Co-translational biogenesis of lipid droplet integral membrane proteins.

Membrane proteins destined for lipid droplets (LDs), a major intracellular storage site for neutral lipids, are inserted into the endoplasmic reticulum (ER) and then trafficked to LDs where they reside in a hairpin loop conformation. Here, we show that LD membrane proteins can be delivered to the ER either co- or post-translationally and that their membrane-embedded region specifies pathway selection. The co-translational route for LD membrane protein biogenesis is insensitive to a small molecule inhibitor of the Sec61 translocon, Ipomoeassin F, and instead relies on the ER membrane protein complex (EMC) for membrane insertion. This route may even result in a transient exposure of the short N termini of some LD membrane proteins to the ER lumen, followed by putative topological rearrangements that would enable their transmembrane segment to form a hairpin loop and N termini to face the cytosol. Our study reveals an unexpected complexity to LD membrane protein biogenesis and identifies a role for the EMC during their co-translational insertion into the ER.

SMIM1, carrier of the Vel blood group, is a tail-anchored transmembrane protein and readily forms homodimers in a cell-free system.

Antibodies to the Vel blood group antigen can cause adverse hemolytic reactions unless Vel-negative blood units are transfused. Since the genetic background of Vel-negativity was discovered in 2013, DNA-based typing of the 17-bp deletion causing the phenotype has facilitated identification of Vel-negative blood donors. SMIM1, the gene underlying Vel, encodes a 78-amino acid erythroid transmembrane protein of unknown function. The transmembrane orientation of SMIM1 has been debated since experimental data supported both the N- and C-termini being extracellular. Likewise, computational predictions of its orientation were divided and potential alternatives such as monotopic or dual-topology have been discussed but not investigated. We used a cell-free system to explore the topology of SMIM1 when synthesized in the endoplasmic reticulum (ER). SMIM1 was tagged with an opsin-derived N-glycosylation reporter at either the N- or C-terminus and synthesized in vitro using rabbit reticulocyte lysate supplemented with canine pancreatic microsomes as a source of ER membrane. SMIM1 topology was then determined by assessing the N-glycosylation of its N- or C-terminal tags. Complementary experiments were carried out by expressing the same SMIM1 variants in HEK293T/17 cells and establishing their membrane orientation by immunoblotting and flow cytometry. Our data consistently indicate that SMIM1 has its short C-terminus located extracellularly and that it most likely belongs to the tail-anchored class of membrane proteins with the bulk of the polypeptide located in the cytoplasm. Having established its membrane orientation in an independent model system, future work can now focus on functional aspects of SMIM1 as a potential regulator of erythropoiesis.

SGTA associates with nascent membrane protein precursors.

The endoplasmic reticulum (ER) is a major site for membrane protein synthesis in eukaryotes. The majority of integral membrane proteins are delivered to the ER membrane via the co-translational, signal recognition particle (SRP)-dependent route. However, tail-anchored proteins employ an alternative, post-translational route(s) that relies on distinct factors such as a cytosolic protein quality control component, SGTA. We now show that SGTA is selectively recruited to ribosomes synthesising a diverse range of membrane proteins, suggesting that its biosynthetic client base also includes precursors on the co-translational ER delivery pathway. Strikingly, SGTA is recruited to nascent membrane proteins before their transmembrane domain emerges from the ribosome. Hence, SGTA is ideally placed to capture these aggregation prone regions shortly after their synthesis. For nascent membrane proteins on the co-translational pathway, SGTA complements the role of SRP by reducing the co-translational ubiquitination of clients with multiple hydrophobic signal sequences. On this basis, we propose that SGTA acts to mask specific transmembrane domains located in complex membrane proteins until they can engage the ER translocon and become membrane inserted.

Expansion of DUB functionality generated by alternative isoforms - USP35, a case study.

Protein ubiquitylation is a dynamic post-translational modification that can be reversed by deubiquitylating enzymes (DUBs). It is unclear how the small number (∼100) of DUBs present in mammalian cells regulate the thousands of different ubiquitylation events. Here, we analysed annotated transcripts of human DUBs and found ∼300 ribosome-associated transcripts annotated as protein coding, which thus increases the total number of DUBs. By using USP35, a poorly studied DUB, as a case study, we provide evidence that alternative isoforms contribute to the functional expansion of DUBs. We show that there are two different USP35 isoforms that localise to different intracellular compartments and have distinct functions. Our results reveal that isoform 1 is an anti-apoptotic factor that inhibits staurosporine- and TNF-related apoptosis-inducing ligand (TRAIL; also known as TNFSF10)-induced apoptosis. In contrast, USP35 isoform 2 is an integral membrane protein of the endoplasmic reticulum (ER) that is also present at lipid droplets. Manipulations of isoform 2 levels cause rapid ER stress, likely through deregulation of lipid homeostasis, and lead to cell death. Our work highlights how alternative isoforms provide functional expansion of DUBs and sets directions for future research.This article has an associated First Person interview with the first author of the paper.

