[ { "id": "authors:aeex6-fvw30", "collection": "authors", "collection_id": "aeex6-fvw30", "cite_using_url": "https://resolver.caltech.edu/CaltechAUTHORS:20230519-1772000.24", "type": "article", "title": "Dodecamer assembly of a metazoan AAA\u207a chaperone couples substrate extraction to refolding", "author": [ { "family_name": "Gupta", "given_name": "Arpit", "orcid": "0000-0002-8573-8818", "clpid": "Gupta-Arpit" }, { "family_name": "Lentzsch", "given_name": "Alfred M.", "orcid": "0000-0002-8150-0511", "clpid": "Lentzsch-Alfred-M" }, { "family_name": "Siegel", "given_name": "Alex", "orcid": "0000-0003-3601-5178", "clpid": "Siegel-Alex" }, { "family_name": "Yu", "given_name": "Zanlin", "orcid": "0000-0001-8381-8788", "clpid": "Yu-Zanlin" }, { "family_name": "Chio", "given_name": "Un Seng", "orcid": "0000-0002-5295-2690", "clpid": "Chio-Un-Seng" }, { "family_name": "Cheng", "given_name": "Yifan", "orcid": "0000-0001-9535-0369", "clpid": "Cheng-Yifan" }, { "family_name": "Shan", "given_name": "Shu-ou", "orcid": "0000-0002-6526-1733", "clpid": "Shan-S-O" } ], "abstract": "Ring-forming AAA\u207a chaperones solubilize protein aggregates and protect organisms from proteostatic stress. In metazoans, the AAA\u207a chaperone Skd3 in the mitochondrial intermembrane space (IMS) is critical for human health and efficiently refolds aggregated proteins, but its underlying mechanism is poorly understood. Here, we show that Skd3 harbors both disaggregase and protein refolding activities enabled by distinct assembly states. High-resolution structures of Skd3 hexamers in distinct conformations capture ratchet-like motions that mediate substrate extraction. Unlike previously described disaggregases, Skd3 hexamers further assemble into dodecameric cages in which solubilized substrate proteins can attain near-native states. Skd3 mutants defective in dodecamer assembly retain disaggregase activity but are impaired in client refolding, linking the disaggregase and refolding activities to the hexameric and dodecameric states of Skd3, respectively. We suggest that Skd3 is a combined disaggregase and foldase, and this property is particularly suited to meet the complex proteostatic demands in the mitochondrial IMS.", "doi": "10.1126/sciadv.adf5336", "pmcid": "PMC10171807", "issn": "2375-2548", "publisher": "American Association for the Advancement of Science", "publication": "Science Advances", "publication_date": "2023-05-10", "series_number": "19", "volume": "9", "issue": "19", "pages": "Art. No. eadf5336" }, { "id": "authors:an1c4-dyw75", "collection": "authors", "collection_id": "an1c4-dyw75", "cite_using_url": "https://resolver.caltech.edu/CaltechAUTHORS:20211022-170110641", "type": "article", "title": "Subunit cooperation in the Get1/2 receptor promotes tail-anchored membrane protein insertion", "author": [ { "family_name": "Chio", "given_name": "Un Seng", "orcid": "0000-0002-5295-2690", "clpid": "Chio-Un-Seng" }, { "family_name": "Liu", "given_name": "Yumeng", "clpid": "Liu-Yumeng" }, { "family_name": "Chung", "given_name": "SangYoon", "orcid": "0000-0002-0592-4099", "clpid": "Chung-SangYoon" }, { "family_name": "Shim", "given_name": "Woo Jun", "orcid": "0000-0001-9596-7393", "clpid": "Shim-Woo-Jun" }, { "family_name": "Chandrasekar", "given_name": "Sowmya", "clpid": "Chandrasekar-Sowmya" }, { "family_name": "Weiss", "given_name": "Shimon", "orcid": "0000-0002-0720-5426", "clpid": "Weiss-Shimon" }, { "family_name": "Shan", "given_name": "Shu-ou", "orcid": "0000-0002-6526-1733", "clpid": "Shan-S-O" } ], "abstract": "The guided entry of tail-anchored protein (GET) pathway, in which the Get3 ATPase delivers an essential class of tail-anchored membrane proteins (TAs) to the Get1/2 receptor at the endoplasmic reticulum, provides a conserved mechanism for TA biogenesis in eukaryotic cells. The membrane-associated events of this pathway remain poorly understood. Here we show that complex assembly between the cytosolic domains (CDs) of Get1 and Get2 strongly enhances the affinity of the individual subunits for Get3\u2022TA, thus enabling efficient capture of the targeting complex. In addition to the known role of Get1CD in remodeling Get3 conformation, two molecular recognition features (MoRFs) in Get2CD induce Get3 opening, and both subunits are required for optimal TA release from Get3. Mutation of the MoRFs attenuates TA insertion into the ER in vivo. Our results demonstrate extensive cooperation between the Get1/2 receptor subunits in the capture and remodeling of the targeting complex, and emphasize the role of MoRFs in receptor function during membrane protein