Hydrophobic amino acids are normally shielded from the cytosol and their exposure is often used as an indicator of protein misfolding to enable the chaperone-mediated recognition and quality control of aberrant polypeptides. Mislocalised membrane proteins (MLPs) represent a particular challenge to cellular quality control, and, in this study, membrane protein fragments have been exploited to study a specialised pathway that underlies the efficient detection and proteasomal degradation of MLPs. Our data show that the BAG6 complex and SGTA compete for cytosolic MLPs by recognition of their exposed hydrophobicity, and the data suggest that SGTA acts to maintain these substrates in a non-ubiquitylated state. Hence, SGTA might counter the actions of BAG6 to delay the ubiquitylation of specific precursors and thereby increase their opportunity for successful post-translational delivery to the endoplasmic reticulum. However, when SGTA is overexpressed, the normally efficient removal of aberrant MLPs is delayed, increasing their steady-state level and promoting aggregation. Our data suggest that SGTA regulates the cellular fate of a range of hydrophobic polypeptides should they become exposed to the cytosol.

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.

ESCRT-I is essential for the multivesicular body (MVB) sorting of ubiquitylated cargo such as epidermal growth factor receptor, as well as for several cellular functions, such as cell division and retroviral budding. ESCRT-I has four subunits; TSG101, VPS28, VPS37 and MVB12. There are several members of VPS37 and MVB12 families in mammalian cells, and their differential incorporation into ESCRT-I could provide function-specific variants of the complex. However, it remains unclear whether these different forms of VPS37 and MVB12 combine randomly or generate selective pairings within ESCRT-I, and what the mechanistic basis for such pairing would be. Here, we show that the incorporation of two MVB12 members, UBAP1 and MVB12A, into ESCRT-I is highly selective with respect to their VPS37 partners. We map the region mediating selective assembly of UBAP1–VPS37A to the core ESCRT-I-binding domain of VPS37A. In contrast, selective integration of UBAP1 requires both the minimal ESCRT-I-binding region and a neighbouring predicted helix. The biochemical specificity in ESCRT-I assembly is matched by functional specialisation as siRNA-mediated depletion of UBAP1, but not MVB12A and MVB12B, disrupts ubiquitin-dependent sorting at the MVB.

LITAF (LPS-induced TNF-activating factor) is an endosome-associated integral membrane protein important for multivesicular body sorting. Several mutations in LITAF cause autosomal-dominant Charcot Marie Tooth disease type 1C. These mutations map to a highly conserved C-terminal region, termed the LITAF domain, which includes a 22 residue hydrophobic sequence and flanking cysteine-rich regions that contain peptide motifs found in zinc fingers. Although the LITAF domain is thought to be responsible for membrane integration, the membrane topology of LITAF has not been established. Here, we have investigated whether LITAF is a tail-anchored (TA) membrane-spanning protein or monotopic membrane protein. When translated in vitro, LITAF integrates poorly into ER-derived microsomes compared with Sec61β, a bona fide TA protein. Furthermore, introduction of N-linked glycosylation reporters shows that neither the N-terminal nor C-terminal domains of LITAF translocate into the ER lumen. Expression in cells of an LITAF construct containing C-terminal glycosylation sites confirms that LITAF is not a TA protein in cells. Finally, an immunofluorescence-based latency assay showed that both the N- and C-termini of LITAF are exposed to the cytoplasm. Recombinant LITAF contains 1 mol/mol zinc, while mutation of predicted zinc-binding residues disrupts LITAF membrane association. Hence, we conclude that LITAF is a monotopic membrane protein whose membrane integration is stabilised by a zinc finger. The related human protein, CDIP1 (cell death involved p53 target 1), displays identical membrane topology, suggesting that this mode of membrane integration is conserved in LITAF family proteins.

SummaryEndosomal sorting complexes required for transport (ESCRTs) are essential for ubiquitin-dependent degradation of mitogenic receptors, a process often compromised in cancer pathologies. Sorting of ubiquinated receptors via ESCRTs is controlled by the tumor suppressor phosphatase HD-PTP. The specific interaction between HD-PTP and the ESCRT-I subunit UBAP1 is critical for degradation of growth factor receptors and integrins. Here, we present the structural characterization by X-ray crystallography and double electron-electron resonance spectroscopy of the coiled-coil domain of HD-PTP and its complex with UBAP1. The coiled-coil domain adopts an unexpected open and rigid conformation that contrasts with the closed and flexible coiled-coil domain of the related ESCRT regulator Alix. The HD-PTP:UBAP1 structure identifies the molecular determinants of the interaction and provides a molecular basis for the specific functional cooperation between HD-PTP and UBAP1. Our findings provide insights into the molecular mechanisms of regulation of ESCRT pathways that could be relevant to anticancer therapies.