Quality Control of Protein Complex Assembly by a Transmembrane Recognition Factor.

The inner nuclear membrane (INM) is continuous with the endoplasmic reticulum (ER) but harbors a distinctive proteome essential for nuclear functions. In yeast, the Asi1/Asi2/Asi3 ubiquitin ligase complex safeguards the INM proteome through the clearance of mislocalized ER membrane proteins. How the Asi complex selectively targets mislocalized proteins and coordinates its activity with other ER functions, such as protein biogenesis, is unclear. Here, we uncover a link between INM proteome identity and membrane protein complex assembly in the remaining ER. We show that lone proteins and complex subunits failing to assemble in the ER access the INM for Asi-mediated degradation. Substrates are recognized by direct binding of Asi2 to their transmembrane domains for subsequent ubiquitination by Asi1/Asi3 and membrane extraction. Our data suggest a model in which spatial segregation of membrane protein complex assembly and quality control improves assembly efficiency and reduces the levels of orphan subunits.

Predominant Golgi Residency of the Plant K/HDEL Receptor Is Essential for Its Function in Mediating ER Retention.

Accumulation of soluble proteins in the endoplasmic reticulum (ER) of plants is mediated by a receptor termed ER RETENTION DEFECTIVE2 (ERD2) or K/HDEL receptor. Using two gain-of-function assays and by complementing loss of function in Nicotiana benthamiana, we discovered that compromising the lumenal N terminus or the cytosolic C terminus with fluorescent fusions abolishes its biological function and profoundly affects its subcellular localization. Based on the confirmed asymmetrical topology of ERD2, we engineered a new fluorescent ERD2 fusion protein that retains biological activity. Using this fusion, we show that ERD2 is exclusively detected at the Golgi apparatus, unlike nonfunctional C-terminal fusions, which also label the ER. Moreover, ERD2 is confined to early Golgi compartments and does not show ligand-induced redistribution to the ER. We show that the cytosolic C terminus of ERD2 plays a crucial role in its function. Two conserved leucine residues that do not correspond to any known targeting motifs for ER-Golgi trafficking were shown to be essential for both ERD2 Golgi residency and its ability to mediate ER retention of soluble ligands. The results suggest that anterograde ER to Golgi transport of ERD2 is either extremely fast, well in excess of the bulk flow rate, or that ERD2 does not recycle in the way originally proposed.

Golgi-dependent transport of vacuolar sorting receptors is regulated by COPII, AP1, and AP4 protein complexes in tobacco.

The cycling of vacuolar sorting receptors (VSRs) between early and late secretory pathway compartments is regulated by signals in the cytosolic tail, but the exact pathway is controversial. Here, we show that receptor targeting in tobacco (Nicotiana tabacum) initially involves a canonical coat protein complex II-dependent endoplasmic reticulum-to-Golgi bulk flow route and that VSR-ligand interactions in the cis-Golgi play an important role in vacuolar sorting. We also show that a conserved Glu is required but not sufficient for rate-limiting YXX-mediated receptor trafficking. Protein-protein interaction studies show that the VSR tail interacts with the µ-subunits of plant or mammalian clathrin adaptor complex AP1 and plant AP4 but not that of plant and mammalian AP2. Mutants causing a detour of full-length receptors via the cell surface invariantly cause the secretion of VSR ligands. Therefore, we propose that cycling via the plasma membrane is unlikely to play a role in biosynthetic vacuolar sorting under normal physiological conditions and that the conserved Ile-Met motif is mainly used to recover mistargeted receptors. This occurs via a fundamentally different pathway from the prevacuolar compartment that does not mediate recycling. The role of clathrin and clathrin-independent pathways in vacuolar targeting is discussed.

Vacuolar transport in tobacco leaf epidermis cells involves a single route for soluble cargo and multiple routes for membrane cargo.