biogenesis.", "doi": "10.1083/jcb.202103079", "issn": "0021-9525", "publisher": "Rockefeller University Press", "publication": "Journal of Cell Biology", "publication_date": "2021-11", "series_number": "11", "volume": "220", "issue": "11", "pages": "Art. No. e202103079" }, { "id": "authors:g7913-0z550", "collection": "authors", "collection_id": "g7913-0z550", "cite_using_url": "https://resolver.caltech.edu/CaltechAUTHORS:20190102-135256811", "type": "article", "title": "A Chaperone Lid Ensures Efficient and Privileged Client Transfer during Tail-Anchored Protein Targeting", "author": [ { "family_name": "Chio", "given_name": "Un Seng", "orcid": "0000-0002-5295-2690", "clpid": "Chio-Un-Seng" }, { "family_name": "Chung", "given_name": "SangYoon", "orcid": "0000-0002-0592-4099", "clpid": "Chung-SangYoon" }, { "family_name": "Weiss", "given_name": "Shimon", "orcid": "0000-0002-0720-5426", "clpid": "Weiss-Shimon" }, { "family_name": "Shan", "given_name": "Shu-ou", "orcid": "0000-0002-6526-1733", "clpid": "Shan-S-O" } ], "abstract": "Molecular chaperones play key roles in maintaining cellular proteostasis. In addition to preventing client aggregation, chaperones often relay substrates within a network while preventing off-pathway chaperones from accessing the substrate. Here we show that a conserved lid motif lining the substrate-binding groove of the Get3 ATPase enables these important functions during the targeted delivery of tail-anchored membrane proteins (TAs) to the endoplasmic reticulum. The lid prevents promiscuous TA handoff to off-pathway chaperones, and more importantly, it cooperates with the Get4/5 scaffolding complex to enable rapid and privileged TA transfer from the upstream co-chaperone Sgt2 to Get3. These findings provide a molecular mechanism by which chaperones maintain the pathway specificity of client proteins in the crowded cytosolic environment.", "doi": "10.1016/j.celrep.2018.12.035", "pmcid": "PMC6689467", "issn": "2211-1247", "publisher": "Cell Press", "publication": "Cell Reports", "publication_date": "2019-01-02", "series_number": "1", "volume": "26", "issue": "1", "pages": "37-44" }, { "id": "authors:bxgje-prz14", "collection": "authors", "collection_id": "bxgje-prz14", "cite_using_url": "https://resolver.caltech.edu/CaltechAUTHORS:20181008-095135080", "type": "article", "title": "In vitro Assays for Targeting and Insertion of Tail\u2010Anchored Proteins Into the ER Membrane", "author": [ { "family_name": "Cho", "given_name": "Hyunju", "orcid": "0000-0001-7393-657X", "clpid": "Cho-Hyunju" }, { "family_name": "Chio", "given_name": "Un Seng", "orcid": "0000-0002-5295-2690", "clpid": "Chio-Un-Seng" }, { "family_name": "Shan", "given_name": "Shu-ou", "orcid": "0000-0002-6526-1733", "clpid": "Shan-S-O" } ], "abstract": "Membrane proteins mediate numerous essential cellular functions. Due to the aggregation propensity of hydrophobic transmembrane domains in aqueous environments, the targeting and insertion of membrane proteins pose major challenges to cells. In the Guided Entry of Tail\u2010anchored protein (GET) pathway, an essential class of newly synthesized tail\u2010anchored proteins (TAs) are chaperoned and guided by multiple targeting factors to the endoplasmic reticulum (ER) membrane. Deciphering the molecular mechanism of this cellular process has benefitted from successful in vitro reconstitution of individual molecular events in the GET pathway with purified components. Here we describe recently developed protocols for in vitro reconstitution of functional complexes of TA substrates with their targeting factors, for monitoring the transfer of TAs between targeting factors, and for the insertion of TA into the microsomal membrane. These procedures are generally applicable to the interrogation of other post\u2010translational membrane protein targeting pathways.", "doi": "10.1002/cpcb.63", "pmcid": "PMC6263799", "issn": "1934-2616", "publisher": "Wiley", "publication": "Current Protocols in Cell Biology", "publication_date": "2018-12", "series_number": "1", "volume": "81", "issue": "1", "pages": "Art. No. e63" }, { "id": "authors:09chm-xpa03", "collection": "authors", "collection_id": "09chm-xpa03", "cite_using_url": "https://resolver.caltech.edu/CaltechAUTHORS:20180510-133648234", "type": "article", "title": "A Protean Clamp Guides Membrane Targeting of Tail-Anchored Proteins", "author": [ { "family_name": "Shan", "given_name": "Shu-ou", "orcid": "0000-0002-6526-1733", "clpid": "Shan-S-O" }, { "family_name": "Chio", "given_name": "Un Seng", "orcid": "0000-0002-5295-2690", "clpid": "Chio-Un-Seng" } ], "abstract": "To maintain