Abstract
BackgroundThe 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.Methodology and Principal FindingsBAG6 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.SignificanceOn 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.
Highlights
Tail-anchored (TA) proteins have provided a convenient paradigm for studying post-translational membrane insertion at the endoplasmic reticulum [1]
The behavior of the BAG6 deletion constructs in this in vitro binding assay was highly reproducible, and we studied several additional truncated forms of BAG6 which behaved in an entirely consistent manner (Figures S3A to S3D), On the basis of this analysis, we conclude that the N-terminal region of BAG6, and the Nterminal UBL domain is required for its efficient binding to SGTA
A role for BAG6 in the recognition and removal of hydrophobic substrates from the cytosol was first apparent when it was identified as part of an upstream loading complex for the TRC40 dependent delivery of TA proteins to the ER membrane [14,15,18]
Summary
Tail-anchored (TA) proteins have provided a convenient paradigm for studying post-translational membrane insertion at the endoplasmic reticulum [1]. Mammalian TRC40 was first identified as a TA protein interacting partner using in vitro assays [7,8], and its binding to a precursor reflects commitment to ER delivery via an interaction that relies on a membrane protein receptor composed of the tryptophan-rich basic protein (WRB) [9], and the calcium-modulating cyclophilin ligand (CAML) [10]. This part of the TA protein delivery pathway to the ER is highly conserved, and in Saccharomyces cerevisiae a similar process is mediated by the TRC40 homolog Get, and the heteromeric Get1/Get membrane receptor [1,6,11,12,13]. 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
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