We tested if different classes of vacuolar cargo reach the vacuole via distinct mechanisms by interference at multiple steps along the transport route. We show that nucleotide-free mutants of low molecular weight GTPases, including Rab11, the Rab5 members Rha1 and Ara6, and the tonoplast-resident Rab7, caused induced secretion of both lytic and storage vacuolar cargo. In situ analysis in leaf epidermis cells indicates a sequential action of Rab11, Rab5, and Rab7 GTPases. Compared with Rab5 members, mutant Rab11 mediates an early transport defect interfering with the arrival of cargo at prevacuoles, while mutant Rab7 inhibits the final delivery to the vacuole and increases cargo levels in prevacuoles. In contrast with soluble cargo, membrane cargo may follow different routes. Tonoplast targeting of an α-TIP chimera was impaired by nucleotide-free Rha1, Ara6, and Rab7 similar to soluble cargo. By contrast, the tail-anchored tonoplast SNARE Vam3 shares only the Rab7-mediated vacuolar deposition step. The most marked difference was observed for the calcineurin binding protein CBL6, which was insensitive to all Rab mutants tested. Unlike soluble cargo, α-TIP and Vam3, CBL6 transport to the vacuole was COPII independent. The results indicate that soluble vacuolar proteins follow a single route to vacuoles, while membrane spanning proteins may use at least three different transport mechanisms.

Endoplasmic reticulum exit sites are segregated for secretion based on cargo size.

TANGO1, TANGO1-Short, and cTAGE5 form stable complexes at the endoplasmic reticulum exit sites (ERES) to preferably export bulky cargoes. Their C-terminal proline-rich domain (PRD) binds Sec23A and affects COPII assembly. The PRD in TANGO1-Short was replaced with light-responsive domains to control its binding to Sec23A in U2OS cells (human osteosarcoma). TANGO1-ShortΔPRD was dispersed in the ER membrane but relocated rapidly, reversibly, to pre-existing ERES by binding to Sec23A upon light activation. Prolonged binding between the two, concentrated ERES in the juxtanuclear region, blocked cargo export and relocated ERGIC53 into the ER, minimally impacting the Golgi complex organization. Bulky collagen VII and endogenous collagen I were collected at less than 47% of the stalled ERES, whereas small cargo molecules were retained uniformly at almost all the ERES. We suggest that ERES are segregated to handle cargoes based on their size, permitting cells to traffic them simultaneously for optimal secretion.

Pla2g12b drives expansion of triglyceride-rich lipoproteins.

Vertebrates transport hydrophobic triglycerides through the circulatory system by packaging them within amphipathic particles called Triglyceride-Rich Lipoproteins. Yet, it remains largely unknown how triglycerides are loaded onto these particles. Mutations in Phospholipase A2 group 12B (PLA2G12B) are known to disrupt lipoprotein homeostasis, but its mechanistic role in this process remains unclear. Here we report that PLA2G12B channels lipids within the lumen of the endoplasmic reticulum into nascent lipoproteins. This activity promotes efficient lipid secretion while preventing excess accumulation of intracellular lipids. We characterize the functional domains, subcellular localization, and interacting partners of PLA2G12B, demonstrating that PLA2G12B is calcium-dependent and tightly associated with the membrane of the endoplasmic reticulum. We also detect profound resistance to atherosclerosis in PLA2G12B mutant mice, suggesting an evolutionary tradeoff between triglyceride transport and cardiovascular disease risk. Here we identify PLA2G12B as a key driver of triglyceride incorporation into vertebrate lipoproteins.

A recycling-defective vacuolar sorting receptor reveals an intermediate compartment situated between prevacuoles and vacuoles in tobacco.

Plant vacuolar sorting receptors (VSRs) display cytosolic Tyr motifs (YMPL) for clathrin-mediated anterograde transport to the prevacuolar compartment. Here, we show that the same motif is also required for VSR recycling. A Y612A point mutation in Arabidopsis thaliana VSR2 leads to a quantitative shift in VSR2 steady state levels from the prevacuolar compartment to the trans-Golgi network when expressed in Nicotiana tabacum. By contrast, the L615A mutant VSR2 leaks strongly to vacuoles and accumulates in a previously undiscovered compartment. The latter is shown to be distinct from the Golgi stacks, the trans-Golgi network, and the prevacuolar compartment but is characterized by high concentrations of soluble vacuolar cargo and the rab5 GTPase Rha1(RabF2a). The results suggest that the prevacuolar compartment matures by gradual receptor depletion, leading to the formation of a late prevacuolar compartment situated between the prevacuolar compartment and the vacuole.

Tetraspanin-8 sequesters syntaxin-2 to control biphasic release propensity of mucin granules.