cellular organization, many chaperones and targeting factors escort nascent proteins to membrane destinations. It was generally thought that substrate proteins preferably bind conformationally closed chaperones. However, how the nature and dynamics of the targeting complex help guide substrate proteins to the target membrane is not understood for most pathways. We addressed this question for the conserved ATPase Get3, which targets the essential class of tail-anchored proteins (TAs) to the endoplasmic reticulum (ER).", "doi": "10.1016/j.bpj.2017.11.3022", "issn": "0006-3495", "publisher": "Biophysical Society", "publication": "Biophysical Journal", "publication_date": "2018-02-02", "series_number": "3", "volume": "114", "issue": "3", "pages": "553a" }, { "id": "authors:4ds6e-83b64", "collection": "authors", "collection_id": "4ds6e-83b64", "cite_using_url": "https://resolver.caltech.edu/CaltechAUTHORS:20170926-110202110", "type": "article", "title": "A protean clamp guides membrane targeting of tail-anchored proteins", "author": [ { "family_name": "Chio", "given_name": "Un Seng", "orcid": "0000-0002-5295-2690", "clpid": "Chio-Un-Seng" }, { "family_name": "Chung", "given_name": "SangYoon", "orcid": "0000-0002-0592-4099", "clpid": "Chung-SangYoon" }, { "family_name": "Weiss", "given_name": "Shimon", "orcid": "0000-0002-0720-5426", "clpid": "Weiss-Shimon" }, { "family_name": "Shan", "given_name": "Shu-ou", "orcid": "0000-0002-6526-1733", "clpid": "Shan-S-O" } ], "abstract": "Proper localization of proteins to target membranes is a fundamental cellular process. How the nature and dynamics of the targeting complex help guide substrate proteins to the target membrane is not understood for most pathways. Here, we address this question for the conserved ATPase guided entry of tail-anchored protein 3 (Get3), which targets the essential class of tail-anchored proteins (TAs) to the endoplasmic reticulum (ER). Single-molecule fluorescence spectroscopy showed that, contrary to previous models of a static closed Get3\u2022TA complex, Get3 samples open conformations on the submillisecond timescale upon TA binding, generating a fluctuating \"protean clamp\" that stably traps the substrate. Point mutations at the ATPase site bias Get3 toward closed conformations, uncouple TA binding from induced Get3\u2022Get4/5 disassembly, and inhibit the ER targeting of the Get3\u2022TA complex. These results demonstrate an essential role of substrate-induced Get3 dynamics in driving TA targeting to the membrane, and reveal a tightly coupled channel of communication between the TA-binding site, ATPase site, and effector interaction surfaces of Get3. Our results provide a precedent for large-scale dynamics in a substrate-bound chaperone, which provides an effective mechanism to retain substrate proteins with high affinity while also generating functional switches to drive vectorial cellular processes.", "doi": "10.1073/pnas.1708731114", "pmcid": "PMC5642712", "issn": "0027-8424", "publisher": "National Academy of Sciences", "publication": "Proceedings of the National Academy of Sciences of the United States of America", "publication_date": "2017-10-10", "series_number": "41", "volume": "114", "issue": "41", "pages": "E8585-E8594" }, { "id": "authors:w6vr2-95t93", "collection": "authors", "collection_id": "w6vr2-95t93", "cite_using_url": "https://resolver.caltech.edu/CaltechAUTHORS:20171012-133314474", "type": "article", "title": "Mechanisms of Tail-Anchored Membrane Protein Targeting and Insertion", "author": [ { "family_name": "Chio", "given_name": "Un Seng", "orcid": "0000-0002-5295-2690", "clpid": "Chio-Un-Seng" }, { "family_name": "Cho", "given_name": "Hyunjun", "orcid": "0000-0002-8963-5525", "clpid": "Cho-Hyunjun" }, { "family_name": "Shan", "given_name": "Shu-ou", "orcid": "0000-0002-6526-1733", "clpid": "Shan-S-O" } ], "abstract": "Proper localization of membrane proteins is essential for the function of biological membranes and for the establishment of organelle identity within a cell. Molecular machineries that mediate membrane protein biogenesis need to not only achieve a high degree of efficiency and accuracy, but also prevent off-pathway aggregation events that can be detrimental to cells. The posttranslational targeting of tail-anchored proteins (TAs) provides tractable model systems to probe these fundamental issues. Recent advances in understanding TA-targeting pathways reveal sophisticated molecular machineries that drive and regulate these processes. These findings also suggest how an interconnected network of targeting factors, cochaperones, and quality control