp70-cochaperone cascade safeguards tail-anchored membrane protein targeting.EMBO J. 2018; 37 (29973361): e9926410.15252/embj.201899264Crossref PubMed Scopus (30) Google Scholar), who demonstrated that the major cytosolic Hsp70 in yeast, Ssa1, is highly effective in capturing newly synthesized TAs and efficiently transfers the bound TAs to Sgt2, in a manner dependent on its interaction with the Sgt2 TPR motif. In vivo, transient inactivation of Ssa1 severely disrupted TA insertion into the ER, analogous to observations with GET gene deletions (23Cho H. Shan S.O. Substrate relay in an Hsp70-cochaperone cascade safeguards tail-anchored membrane protein targeting.EMBO J. 2018; 37 (29973361): e9926410.15252/embj.201899264Crossref PubMed Scopus (30) Google Scholar). Together, Hsp70, Sgt2, Get4/5, and Get3 form the minimal components that allow reconstitution of the molecular events required to generate a soluble, translocation-competent targeting complex in the cytosol (23Cho H. Shan S.O. Substrate relay in an Hsp70-cochaperone cascade safeguards tail-anchored membrane protein targeting.EMBO J. 2018; 37 (29973361): e9926410.15252/embj.201899264Crossref PubMed Scopus (30) Google Scholar). Collectively, these works demonstrate that even a compositionally simple integral membrane protein, such as the TA, is sequentially funneled through a multicomponent Hsp70-cochaperone cascade (Fig. 2). Newly synthesized TAs released from the ribosome are captured by Ssa1, which effectively shields the TA-TMD from aggregation in the aqueous cytosol (steps 1–2). Ssa1 assembles the first transfer complex via interaction of its C terminus with the TPR domain of Sgt2, in which TA is rapidly transferred (steps 3–4). A second client transfer complex is assembled via the Get4/5 scaffold complex, which bridges between Sgt2 and Get3 to facilitate TA transfer onto Get3 (step 5). The TA is then delivered to the ER membrane via the interaction of Get3 with the Get1/2 receptors (steps 6–7). Although the complexity of the GET pathway is counterintuitive, many observations in this pathway suggest potential chemical and biological rationales for the evolution of this elaborate chaperone cascade, and their further investigation could provide valuable insights into the roles and organization principles of chaperone networks in general. Below, I highlight and discuss the implications of some of these observations, with the hope to stimulate additional studies into this and conceptually analogous multicomponent chaperone systems. What drives the directional substrate transfers in the GET pathway? Quantitative measurements suggested that both the Ssa1-to-Sgt2 and Sgt2-to-Get3 TA transfers are energetically downhill, with the transfer equilibrium ∼100- and ∼20-fold in favor of the downstream chaperone in the respective transfer complexes (23Cho H. Shan S.O. Substrate relay in an Hsp70-cochaperone cascade safeguards tail-anchored membrane protein targeting.EMBO J. 2018; 37 (29973361): e9926410.15252/embj.201899264Crossref PubMed Scopus (30) Google Scholar, 33Rao M. Okreglak V. Chio U.S. Cho H. Walter P. Shan S.O. Multiple selection filters ensure accurate tail-anchored membrane protein targeting.Elife. 2016; 5 (27925580): e2130110.7554/eLife.21301Crossref PubMed Scopus (52) Google Scholar). This implies that the downstream chaperones bind TAs much more strongly than their respective upstream chaperones, and TA transfer in the reverse direction is unfavorable under physiological conditions. Measurements of the kine" @default.
• W2979161746 created "2019-10-10" @default.
• W2979161746 creator A5000584662 @default.
• W2979161746 date "2019-11-01" @default.
• W2979161746 modified "2023-10-16" @default.
• W2979161746 title "Guiding tail-anchored membrane proteins to the endoplasmic reticulum in a chaperone cascade" @default.
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