Agonist-mediated stimulated pathway of mucin and insulin release are biphasic in which rapid fusion of pre-docked granules is followed by slow docking and fusion of granules from the reserve pool. Here, based on a cell-culture system, we show that plasma membrane-located tetraspanin-8 sequesters syntaxin-2 to control mucin release. Tetraspanin-8 affects fusion of granules during the second phase of stimulated mucin release. The tetraspanin-8/syntaxin-2 complex does not contain VAMP-8, which functions with syntaxin-2 to mediate granule fusion. We suggest that by sequestering syntaxin-2, tetraspanin-8 prevents docking of granules from the reserve pool. In the absence of tetraspanin-8, more syntaxin-2 is available for docking and fusion of granules and thus doubles the quantities of mucins secreted. This principle also applies to insulin release and we suggest a cell type specific Tetraspanin/Syntaxin combination is a general mechanism regulating the fusion of dense core granules.

Defects in lipid homeostasis reflect the function of TANGO2 in phospholipid and neutral lipid metabolism.

We show that TANGO2 in mammalian cells localizes predominantly to mitochondria and partially at mitochondria sites juxtaposed to lipid droplets (LDs) and the endoplasmic reticulum. HepG2 cells and fibroblasts of patients lacking TANGO2 exhibit enlarged LDs. Quantitative lipidomics revealed a marked increase in lysophosphatidic acid (LPA) and a concomitant decrease in its biosynthetic precursor phosphatidic acid (PA). These changes were exacerbated in nutrient-starved cells. Based on our data, we suggest that TANGO2 function is linked to acyl-CoA metabolism, which is necessary for the acylation of LPA to generate PA. The defect in acyl-CoA availability impacts the metabolism of many other fatty acids, generates high levels of reactive oxygen species, and promotes lipid peroxidation. We suggest that the increased size of LDs is a combination of enrichment in peroxidized lipids and a defect in their catabolism. Our findings help explain the physiological consequence of mutations in TANGO2 that induce acute metabolic crises, including rhabdomyolysis, cardiomyopathy, and cardiac arrhythmias, often leading to fatality upon starvation and stress.

Intermediate organelles of the plant secretory pathway: identity and function.

The secretory pathway of eukaryotic cells comprises a network of organelles that connects three large membranes, the plasma membrane, the vacuole and the endoplasmic reticulum. The Golgi apparatus and the various post-Golgi organelles that control vacuolar sorting, secretion and endocytosis can be regarded as intermediate organelles of the endocytic and biosynthetic routes. Many processes in the secretory pathway have evolved differently in plants and cannot be studied using yeast or mammalian cells as models. The best characterized organelles are the Golgi apparatus and the prevacuolar compartment, but recent work has shed light on the role of the trans Golgi network, which has to be regarded as a separate organelle in plants. In this study, we wish to highlight recent findings regarding the late secretory pathway and its crosstalk with the early secretory pathway as well as the endocytic route in plants. Recently published findings and suggested models are discussed within the context of known features of the equivalent pathway in other eukaryotes.

The syntaxins SYP31 and SYP81 control ER-Golgi trafficking in the plant secretory pathway.

Overexpression of the Golgi and endoplasmic reticulum (ER) syntaxins SYP31 and SYP81 strongly inhibits constitutive secretion. By comparing the secreted reporter alpha-amylase with the ER-retained reporter alpha-amylase-HDEL, it was concluded that SYP81 overexpression inhibits both retrograde and anterograde transport, while SYP31 overexpression mainly affected anterograde transport. Of the other interacting SNAREs investigated, only the overexpression of MEMB11 led to an inhibition of protein secretion. Although the position of a fluorescent tag does not influence the correct localization of the fusion protein, only N-terminal-tagged SYP31 retained the ability of the untagged SNARE to inhibit transport. C-terminal-tagged SYP31 failed to exhibit this effect. Overexpression of both wild-type and N-terminal-tagged syntaxins caused standard Golgi marker proteins to redistribute into the ER. Nevertheless, green fluorescent protein (GFP)-SYP31 was still visible as fluorescent punctae, which, unlike SYP31-GFP, were resistant to brefeldin A treatment. Immunogold electron microscopy showed that endogenous SYP81 is not only present at the ER but also in the cis Golgi, indicating that this syntaxin cycles between these two organelles. However, when expressed at non-inhibitory levels, YFP-SYP81 was seen to locate principally to subdomains of the ER. These punctate structures were physically separated from the Golgi, suggesting that they might possibly reflect the position of ER import sites.

