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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.

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.

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. Thierer and colleagues identify PLA2G12B as a key gene driving triglyceride incorporation into lipoproteins and show that disruption of this activity provides protection from atherosclerosis.

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. All cells are enclosed by a membrane that is made up of fatty molecules called lipids and is studded with proteins. This membrane allows cells to detect and react to outside events. Since external conditions, such as temperature, can vary dramatically, membranes need to be able to adjust their properties. For example, lipids become more fluid as the temperature rises, so membranes respond to heat stress by incorporating molecules called sterols to increase their rigidity. In fact, sterols have profound effects on membrane properties and are essential to regulate a number of cellular processes. But high levels of sterols can become toxic, so it is essential that they are carefully controlled. Sterols, such as ergosterol in yeast or cholesterol in mammals, are synthesized in a tightly regulated multi-step process; some of the early steps in sterol production also make common building blocks for other key molecules in the cell. A mechanism to control sterol levels is the regulated destruction of an enzyme that carries out an early step of their synthesis. This occurs via one branch of the ER-associated degradation (ERAD) pathway, which also destroys non-functional proteins. Now, Foresti et al. have found that sterol synthesis is also regulated by another branch of the ERAD pathway. This second control point, which occurs later in the biosynthetic process, allows cells to regulate sterol levels independent of the other products of the pathway that are derived from the same preliminary compounds. In yeast, the two ERAD branches are directed by Hrd1 and Doa10. These are both ubiquitin ligases—proteins that attach a tag called ubiquitin to other proteins, thus labeling them for recycling by the proteasome (essentially a waste-disposal complex in the cell). To identify the proteins that are tagged by Doa10, Foresti et al. compared protein levels in strains lacking Doa10 with those in wild type yeast. Unexpectedly, the enzyme Erg1, which helps to synthesize ergosterol, was more abundant in cells lacking Doa10. Foresti et al. found that Doa10 tagged Erg1 for destruction when levels of the building blocks of ergosterol rose inside the cell. These ergosterol intermediates are toxic to yeast, which converts them into less harmful molecules known as sterol esters using the proteins Are1/2. When the DOA10 or ARE1/2 genes were deleted, these intermediates were more abundant; strikingly, they became even more prevalent when all three genes were knocked out in the same strain. In contrast, blocking the other ERAD branch by deleting HRD1 did not cause ergosterol intermediates to accumulate, nor did it exacerbate the effects of ARE1/2 knockout. When combined with previous findings, these results provide evidence that the different branches of the ERAD pathway regulate ergosterol synthesis at distinct steps. The same mechanism is observed in human cells when high levels of cholesterol are detected. By identifying parallel routes to control sterol levels, this work reinforces the importance of membrane integrity to life.

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. The mechanisms regulating granule secretion are not fully understood. Here the authors show that tetraspanin-8 sequesters syntaxin-2 at the cell surface, limiting the syntaxin available for fusion and the quantities of mucin secreted. This mechanism may also apply to insulin release

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.

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.

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.

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.