machineries together ensures robust membrane protein biogenesis.", "doi": "10.1146/annurev-cellbio-100616-060839", "pmcid": "PMC6343671", "issn": "1081-0706", "publisher": "Annual Reviews", "publication": "Annual Review of Cell and Developmental Biology", "publication_date": "2017-10", "volume": "33", "pages": "417-438" }, { "id": "authors:mqs9g-yn993", "collection": "authors", "collection_id": "mqs9g-yn993", "cite_using_url": "https://resolver.caltech.edu/CaltechAUTHORS:20161212-104646015", "type": "article", "title": "Multiple selection filters ensure accurate tail-anchored membrane protein targeting", "author": [ { "family_name": "Rao", "given_name": "Meera", "clpid": "Rao-Meera" }, { "family_name": "Okreglak", "given_name": "Voytek", "clpid": "Okreglak-V" }, { "family_name": "Chio", "given_name": "Un Seng", "orcid": "0000-0002-5295-2690", "clpid": "Chio-Un-Seng" }, { "family_name": "Cho", "given_name": "Hyunjun", "clpid": "Cho-Hyunjun" }, { "family_name": "Walter", "given_name": "Peter", "clpid": "Walter-Peter" }, { "family_name": "Shan", "given_name": "Shu-ou", "orcid": "0000-0002-6526-1733", "clpid": "Shan-S-O" } ], "abstract": "Accurate protein localization is crucial to generate and maintain organization in all cells. Achieving accuracy is challenging, as the molecular signals that dictate a protein's cellular destination are often promiscuous. A salient example is the targeting of an essential class of tail-anchored (TA) proteins, whose sole defining feature is a transmembrane domain near their C-terminus. Here we show that the Guided Entry of Tail-anchored protein (GET) pathway selects TA proteins destined to the endoplasmic reticulum (ER) utilizing distinct molecular steps, including differential binding by the co-chaperone Sgt2 and kinetic proofreading after ATP hydrolysis by the targeting factor Get3. Further, the different steps select for distinct physicochemical features of the TA substrate. The use of multiple selection filters may be general to protein biogenesis pathways that must distinguish correct and incorrect substrates based on minor differences.", "doi": "10.7554/eLife.21301", "pmcid": "PMC5214336", "issn": "2050-084X", "publisher": "eLife Sciences Publications", "publication": "eLife", "publication_date": "2016-12-07", "volume": "5", "pages": "Art. No. e21301" }, { "id": "authors:aeabp-gfj80", "collection": "authors", "collection_id": "aeabp-gfj80", "cite_using_url": "https://resolver.caltech.edu/CaltechAUTHORS:20141105-102115424", "type": "article", "title": "Differential gradients of interaction affinities drive efficient targeting and recycling in the GET pathway", "author": [ { "family_name": "Rome", "given_name": "Michael E.", "clpid": "Rome-M-E" }, { "family_name": "Chio", "given_name": "Un Seng", "orcid": "0000-0002-5295-2690", "clpid": "Chio-Un-Seng" }, { "family_name": "Rao", "given_name": "Meera", "clpid": "Rao-Meera" }, { "family_name": "Gristick", "given_name": "Harry", "clpid": "Gristick-H-B" }, { "family_name": "Shan", "given_name": "Shu-ou", "orcid": "0000-0002-6526-1733", "clpid": "Shan-S-O" } ], "abstract": "Efficient and accurate localization of membrane proteins requires a complex cascade of interactions between protein machineries. This requirement is exemplified in the guided entry of tail-anchored (TA) protein (GET) pathway, where the central targeting factor Get3 must sequentially interact with three distinct binding partners to ensure the delivery of TA proteins to the endoplasmic reticulum (ER) membrane. To understand the molecular principles that provide the vectorial driving force of these interactions, we developed quantitative fluorescence assays to monitor Get3\u2013effector interactions at each stage of targeting. We show that nucleotide and substrate generate differential gradients of interaction energies that drive the ordered interaction of Get3 with successive effectors. These data also provide more molecular details on how the targeting complex is captured and disassembled by the ER receptor and reveal a previously unidentified role for Get4/5 in recycling Get3 from the ER membrane at the end of the targeting reaction. These results provide general insights into how complex protein interaction cascades are coupled to energy inputs in biological systems.", "doi": "10.1073/pnas.1411284111", "pmcid": "PMC4246279", "issn": "0027-8424", "publisher": "National Academy of Sciences", "publication": "Proceedings of the National Academy of Sciences of the United States of America", "publication_date": "2014-11-06", "series_number": "46", "volume": "111", "issue": "46", "pages": "E4929-E4935" } ]