title: The association of BAG6 with SGTA and tail-anchored proteins.
abstract: BACKGROUND
  The BAG6 protein is a subunit of a heterotrimeric complex that binds a range of membrane and secretory protein precursors localized to the cytosol, enforcing quality control and influencing their subsequent fate.
  
  
  RESULTS
  BAG6 has an N-terminal ubiquitin-like domain, and a C-terminal Bcl-2-associated athanogene domain, separated by a large central proline-rich region. We have used in vitro binding approaches to identify regions of BAG6 important for its interactions with: i) the small-glutamine rich tetratricopeptide repeat-containing protein alpha (SGTA) and ii) two model tail-anchored membrane proteins as a paradigm for its hydrophobic substrates. We show that the BAG6-UBL is essential for binding to SGTA, and find that the UBL of a second subunit of the BAG6-complex, ubiquitin-like protein 4A (UBL4A), competes for SGTA binding. Our data show that this binding is selective, and suggest that SGTA can bind either BAG6, or UBL4A, but not both at the same time. We adapted our in vitro binding assay to study the association of BAG6 with an immobilized tail-anchored protein, Sec61β, and find both the UBL and BAG domains are dispensable for binding this substrate. This conclusion was further supported using a heterologous subcellular localization assay in yeast, where the BAG6-dependent nuclear relocalization of a second tail-anchored protein, GFP-Sed5, also required neither the UBL, nor the BAG domain of BAG6.
  
  
  CONCLUSIONS
  On the basis of these findings, we propose a working model where the large central region of the BAG6 protein provides a binding site for a diverse group of substrates, many of which expose a hydrophobic stretch of polypeptide. This arrangement would enable the BAG6 complex to bring together its substrates with potential effectors including those recruited via its N-terminal UBL. Such effectors may include SGTA, and the resulting assemblies influence the subsequent fate of the hydrophobic BAG6 substrates.
authors.affiliation: Faculty of Life Sciences, University of Manchester, Manchester, United Kingdom.
authors.forename: Pawel
authors.initials: P
authors.lastname: Leznicki
authors.forename: Quentin P
authors.initials: QP
authors.lastname: Roebuck
authors.forename: Lydia
authors.initials: L
authors.lastname: Wunderley
authors.forename: Anne
authors.initials: A
authors.lastname: Clancy
authors.forename: Ewelina M
authors.initials: EM
authors.lastname: Krysztofinska
authors.forename: Rivka L
authors.initials: RL
authors.lastname: Isaacson
authors.forename: Jim
authors.initials: J
authors.lastname: Warwicker
authors.forename: Blanche
authors.initials: B
authors.lastname: Schwappach
authors.forename: Stephen
authors.initials: S
authors.lastname: High
mesh_terms: D002352:Carrier Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism*; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism
chemical_list: D002352:Carrier Proteins; D008565:Membrane Proteins; D029701:Saccharomyces cerevisiae Proteins