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.

We have characterized the requirements to inhibit the function of the plant vacuolar sorting receptor BP80 in vivo and gained insight into the crucial role of receptor recycling between the prevacuolar compartment and the Golgi apparatus. The drug wortmannin interferes with the BP80-mediated route to the vacuole and induces hypersecretion of a soluble BP80-ligand. Wortmannin does not prevent receptor-ligand binding itself but causes BP80 levels to be limiting. Consequently, overexpression of BP80 partially restores vacuolar cargo transport. To simulate receptor traffic, we tested a truncated BP80 derivative in which the entire lumenal domain of BP80 has been replaced by the green fluorescent protein (GFP). The resulting chimeric protein (GFP-BP80) accumulates in the prevacuolar compartment as expected, but a soluble GFP fragment can also be detected in purified vacuoles. Interestingly, GFP-BP80 coexpression interferes with the correct sorting of a BP80-ligand and causes hypersecretion that is reversible by expressing a 10-fold excess of full-length BP80. This suggests that GFP-BP80 competes with endogenous BP80 mainly at the retrograde transport route that rescues receptors from the prevacuolar compartment. Treatment with wortmannin causes further leakage of GFP-BP80 from the prevacuolar compartment to the vacuoles, whereas BP80-ligands are secreted. We propose that recycling of the vacuolar sorting receptor from the prevacuolar compartment to the Golgi apparatus is an essential process that is saturable and wortmannin sensitive.

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.

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

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.

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.

Golgi-mediated transport to the lytic vacuole involves passage through the prevacuolar compartment (PVC), but little is known about how vacuolar proteins exit the PVC. We show that this last step is inhibited by overexpression of Arabidopsis thaliana syntaxin PEP12/SYP21, causing an accumulation of soluble and membrane cargo and the plant vacuolar sorting receptor BP80 in the PVC. Anterograde transport proceeds normally from the endoplasmic reticulum to the Golgi and the PVC, although export from the PVC appears to be compromised, affecting both anterograde membrane flow to the vacuole and the recycling route of BP80 to the Golgi. However, Golgi-mediated transport of soluble and membrane cargo toward the plasma membrane is not affected, but a soluble BP80 ligand is partially mis-sorted to the culture medium. We also observe clustering of individual PVC bodies that move together and possibly fuse with each other, forming enlarged compartments. We conclude that PEP12/SYP21 overexpression specifically inhibits export from the PVC without affecting the Golgi complex or compromising the secretory branch of the endomembrane system. The results provide a functional in vivo assay that confirms PEP12/SYP21 involvement in vacuolar sorting and indicates that excess of this syntaxin in the PVC can be detrimental for further transport from this organelle.

Although signals for vacuolar sorting of soluble proteins are well described, we have yet to learn how the plant vacuolar sorting receptor BP80 reaches its correct destination and recycles. To shed light on receptor targeting, we used an in vivo competition assay in which a truncated receptor (green fluorescent protein-BP80) specifically competes with sorting machinery and causes hypersecretion of BP80-ligands from tobacco (Nicotiana tabacum) leaf protoplasts. We show that both the transmembrane domain and the cytosolic tail of BP80 contain information necessary for efficient progress to the prevacuolar compartment (PVC). Furthermore, the tail must be exposed on the correct membrane surface to compete with sorting machinery. Mutational analysis of conserved residues revealed that multiple sequence motifs are necessary for competition, one of which is a typical Tyr-based motif (YXXF). Substitution of Tyr-612 for Ala causes partial retention in the Golgi apparatus, mistargeting to the plasma membrane (PM), and slower progress to the PVC. A role in Golgi-to-PVC transport was confirmed by generating the corresponding mutation on full-length BP80. The mutant receptor was partially mistargeted to the PM and induced the secretion of a coexpressed BP80-ligand. Further mutants indicate that the cytosolic tail is likely to contain other information besides the YXXF motif, possibly for endoplasmic reticulum export, endocytosis from the PM, and PVC-to-Golgi recycling.