Mechanisms of regulation and diversification of deubiquitylating enzyme function.

Deubiquitylating (or deubiquitinating) enzymes (DUBs) are proteases that reverse protein ubiquitylation and therefore modulate the outcome of this post-translational modification. DUBs regulate a variety of intracellular processes, including protein turnover, signalling pathways and the DNA damage response. They have also been linked to a number of human diseases, such as cancer, and inflammatory and neurodegenerative disorders. Although we are beginning to better appreciate the role of DUBs in basic cell biology and their importance for human health, there are still many unknowns. Central among these is the conundrum of how the small number of ∼100 DUBs encoded in the human genome is capable of regulating the thousands of ubiquitin modification sites detected in human cells. This Commentary addresses the biological mechanisms employed to modulate and expand the functions of DUBs, and sets directions for future research aimed at elucidating the details of these fascinating processes.This article is part of a Minifocus on Ubiquitin Regulation and Function. For further reading, please see related articles: 'Exploitation of the host cell ubiquitin machinery by microbial effector proteins' by Yi-Han Lin and Matthias P. Machner (J. Cell Sci.130, 1985-1996). 'Cell scientist to watch - Mads Gyrd-Hansen' (J. Cell Sci.130, 1981-1983).

Binding of SGTA to Rpn13 selectively modulates protein quality control.

Rpn13 is an intrinsic ubiquitin receptor of the 26S proteasome regulatory subunit that facilitates substrate capture prior to degradation. Here we show that the C-terminal region of Rpn13 binds to the tetratricopeptide repeat (TPR) domain of SGTA, a cytosolic factor implicated in the quality control of mislocalised membrane proteins (MLPs). The overexpression of SGTA results in a substantial increase in steady-state MLP levels, consistent with an effect on proteasomal degradation. However, this effect is strongly dependent upon the interaction of SGTA with the proteasomal component Rpn13. Hence, overexpression of the SGTA-binding region of Rpn13 or point mutations within the SGTA TPR domain both inhibit SGTA binding to the proteasome and substantially reduce MLP levels. These findings suggest that SGTA can regulate the access of MLPs to the proteolytic core of the proteasome, implying that a protein quality control cycle that involves SGTA and the BAG6 complex can operate at the 19S regulatory particle. We speculate that the binding of SGTA to Rpn13 enables specific polypeptides to escape proteasomal degradation and/or selectively modulates substrate degradation.

Solution structure of the SGTA dimerisation domain and investigation of its interactions with the ubiquitin-like domains of BAG6 and UBL4A.

BACKGROUND: The BAG6 complex resides in the cytosol and acts as a sorting point to target diverse hydrophobic protein substrates along their appropriate paths, including proteasomal degradation and ER membrane insertion. Composed of a trimeric complex of BAG6, TRC35 and UBL4A, the BAG6 complex is closely associated with SGTA, a co-chaperone from which it can obtain hydrophobic substrates. METHODOLOGY AND PRINCIPAL FINDINGS: SGTA consists of an N-terminal dimerisation domain (SGTA_NT), a central tetratricopeptide repeat (TPR) domain, and a glutamine rich region towards the C-terminus. Here we solve a solution structure of the SGTA dimerisation domain and use biophysical techniques to investigate its interaction with two different UBL domains from the BAG6 complex. The SGTA_NT structure is a dimer with a tight hydrophobic interface connecting two sets of four alpha helices. Using a combination of NMR chemical shift perturbation, isothermal titration calorimetry (ITC) and microscale thermophoresis (MST) experiments we have biochemically characterised the interactions of SGTA with components of the BAG6 complex, the ubiquitin-like domain (UBL) containing proteins UBL4A and BAG6. We demonstrate that the UBL domains from UBL4A and BAG6 directly compete for binding to SGTA at the same site. Using a combination of structural and interaction data we have implemented the HADDOCK protein-protein interaction docking tool to generate models of the SGTA-UBL complexes. SIGNIFICANCE: This atomic level information contributes to our understanding of the way in which hydrophobic proteins have their fate decided by the collaboration between SGTA and the BAG6 complex.

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.

The association of BAG6 with SGTA and tail-anchored proteins.

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. METHODOLOGY AND PRINCIPAL FINDINGS: 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, Sec61ß, 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. SIGNIFICANCE: 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.

