SGTA regulates the cytosolic quality control of hydrophobic substrates

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

Characterisation of The Novel AAA+ atpase TorsinA

EThOS - Electronic Theses Online ServiceGBUnited Kingdo

The molecular basis for selective assembly of the UBAP1-containing endosome-specific ESCRT-I complex.

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

The Charcot Marie Tooth disease protein LITAF is a zinc-binding monotopic membrane protein

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

Reorientation of the first signal-anchor sequence during potassium channel biogenesis at the Sec61 complex.

The majority of the polytopic proteins that are synthesized at the ER (endoplasmic reticulum) are integrated co-translationally via the Sec61 translocon, which provides lateral access for their hydrophobic TMs (transmembrane regions) to the phospholipid bilayer. A prolonged association between TMs of the potassium channel subunit, TASK-1 [TWIK (tandem-pore weak inwardly rectifying potassium channel)-related acid-sensitive potassium channel 1], and the Sec61 complex suggests that the ER translocon co-ordinates the folding/assembly of the TMs present in the nascent chain. The N-terminus of both TASK-1 and Kcv (potassium channel protein of chlorella virus), another potassium channel subunit of viral origin, has access to the N-glycosylation machinery located in the ER lumen, indicating that the Sec61 complex can accommodate multiple arrangements/orientations of TMs within the nascent chain, both in vitro and in vivo. Hence the ER translocon can provide the ribosome-bound nascent chain with a dynamic environment in which it can explore a range of different conformations en route to its correct transmembrane topology and final native structure

Structural basis for specific interaction of TGFβ signalling regulators SARA/endofin with HD-PTP

SARA and endofin are endosomal adaptor proteins that drive Smad phosphorylation by ligand-activated TGFβ/BMP receptors. We show in this study that SARA and endofin also recruit the tumour supressor HD-PTP, a master regulator of endosomal sorting and ESCRT-dependent receptor down-regulation. High affinity interactions occur between the SARA/endofin N-termini, and the conserved hydrophobic region in the HD-PTP Bro1 domain that binds CHMP4/ESCRT-III. CHMP4 engagement is a universal feature of Bro1 proteins, but SARA/endofin binding is specific to HD-PTP. Crystallographic structures of HD-PTPBro1 in complex with SARA, endofin and three CHMP4 isoformsrevealed that all ligands bind similarly to the conserved site, but critically, onlySARA/endofin interact at a neighbouring pocket unique to HD-PTP. The structures, together with mutagenesis and binding analysis, explain the high affinity and specific binding of SARA/endofin, and why they compete so effectively with CHMP4. Our data invoke models for how endocytic regulation of TGFβ/BMP signalling is controlled

Endosomal recycling tubule scission and integrin recycling involve the membrane curvature-supporting protein LITAF

ABSTRACT Recycling to the cell surface requires the scission of tubular membrane intermediates emanating from endosomes. Here, we identify the monotopic membrane protein LPS-induced TNF-activating factor (LITAF) and the related protein cell death involved p53 target 1 (CDIP1) as novel membrane curvature proteins that contribute to recycling tubule scission. Recombinant LITAF supports high membrane curvature, shown by its ability to reduce proteoliposome size. The membrane domains of LITAF and CDIP1 partition strongly into ∼50 nm diameter tubules labelled with the recycling markers Pacsin2, ARF6 and SNX1, and the recycling cargoes MHC class I and CD59. Partitioning of LITAF into tubules is impaired by mutations linked to Charcot Marie Tooth disease type 1C. Meanwhile, co-depletion of LITAF and CDIP1 results in the expansion of tubular recycling compartments and stabilised Rab11 tubules, pointing to a function for LITAF and CDIP1 in membrane scission. Consistent with this, co-depletion of LITAF and CDIP1 impairs integrin recycling and cell migration.</jats:p

Endosomal recycling tubule scission and integrin recycling involve the membrane curvature supporting protein LITAF

Recycling to the cell surface requires the scission of tubular membrane intermediates emanating from endosomes. Here, we identify the monotopic membrane protein LPS-induced TNF-activating factor (LITAF) and the related protein cell death involved p53 target 1 (CDIP1) as novel membrane curvature proteins that contribute to recycling tubule scission. Recombinant LITAF supports high membrane curvature, shown by its ability to reduce proteoliposome size. The membrane domains of LITAF and CDIP1 partition strongly into ∼50 nm diameter tubules labelled with the recycling markers Pacsin2, ARF6 and SNX1, and the recycling cargoes MHC class I and CD59. Partitioning of LITAF into tubules is impaired by mutations linked to Charcot Marie Tooth disease type 1C. Meanwhile, co-depletion of LITAF and CDIP1 results in the expansion of tubular recycling compartments and stabilised Rab11 tubules, pointing to a function for LITAF and CDIP1 in membrane scission. Consistent with this, co-depletion of LITAF and CDIP1 impairs integrin recycling and cell migration

Structural Basis for Selective Interaction between the ESCRT Regulator HD-PTP and UBAP1

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