Structural basis for regulation of the nucleo-cytoplasmic distribution of Bag6 by TRC35

Abstract

The metazoan protein BCL2-associated athanogene cochaperone 6 (Bag6) forms a hetero-trimeric complex with ubiquitin-like 4A and transmembrane domain recognition complex 35 (TRC35). This Bag6 complex is involved in tail-anchored protein targeting and various protein quality-control pathways in the cytosol as well as regulating transcription and histone methylation in the nucleus. Here we present a crystal structure of Bag6 and its cytoplasmic retention factor TRC35, revealing that TRC35 is remarkably conserved throughout the opisthokont lineage except at the C-terminal Bag6-binding groove, which evolved to accommodate Bag6, a unique metazoan factor. While TRC35 and its fungal homolog, guided entry of tail-anchored protein 4 (Get4), utilize a conserved hydrophobic patch to bind their respective partners, Bag6 wraps around TRC35 on the opposite face relative to the Get4-5 interface. We further demonstrate that TRC35 binding is critical not only for occluding the Bag6 nuclear localization sequence from karyopherin α to retain Bag6 in the cytosol but also for preventing TRC35 from succumbing to RNF126-mediated ubiquitylation and degradation. The results provide a mechanism for regulation of Bag6 nuclear localization and the functional integrity of the Bag6 complex in the cytosol.tail-anchor targeting | X-ray crystallography | tail-anchor recognition complex | proteasome-dependent degradation | GET pathway

The roles of cytosolic quality control proteins, SGTA and the BAG6 complex, in disease

“…Moreover, due to evolutionary pressure on the BAG domain, it has undergone phylogenetic divergence to accommodate for more complex demands in mammals. This indicates that on a functional and sequence level, the BAG6 C-terminal BAG domain is unique (Kuwabara et al, 2015;Mock et al, 2017).…”

“…The binding of hydrophobic stretches of protein onto BAG6 is imperative for substrate discrimination. Domain I contains ECI1 and ECI2, both of which are capable of binding hydrophobic substrates, but cannot ; NLS (residues 1008-1050, orange) and TRC35 (residues 23-305, gray) from PDB: 6AU8 (Mock, Xu, Ye, & Clemons, 2017); BAG domain (blue) in complex with UBL4A (residues 95-147, gray), from PDB: 4X86 (Kuwabara et al, 2015).…”

“…BAG6 variants lacking the NLS due to alternative splicing have shown preferential localization at the cytosol. Although BAG6 is primarily localized in the cytosol, the NLS domain indicates that its presence in the nucleus is important too, and functions of BAG6 as a quality control site have been displayed in both localities (Mock et al, 2017). However, some cases show that increased nuclear levels of BAG6 can be associated with tumorigenesis, for example, in osteosarcoma tissue (Tsukahara et al, 2009).…”

The natural history of Get3‐like chaperones

“…Some phylogenetic aspects of other GET pathway components have been recently discussed, in particular the evolutionary relationships between components of the pretargeting complex comprising Get4, Get5 and Bag6 and the legacy of membrane protein biogenesis factors similar to bacterial Oxa1 that also include Get1 . Here, we combine a review of the literature on Get3 structure and function with a comprehensive bioinformatic analysis of the structural elements of the protein involved in TA protein binding.…”

“…Extensive biochemical and structural studies performed over the last decade have characterized the targeting of TA proteins utilizing the yeast GET pathway (as reviewed in). Initially, a pre‐targeting complex, consisting of a small glutamine‐rich tetratricopeptide repeat containing protein Sgt2, and Get4 and Get5 in yeast, or Bag6, SGTA, TRC35 and UBL4A in mammals, captures the TA protein following its release from the ribosome, then transfers it to the ATPase Get3 in budding yeast, or TRC40 in mammals . The TA‐bound Get3/TRC40 protects and delivers the TA protein to the ER membrane, where its receptor complex comprised of Get1 and Get2 in yeast or WRB and CAML in mammals stimulates its subsequent release into the membrane …”