SGTA antagonizes BAG6-mediated protein triage.

The BAG6 complex was first identified as an upstream loading factor for tail-anchored membrane proteins entering the TRC40-dependent pathway for posttranslational delivery to the endoplasmic reticulum. Subsequently, BAG6 was shown to enhance the proteasomal degradation of mislocalized proteins by selectively promoting their ubiquitination. We now show that the BAG6-dependent ubiquitination of mislocalized proteins is completely reversible and identify a pivotal role for the small glutamine-rich tetratricopeptide repeat-containing protein α (SGTA) in specifically antagonizing this process. SGTA does not simply mask the exposed hydrophobic transmembrane domain of a mislocalized protein, thereby preventing BAG6 recruitment. Rather, SGTA actively promotes the deubiquitination of mislocalized proteins that are already covalently modified, thus reversing the actions of BAG6 and inhibiting its capacity to promote substrate-specific degradation. This SGTA-mediated effect is independent of its tetratricopeptide motifs, suggesting it does not require the actions of Hsp70 and Hsp90 chaperones. These data reveal that, in a cellular context, mislocalized protein ubiquitination is the result of a dynamic equilibrium reflecting competition between pathways that promote either protein maturation or degradation. The targeted perturbation of this equilibrium, achieved by increasing steady-state SGTA levels, results in a specific stabilization of a model mislocalized protein derived from the amyloid precursor protein, an effect that is completely negated by ensuring efficient precursor delivery to the endoplasmic reticulum. We speculate that a BAG6/SGTA cycle operates during protein maturation and quality control in the cytosol and that together these components dictate the fate of a specific subset of newly synthesized proteins.

TRC40 can deliver short secretory proteins to the Sec61 translocon.

Whilst the co-translational translocation of nascent proteins across the mammalian endoplasmic reticulum (ER) is well defined, the capacity of this organelle for post-translational translocation is poorly delineated. Here we identify two human secretory protein precursors, apelin and statherin, as bona fide substrates for post-translational translocation across the ER membrane. Further studies, in combination with Hyalophora cecropia preprocecropin A (ppcecA), show that all three proteins bind to TRC40 and can utilise this component for their delivery to the ER membrane in a well-established in vitro system. However, ppcecA is not an obligate TRC40 substrate, and it can also be delivered to the ER by an alternative TRC40-independent pathway. Upon arrival at the ER membrane, these short secretory proteins appear to be ubiquitously transported across the ER membrane through the Sec61 translocon, apparently irrespective of their delivery route. We speculate that the post-translational translocation of secretory proteins in higher eukaryotes is more prevalent than previously acknowledged.

Role of a conserved aspartic acid in nucleotide binding domain 1 (NBD1) of Hsp100 chaperones in their activities.

Besides its beneficial role in thermotolerance, the chaperone protein Hsp104 is involved in the inheritance of yeast Saccharomyces cerevisiae prions. Guanidine hydrochloride was previously shown to interfere with Hsp104 chaperone activity in vivo, thus impairing thermotolerance and resulting in prion curing. It was also reported that guanidine inhibits Hsp104 ATPase and disaggregation activity. We show that in vitro guanidine significantly inhibits the disaggregation activity of ClpB, the bacterial orthologue of Hsp104. However, guanidine exerts opposite effects on the ATPase activities of Hsp104 and ClpB. While the ATPase activity of Hsp104 is inhibited, the analogous ClpB activity is stimulated several-fold. Mutation of the universally conserved aspartic acid residue in position 184 to serine (D184S) in HSP104 and the analogous mutation in clpB (D178S) resulted in chaperones with lower disaggregating and ATPase activities. The activities of such changed chaperones are not influenced by guanidine, which suggests the role of this residue in the interaction with guanidine.

Ca2+-calmodulin inhibits tail-anchored protein insertion into the mammalian endoplasmic reticulum membrane.

Cytosolic components and pathways have been identified that are involved in inserting tail-anchored (TA) membrane proteins into the yeast or mammalian endoplasmic reticulum (ER) membrane. Searching for regulatory mechanisms of TA protein biogenesis, we found that Ca(2+)-calmodulin (CaM) inhibits the insertion of TA proteins into mammalian ER membranes and that this inhibition is prevented by trifluoperazine, a CaM antagonist that interferes with substrate binding of Ca(2+)-CaM. The effects of Ca(2+)-CaM on cytochrome b(5) and Synaptobrevin 2 suggest a direct interaction between Ca(2+)-CaM and TA proteins. Thus, CaM appears to regulate TA insertion into the ER membrane in a Ca(2+) dependent manner.