Biallelic TANGO1 mutations cause a novel syndromal disease due to hampered cellular collagen secretion.

The transport and Golgi organization 1 (TANGO1) proteins play pivotal roles in the secretory pathway. Full length TANGO1 is a transmembrane protein localised at endoplasmic reticulum (ER) exit sites, where it binds bulky cargo within the ER lumen and recruits membranes from the ER Golgi intermediate compartment to create an exit route for their export. Here we report the first TANGO1-associated syndrome in humans. A synonymous substitution that results in exon eight skipping in most mRNA molecules, ultimately leading to a truncated TANGO1 protein was identified as disease-causing mutation. The four homozygously affected sons of a consanguineous family display severe dentinogenesis imperfecta, short stature, various skeletal abnormalities, insulin-dependent diabetes mellitus, sensorineural hearing loss, and mild intellectual disability. Functional studies in HeLa and U2OS cells revealed that the corresponding truncated TANGO1 protein is dispersed in the ER and its expression in cells with intact endogenous TANGO1 impairs cellular collagen I secretion.

TANGO1 builds a machine for collagen export by recruiting and spatially organizing COPII, tethers and membranes.

Collagen export from the endoplasmic reticulum (ER) requires TANGO1, COPII coats, and retrograde fusion of ERGIC membranes. How do these components come together to produce a transport carrier commensurate with the bulky cargo collagen? TANGO1 is known to form a ring that corrals COPII coats, and we show here how this ring or fence is assembled. Our data reveal that a TANGO1 ring is organized by its radial interaction with COPII, and lateral interactions with cTAGE5, TANGO1-short or itself. Of particular interest is the finding that TANGO1 recruits ERGIC membranes for collagen export via the NRZ (NBAS/RINT1/ZW10) tether complex. Therefore, TANGO1 couples retrograde membrane flow to anterograde cargo transport. Without the NRZ complex, the TANGO1 ring does not assemble, suggesting its role in nucleating or stabilising this process. Thus, coordinated capture of COPII coats, cTAGE5, TANGO1-short, and tethers by TANGO1 assembles a collagen export machine at the ER.

Quality control: ER-associated degradation: protein quality control and beyond.

Even with the assistance of many cellular factors, a significant fraction of newly synthesized proteins ends up misfolded. Cells evolved protein quality control systems to ensure that these potentially toxic species are detected and eliminated. The best characterized of these pathways, the ER-associated protein degradation (ERAD), monitors the folding of membrane and secretory proteins whose biogenesis takes place in the endoplasmic reticulum (ER). There is also increasing evidence that ERAD controls other ER-related functions through regulated degradation of certain folded ER proteins, further highlighting the role of ERAD in cellular homeostasis.

Quality control of inner nuclear membrane proteins by the Asi complex.

Misfolded proteins in the endoplasmic reticulum (ER) are eliminated by a quality control system called ER-associated protein degradation (ERAD). However, it is unknown how misfolded proteins in the inner nuclear membrane (INM), a specialized ER subdomain, are degraded. We used a quantitative proteomics approach to reveal an ERAD branch required for INM protein quality control in yeast. This branch involved the integral membrane proteins Asi1, Asi2, and Asi3, which assembled into an Asi complex. Besides INM misfolded proteins, the Asi complex promoted the degradation of functional regulators of sterol biosynthesis. Asi-mediated ERAD was required for ER homeostasis, which suggests that spatial segregation of protein quality control systems contributes to ER function.

Analysis of familial hemophagocytic lymphohistiocytosis type 4 (FHL-4) mutant proteins reveals that S-acylation is required for the function of syntaxin 11 in natural killer cells.

Natural killer (NK) cell secretory lysosome exocytosis and cytotoxicity are impaired in familial hemophagocytic lymphohistiocytosis type 4 (FHL-4), a disorder caused by mutations in the gene encoding the SNARE protein syntaxin 11. We show that syntaxin 11 binds to SNAP23 in NK cells and that this interaction is reduced by FHL-4 truncation and frameshift mutation proteins that delete all or part of the SNARE domain of syntaxin 11. In contrast the FHL-4 mutant proteins bound to the Sec-1/Munc18-like (SM) protein Munc18-2. We demonstrate that the C-terminal cysteine rich region of syntaxin 11, which is deleted in the FHL-4 mutants, is S-acylated. This posttranslational modification is required for the membrane association of syntaxin 11 and for its polarization to the immunological synapse in NK cells conjugated to target cells. Moreover, we show that Munc18-2 is recruited by syntaxin 11 to intracellular membranes in resting NK cells and to the immunological synapse in activated NK cells. This recruitment of Munc18-2 is abolished by deletion of the C-terminal cysteine rich region of syntaxin 11. These results suggest a pivotal role for S-acylation in the function of syntaxin 11 in NK cells.

