Gating motions underlie AMPA receptor secretion from the endoplasmic reticulum

Ion channel biogenesis involves an intricate interplay between subunit folding and assembly. Channel stoichiometries vary and give rise to diverse functions, which impacts on neuronal signalling. AMPA glutamate receptor (AMPAR) assembly is modulated by RNA processing. Here, we provide mechanistic insight into this process. First, we show that a single alternatively spliced residue within the ligand-binding domain alters AMPAR secretion from the ER. Local contacts differ between Leu758 of the GluR2-flop splice form as compared with the flip-specific Val758, which is transmitted globally to alter resensitization kinetics. Detailed biochemical and functional analysis of mutants suggest that AMPARs sample the gating cascade prior to ER export. Irreversibly locking the receptor within various states of the cascade severely attenuates ER transit. Alternative RNA processing by contrast, shifts equilibria between transition states reversibly and thereby modulates secretion kinetics. These data reveal how RNA processing tunes AMPAR biogenesis, and imply that gating transitions in the ER determine iGluR secretory traffic.     

Protein sorting upon exit from the endoplasmic reticulum

It is currently thought that all secretory proteins travel together to the Golgi apparatus where they are sorted to different destinations. However, the specific requirements for transport of GPI-anchored proteins from the endoplasmic reticulum to the Golgi apparatus in yeast could be explained if protein sorting occurs earlier in the pathway. Using an in vitro assay that reconstitutes a single round of budding from the endoplasmic reticulum, we found that GPI-anchored proteins and other secretory proteins exit the endoplasmic reticulum in distinct vesicles. Therefore, GPI-anchored proteins are sorted from other proteins, in particular other plasma membrane proteins, at an early stage of the secretory pathway. These results have wide implications for the mechanism of protein exit from the endoplasmic reticulum.     

Legionella phagosomes intercept vesicular traffic from endoplasmic reticulum exit sites

It is unknown how Legionella pneumophila cells escape the degradative lysosomal pathway after phagocytosis by macrophages and replicate in an organelle derived from the endoplasmic reticulum. Here we show that, after internalization, L. pneumophila-containing phagosomes recruit early secretory vesicles. Once L. pneumophila phagosomes have intercepted early secretory vesicles they begin to acquire proteins residing in transitional and rough endoplasmic reticulum. The functions of Sar1 and ADP-ribosylation factor-1 are important for biogenesis of the L. pneumophila replicative organelle. These data indicate that L. pneumophila intercepts vesicular traffic from endoplasmic-reticulum exit sites to create an organelle that permits intracellular replication and prevents destruction by the host cell.     

Characterization of residues in the cytoplasmic domain of the LDL receptor required for exit from the endoplasmic reticulum

Newly synthesized low density lipoprotein receptors (LDLRs) exit the endoplasmic reticulum (ER) as the first step in the secretory pathway. In this study we have generated truncating deletions and substitutions within the 50 amino acid cytoplasmic domain of the LDLR in order to identify residues required for the exit from the ER. Western blot analysis was used to determine the relative amounts of the 120 kDa precursor form of the LDLR located in the ER and the 160 kDa mature form that has exited the ER. These studies have shown that the exit of an LDLR lacking the cytoplasmic domain, is markedly reduced. Moreover, the longer the cytoplasmic domain, the more efficient is the exit from the ER. At least 30 residues were required for the LDLR to efficiently exit the ER. Mutations in the two di-acidic motifs ExE₈₁₄ and/or ExD₈₃₇ had only a small effect on the exit from the ER. The requirement for a certain length of the cytoplasmic domain for efficient exit from the ER, could reflect the distance needed to interact with the COPII complex of the ER membrane or the requirement for the LDLR to undergo dimerization.     

Cutting edge: Tapasin is retained in the endoplasmic reticulum by dynamic clustering and exclusion from endoplasmic reticulum exit sites

Tapasin retains empty or suboptimally loaded MHC class I molecules in the endoplasmic reticulum (ER). However, the molecular mechanism of this process and how tapasin itself is retained in the ER are unknown. These questions were addressed by tagging tapasin with the cyan fluorescent protein or yellow fluorescent protein (YFP) and probing the distribution and mobility of the tagged proteins. YFP-tapasin molecules were functional and could be isolated in association with TAP, as reported for native tapasin. YFP-tapasin was excluded from ER exit sites even after accumulation of secretory cargo due to disrupted anterograde traffic. Almost all tapasin molecules were clustered, and these clusters diffused freely in the ER. Tapasin oligomers appear to be retained by the failure of the export machinery to recognize them as cargo.     

Assembly,sorting, and exit of oligomeric proteins from the endoplasmic reticulum

The endoplasmic reticulum (ER) uses various mechanisms to ensure that only properly folded proteins enter the secretory pathway. For proteins that oligomerize in the ER, the proper tertiary and quaternary structures must be achieved before their release. Although some proteins fold before oligomerization, others initiate oligomerization cotranslationally. Here, we discuss these different strategies and some of the unique problems they present for the ER quality control system. One mechanism used by the ER is thiol retention. Thiol retention operates by monitoring the redox state of specific cysteine residue(s) and was discovered in studies on the assembly of IgM, a complex oligomeric glycoprotein. This system is also involved in retaining other unassembled proteins in the ER. Mutations that result in uneven numbers of cysteine residues can subject yet other proteins to thiol retention, altering their oligomerization status and function. The implications of these results on the effects of thiol retention on protein function and cell fate are discussed. BioEssays 20 :546–554, 1998. © 1998 John Wiley & Sons Inc.     