A biochemical analysis of the constraints of tail-anchored protein biogenesis.

TA (tail-anchored) proteins utilize distinct biosynthetic pathways, including TRC40 (transmembrane domain recognition complex of 40 kDa)-mediated, chaperone-dependent and/or unassisted routes to the ER (endoplasmic reticulum) membrane. We have addressed the flexibility of cytosolic components participating in these pathways, and explored the thermodynamic constraints of their membrane insertion, by exploiting recombinant forms of Sec61ß and Cytb5 (cytochrome b5) bearing covalent modifications within their TA region. In both cases, efficient membrane insertion relied on cytosolic factors capable of accommodating a surprising range of covalent modifications to the TA region. For Sec61ß, we found that both SGTA (small glutamine-rich tetratricopeptide repeat-containing protein α) and TRC40 can bind this substrate with a singly PEGylated TA region. However, by introducing two PEG [poly(ethylene glycol)] moieties, TRC40 binding can be prevented, resulting in a block of subsequent membrane integration. Although TRC40 can bind Sec61ß polypeptides singly PEGylated at different locations, membrane insertion is more sensitive to the precise location of PEG attachment. Modelling and experimentation indicate that this post-TRC40 effect results from an increased energetic cost of inserting different PEGylated TA regions into the lipid bilayer. We therefore propose that the membrane integration of TA proteins delivered via TRC40 is strongly dependent upon underlying thermodynamics, and speculate that their insertion is via a phospholipid-mediated process.

Bat3 promotes the membrane integration of tail-anchored proteins.

The membrane integration of tail-anchored proteins at the endoplasmic reticulum (ER) is post-translational, with different tail-anchored proteins exploiting distinct cytosolic factors. For example, mammalian TRC40 has a well-defined role during delivery of tail-anchored proteins to the ER. Although its Saccharomyces cerevisiae equivalent, Get3, is known to function in concert with at least four other components, Get1, Get2, Get4 and Get5 (Mdy2), the role of additional mammalian proteins during tail-anchored protein biogenesis is unclear. To this end, we analysed the cytosolic binding partners of Sec61beta, a well-defined substrate of TRC40, and identified Bat3 as a previously unknown interacting partner. Depletion of Bat3 inhibits the membrane integration of Sec61beta, but not of a second, TRC40-independent, tail-anchored protein, cytochrome b5. Thus, Bat3 influences the in vitro membrane integration of tail-anchored proteins using the TRC40 pathway. When expressed in Saccharomyces cerevisiae lacking a functional GET pathway for tail-anchored protein biogenesis, Bat3 associates with the resulting cytosolic pool of non-targeted chains and diverts it to the nucleus. This Bat3-mediated mislocalisation is not dependent upon Sgt2, a recently identified component of the yeast GET pathway, and we propose that Bat3 either modulates the TRC40 pathway in higher eukaryotes or provides an alternative fate for newly synthesised tail-anchored proteins.

Post-translational integration of tail-anchored proteins is facilitated by defined molecular chaperones.

Tail-anchored (TA) proteins provide an ideal model for studying post-translational integration at the endoplasmic reticulum (ER) of eukaryotes. There are multiple pathways for delivering TA proteins from the cytosol to the ER membrane yet, whereas an ATP-dependent route predominates, none of the cytosolic components involved had been identified. In this study we have directly addressed this issue and identify novel interactions between a model TA protein and the two cytosolic chaperones Hsp40 and Hsc70. To investigate their function, we have reconstituted the membrane integration of TA proteins using purified components. Remarkably, we find that a combination of Hsc70 and Hsp40 can completely substitute for the ATP-dependent factors present in cytosol. On the basis of this in vitro analysis, we conclude that this chaperone pair can efficiently facilitate the ATP-dependent integration of TA proteins.

[Aggregation and toxicity of the proteins with polyQ repeats]. / Agregacja a toksycznosc bialek z powtórzeniami polyQ.

Expansion of CAG triplet repeats is a cause of at least nine late-onset neurodegenerative disorders. The mutation manifests itself as a long stretch of glutamine repeats. The number of approximately 38 repeats is usually a threshold at which the disease develops and the longer the polyglutamine tract, the earlier the onset of disease. A common feature of these disorders is the presence of protein aggregates which are believed to be formed either by the formation of hydrogen bonds between amide residues or through the action of the enzyme transglutaminase. Mutated proteins may cause neurodegeneration by sequestering vital cellular proteins, inhibiting proteasomal system or by inducing apoptosis. It has been proved that molecular chaperones may block the negative effects of expression of mutated genes and for this reason they are a promising object for various therapeutic research.