Sterol homeostasis requires regulated degradation of squalene monooxygenase by the ubiquitin ligase Doa10/Teb4.

Sterol homeostasis is essential for the function of cellular membranes and requires feedback inhibition of HMGR, a rate-limiting enzyme of the mevalonate pathway. As HMGR acts at the beginning of the pathway, its regulation affects the synthesis of sterols and of other essential mevalonate-derived metabolites, such as ubiquinone or dolichol. Here, we describe a novel, evolutionarily conserved feedback system operating at a sterol-specific step of the mevalonate pathway. This involves the sterol-dependent degradation of squalene monooxygenase mediated by the yeast Doa10 or mammalian Teb4, a ubiquitin ligase implicated in a branch of the endoplasmic reticulum (ER)-associated protein degradation (ERAD) pathway. Since the other branch of ERAD is required for HMGR regulation, our results reveal a fundamental role for ERAD in sterol homeostasis, with the two branches of this pathway acting together to control sterol biosynthesis at different levels and thereby allowing independent regulation of multiple products of the mevalonate pathway. DOI:http://dx.doi.org/10.7554/eLife.00953.001.

Protein domains involved in assembly in the endoplasmic reticulum promote vacuolar delivery when fused to secretory GFP, indicating a protein quality control pathway for degradation in the plant vacuole.

The correct folding and assembly of newly synthesized secretory proteins are monitored by the protein quality control system of the endoplasmic reticulum (ER). Through interactions with chaperones such as the binding protein (BiP) and other folding helpers, quality control favors productive folding and sorts for degradation defective proteins. A major route for quality control degradation identified in yeast, plants, and animals is constituted by retrotranslocation from the ER to the cytosol and subsequent disposal by the ubiquitin/proteasome system, but alternative routes involving the vacuole have been identified in yeast. In this study, we have studied the destiny of sGFP418, a fusion between a secretory form of GFP and a domain of the vacuolar protein phaseolin that is involved in the correct assembly of phaseolin and in BiP recognition of unassembled subunits. We show that sGFP418, despite lacking the phaseolin vacuolar sorting signal, is delivered to the vacuole and fragmented, in a process that is inhibited by the secretory traffic inhibitor brefeldin A. Moreover, a fusion between GFP and a domain of the maize storage protein gamma-zein involved in zein polymerization also undergoes post-translational fragmentation similar to that of sGFP418. These results show that defective secretory proteins with permanently exposed sequences normally involved in oligomerization can be delivered to the vacuole by secretory traffic. This strongly suggests the existence of a plant vacuolar sorting mechanism devoted to the disposal of defective secretory proteins.

Tomato spotted wilt virus glycoproteins induce the formation of endoplasmic reticulum- and Golgi-derived pleomorphic membrane structures in plant cells.

Tomato spotted wilt virus (TSWV) particles are spherical and enveloped, an uncommon feature among plant infecting viruses. Previous studies have shown that virus particle formation involves the enwrapment of ribonucleoproteins with viral glycoprotein containing Golgi stacks. In this study, the localization and behaviour of the viral glycoproteins Gn and Gc were analysed, upon transient expression in plant protoplasts. When separately expressed, Gc was solely observed in the endoplasmic reticulum (ER), whereas Gn was found both within the ER and Golgi membranes. Upon co-expression, both glycoproteins were found at ER-export sites and ultimately at the Golgi complex, confirming the ability of Gn to rescue Gc from the ER, possibly due to heterodimerization. Interestingly, both Gc and Gn were shown to induce the deformation of ER and Golgi membranes, respectively, also observed upon co-expression of the two glycoproteins. The behaviour of both glycoproteins within the plant cell and the phenomenon of membrane deformation are discussed in light of the natural process of viral infection.