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Bag6 Complex Contains a Minimal Tail-Anchor–Targeting Module and a Mock BAG Domain

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Bag6 Complex Contains a Minimal Tail-Anchor–Targeting Module and a Mock BAG Domain Bag6 complex contains a minimal tail-anchor–targeting module and a mock BAG domain Jee-Young Mocka, Justin William Chartrona,Ma’ayan Zaslavera,YueXub,YihongYeb, and William Melvon Clemons Jr.a,1 aDivision of Chemistry and Chemical Engineering, California Institute of Technology, Pasadena, CA 91125; and bLaboratory of Molecular Biology, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MD 20892 Edited by Gregory A. Petsko, Weill Cornell Medical College, New York, NY, and approved December 1, 2014 (received for review February 12, 2014) BCL2-associated athanogene cochaperone 6 (Bag6) plays a central analogous yeast complex contains two proteins, Get4 and Get5/ role in cellular homeostasis in a diverse array of processes and is Mdy2, which are homologs of the mammalian proteins TRC35 part of the heterotrimeric Bag6 complex, which also includes and Ubl4A, respectively. In yeast, these two proteins form ubiquitin-like 4A (Ubl4A) and transmembrane domain recognition a heterotetramer that regulates the handoff of the TA protein complex 35 (TRC35). This complex recently has been shown to be from the cochaperone small, glutamine-rich, tetratricopeptide important in the TRC pathway, the mislocalized protein degrada- repeat protein 2 (Sgt2) [small glutamine-rich tetratricopeptide tion pathway, and the endoplasmic reticulum-associated degrada- repeat-containing protein (SGTA) in mammals] to the delivery tion pathway. Here we define the architecture of the Bag6 factor Get3 (TRC40 in mammals) (19–22). It is expected that the complex, demonstrating that both TRC35 and Ubl4A have distinct mammalian homologs, along with Bag6, play a similar role (23– C-terminal binding sites on Bag6 defining a minimal Bag6 complex. 27). Bag6 also interacts with other proteins such as apoptosis- A crystal structure of the Bag6–Ubl4A dimer demonstrates that inducing factor, glycoprotein 78 (gp78), regulatory particle 5, and Bag6–BAG is not a canonical BAG domain, and this finding is sub- brother of regulator of imprinted sites (BORIS) (16, 27–32) and stantiated biochemically. Remarkably, the minimal Bag6 complex can homo-oligomerize, increasing the level of complexity (30). defined here facilitates tail-anchored substrate transfer from small These findings build a picture of Bag6 as a central hub for a diverse glutamine-rich tetratricopeptide repeat-containing protein α to physiological network of proteins. TRC40. These findings provide structural insight into the complex A variety of diseases, ranging from cancer to autoimmune network of proteins coordinated by Bag6. disorders and diabetes, are linked to Bag6 (33-37). Despite this BIOCHEMISTRY demonstrated importance, structural characterization of the Bag6 GET pathway | Scythe | Bat3 | X-ray crystallography | tail-anchored proteins complex is lacking. The longest and most common isoform of the Bag6 gene encodes an 1,132-aa protein (38) with an N-terminal he well-studied BCL2-associated athanogene cochaperone 6 ubiquitin-like (UBL) domain that has been characterized struc- T(Bag6, also known as “BAT3” or “Scythe”) plays a central turally (PDB ID codes 4EEW, 4DWF, and 1WX9), a large pro- role in membrane protein quality control, with additional links to line-rich central domain that is predicted to be unstructured, and apoptosis, gene regulation, and immunoregulation (for reviews, a C-terminal predicted BAG domain (Bag6-BAG). In this study, see refs. 1–3). Recent studies demonstrated that Bag6 forms we map the TRC35- and Ubl4A-binding regions to the C termi- a heterotrimeric Bag6 complex with ubiquitin-like 4A (Ubl4A) nus of Bag6. Based on these results, the structure of the complex and transmembrane domain recognition complex 35 (TRC35) (4, between the heterodimerization domains of Bag6 and Ubl4A was 5) that mediates the fates of membrane proteins in tail-anchor (TA) protein targeting (6), mislocalized protein degradation (7), Significance and endoplasmic reticulum (ER)-associated protein degradation (4). The many roles of the Bag6 complex likely are centered on its Quality control of proteins is critical to cellular homeostasis. ability to bind exposed hydrophobic regions of proteins, such as The BCL2-associated athanogene cochaperone 6 (Bag6) complex, transmembrane domains. In the cytoplasm, the Bag6 complex which contains Bag6, transmembrane domain recognition com- directs substrates either to targeting factors for the ER membrane plex 35 (TRC35), and ubiquitin-like 4A (Ubl4A), plays an essential (for TA proteins) or to ubiquitylation and subsequent proteaso- role in targeting transmembrane domains either to the endo- mal degradation. plasmic reticulum or degradation. Bag6 is a central hub for Bag6 initially was described as part of the gene cluster that numerous activities, functionally linked to an array of cellular included