Abstract
The Bag6-Ubl4A-Trc35 complex is a multifunctional chaperone that regulates various cellular processes. The diverse functions of Bag6 are supported by its ubiquitous localization to the cytoplasm, the nucleus, and membranes of the endoplasmic reticulum (ER) in cells. In ER-associated degradation (ERAD) pathways, Bag6 can interact with the membrane-associated ubiquitin ligase gp78 via its ubiquitin-like (UBL) domain, but the relative low affinity of this interaction does not reconcile with the fact that a fraction of Bag6 is tightly bound to the membranes. Here, we demonstrate that the UBL domain of Bag6 is required for interaction with the ER membranes. We find that in addition to gp78, the Bag6 UBL domain also binds a UBL-binding motif in UbxD8, an essential component of the gp78 ubiquitinating machinery. Importantly, Bag6 contains a proline-rich (PR) domain termed PDP (Proline rich-DUF3587-Proline rich) that forms homo-oligomer, allowing the UBL domain to form multivalent interactions with gp78 and UbxD8, which are essential for recruitment of Bag6 to the ER membrane. Furthermore, the PR domain comprises largely intrinsically disordered segments, which are sufficient for interaction with an unfolded substrate. We propose that simultaneous association with multiple ERAD factors helps to anchor a disordered chaperone oligomer to the site of retrotranslocation to prevent protein aggregation in ERAD.
Highlights
The Bag6-Ubl4A-Trc35 complex is localized to the endoplasmic reticulum (ER) membrane to regulate ER-associated degradation (ERAD)
The precise mechanism by which misfolded proteins are moved across the ER membrane during retrotranslocation is unclear, but it has been proposed that a few large membrane protein complexes, each assembled around a multiple-spanning transmembrane ubiquitin ligase (E3), may play crucial roles in this process [5, 6]
The UBL Domain Is Required for Membrane and Substrate Binding—To test whether the Bag6 UBL domain is required for membrane association, we developed an in vitro membrane binding assay
Summary
The Bag6-Ubl4A-Trc complex is localized to the ER membrane to regulate ERAD. Results: A disordered chaperoning domain in Bag forms homo-oligomer. The precise mechanism by which misfolded proteins are moved across the ER membrane during retrotranslocation is unclear, but it has been proposed that a few large membrane protein complexes, each assembled around a multiple-spanning transmembrane ubiquitin ligase (E3), may play crucial roles in this process [5, 6] These enzymes may participate in the formation of a putative retrotranslocon(s) using their transmembrane segments [7, 8]. Oligomerization allows its UBL domain to interact simultaneously with gp and its functional partner UbxD8, which localize Bag to the ER membrane This ensures that protein ubiquitination, dislocation, targeting to the proteasome take place in a highly coordinated manner. Our study reveals how Bag interacts with the ER membrane and its substrates to promote protein homeostasis in the ER in mammalian cells
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