the human MHC class III on chromosome 6, resulting pathways, from immunoregulation to apoptosis. Here we define in its first name, “HLA-B–associated transcript 3” (BAT3) (8). the molecular architecture of this heterotrimer, revealing dis- The genomic localization suggested a role in immune response, tinct binding sites on Bag6 for TRC35 and Ubl4A. The truncated and this suggestion has been supported by evidence of its roles in Bag6 complex defined in this study is sufficient to facilitate Th1 cell survival (9), natural killer cell cytotoxicity (10), and substrate transfer from small glutamine-rich tetratricopeptide MHC class II molecule presentation (11, 12). The initial Bag6 repeat-containing protein α (SGTA) to TCR40. In addition, struc- link to apoptosis was based on its interaction with Reaper, an tural and biochemical characterization of the BAG domain of apoptosis-inducing Drosophila protein (13). Bag6 was not iden- Bag6 demonstrate that it is not a canonical BAG domain. tified in initial searches for functional homologs of Bag1 (14), which identified four additional proteins (Bag2–5) (15). The Author contributions: J.-Y.M., J.W.C., and W.M.C. designed research; J.-Y.M., J.W.C., M.Z., Y.X., and Y.Y. performed research; M.Z., Y.X., and Y.Y. contributed new reagents/analytic designation of Bag6 as a Bag family member came later from tools; J.-Y.M., Y.Y., and W.M.C. analyzed data; and J.-Y.M. and W.M.C. wrote the paper. limited sequence homology to the defined BAG domain and an The authors declare no conflict of interest. apparent heat shock cognate 70 (Hsc70)-regulating activity This article is a PNAS Direct Submission. found in other Bag family members (16). Data deposition: Crystallography, atomic coordinates, and structure factors reported in this In mammals, Bag6 has been shown to be critical in the tar- paper have been deposited in the Research Collaboratory for Structural Bioinformatics geting of TA proteins to the ER by the transmembrane recog- Protein Data Bank, www.rcsb.org/pdb/ (accession no. 4WWR). nition complex (TRC) pathway (5), a process best understood in 1To whom correspondence should be addressed. Email: [email protected]. the equivalent fungal guided entry of tail-anchored proteins This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. (GET) pathway (17, 18). Although Bag6 is missing in fungi, the 1073/pnas.1402745112/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1402745112 PNAS Early Edition | 1of6 Downloaded by guest on September 25, 2021 solved, revealing unexpected structural homology to Get5 and “ ” A B showing that the Bag6-BAG is a mock BAG domain, as was Get5-N TRC35 demonstrated further biochemically. Finally, a defined minimal FL N C Bag6 complex facilitates the transfer of a TA substrate from Bag6E SGTA to TRC40 in vitro. Bag6EN Results Get4-C Get4-N TRC35 Binds the Bag6 Nuclear Localization Sequence, Whereas Ubl4A Get3 Binding 90° Bag6E Binds the BAG Domain. To define the molecular architecture of the NLS heterotrimeric Bag6 complex (Bag6, Ubl4A, and TRC35), a se- ries of yeast two-hybrid assays were performed. Bag6 was divided Missing in TRC35 Bag6EBAG into five fragments: A (amino acids 1–225), B (amino acids 226– 399), C (amino acids 400–659), D (amino acids 660–950), and E N Bag6EN+NLS (amino acids 951–1,126), with the activating domain attached at the N terminus (Fig. 1A); TRC35 or Ubl4A contained N-ter- C Bag6E minal DNA-binding domains. TRC35 and Ubl4A both showed C (158-327) N (1-157) NLS+BAG a positive interaction with the C-terminal Bag6E fragment that TRC35 contains the nuclear localization sequence (NLS) and the BAG B Fig. 2. Defining the interaction between Bag6 and TRC35 by yeast two- domain (Fig. 1 ). To refine the interfaces, Bag6E was divided hybrid assay. (A, Upper) Diagram of the Get4/Get5-N complex (PDB ID further into an N-terminal domain, an NLS domain, and the code 3LKU). Get4 is shown in gray and red, and Get5 is shown in green. putative BAG domain (EN,ENLS, and EBAG respectively) (Fig. (Lower) A top-view schematic representation of the architecture. The two β 1A). TRC35 showed an interaction with ENLS (Fig. 1B) con- -strands in Get4 that are missing in TRC35 are outlined in blue. Ubl4A firming the in vivo result that TRC35 masks the Bag6-NLS, does not contain a Get5-N equivalent. (B) Bag6E fragments containing the preventing nuclear targeting (4). Ubl4A showed an interaction activating domain were combined with full-length TRC35 (FL), TRC35-N – – with E (Fig. 1B), a surprising result because none of the five (residues 1 157), or TRC35-C (residues 158 327) conjugated to the binding BAG domain. TRC35-N and TRC35-C were defined by sequence alignment previously characterized BAG domains was known to form sta- with Get4. ble interactions with other proteins. According to sequence alignment, Ubl4A lacks the Get5 N- terminal domain, and TRC35 lacks the β-loop in Get4 (Fig. 2A), TRC35-C and no interaction with TRC35-N (Fig. 2B). Surpris- both of which are involved in the Get4 interface with Get5 (19).
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