Palmitylation of viral membrane glycoproteins takes place after exit from the endoplasmic reticulum

Palmitylation of vesicular stomatitis virus G and Sindbis virus E1 glycoproteins has been studied in relation to the transport from the endoplasmic reticulum (ER) to the Golgi complex. Incubation of infected cells at 15 degrees C prevents the transport of newly synthesized membrane proteins from the ER to the Golgi (Saraste, J., and Kuismanen, E. (1984) Cell 38, 535-549). In these conditions, also palmitylation of G protein and of E1 glycoprotein is blocked. When the transport is restored by increasing the temperature, palmitylation occurs quickly and is followed by the complete trimming of peripheral mannose residues due to mannosidase I (a putative cis-Golgi function). Immunofluorescence analysis showed that the G glycoprotein accumulated at 15 degrees C in structures distinct from both ER and Golgi. These studies suggest that transport from the ER to the cis-Golgi involves intermediate compartments.     

AMPA receptor subunit expression in the endoplasmic reticulum in frontal cortex of elderly patients with schizophrenia

Several lines of evidence indicate altered trafficking of α-amino-3-hydroxyl-5-methyl-4-isoxazole-propionate (AMPA) receptors in schizophrenia. Previous reports have shown potential changes in the trafficking of AMPA receptors based on subunit expression of endosomes, subcellular organelles located near post-synaptic sites. We hypothesized that alterations in AMPA receptor trafficking through the endoplasmic reticulum (ER) may also be altered in schizophrenia. Accordingly, we developed a technique to isolate and measure content of the ER from postmortem brain tissue. We used Western blot and electron microscopy to show that we isolated an ER enriched fraction. We found no changes in the expression of the AMPA receptor subunits, GluR1-4, in the ER from the dorsolateral prefrontal cortex in schizophrenia. These data suggest that AMPA receptor trafficking through the ER is largely intact in schizophrenia.     

Alpha2B-adrenergic receptor interaction with tubulin controls its transport from the endoplasmic reticulum to the cell surface

It is well recognized that the C terminus (CT) plays a crucial role in modulating G protein-coupled receptor (GPCR) transport from the endoplasmic reticulum (ER) to the cell surface. However the molecular mechanisms that govern CT-dependent ER export remain elusive. To address this issue, we used α(2B)-adrenergic receptor (α(2B)-AR) as a model GPCR to search for proteins interacting with the CT. By using peptide-conjugated affinity matrix combined with proteomics and glutathione S-transferase fusion protein pull-down assays, we identified tubulin directly interacting with the α(2B)-AR CT. The interaction domains were mapped to the acidic CT of tubulin and the basic Arg residues in the α(2B)-AR CT, particularly Arg-437, Arg-441, and Arg-446. More importantly, mutation of these Arg residues to disrupt tubulin interaction markedly inhibited α(2B)-AR transport to the cell surface and strongly arrested the receptor in the ER. These data provide the first evidence indicating that the α(2B)-AR C-terminal Arg cluster mediates its association with tubulin to coordinate its ER-to-cell surface traffic and suggest a novel mechanism of GPCR export through physical contact with microtubules.     

Specific heterodimer formation is a prerequisite for uroplakins to exit from the endoplasmic reticulum

Much of the lower urinary tract, including the bladder, is lined by a stratified urothelium forming a highly differentiated, superficial umbrella cell layer. The apical plasma membrane as well as abundant cytoplasmic fusiform vesicles of the umbrella cells is covered by two-dimensional crystals that are formed by four membrane proteins named uroplakins (UPs) Ia, Ib, II, and III. UPs are synthesized on membrane-bound polysomes, and after several co- and posttranslational modifications they assemble into planar crystals in a post-Golgi vesicular compartment. Distension of the bladder may cause fusiform vesicles to fuse with the apical plasma membrane. We have investigated the early stages of uroplakin assembly by expressing the four uroplakins in 293T cells. Transfection experiments showed that, when expressed individually, only UPIb can exit from the endoplasmic reticulum (ER) and move to the plasma membrane, whereas UPII and UPIII reach the plasma membrane only when they form heterodimeric complexes with UPIa and UPIb, respectively. Heterodimer formation in the ER was confirmed by pulse-chase experiment followed by coimmunoprecipitation. Our results indicate that the initial building blocks for the assembly of crystalline uroplakin plaques are heterodimeric uroplakin complexes that form in the ER.     

Perturbation of cellular calcium blocks exit of secretory proteins from the rough endoplasmic reticulum

In the cultured human hepatoma HepG2, Ca2+ ionophores block secretion of different secretary proteins to different extents, alpha 1-antitrypsin secretion being more sensitive to A23187 and ionomycin than is alpha 1-antichymotrypsin, and albumin secretion the least of the three proteins studied. As judged by subcellular fractionation experiments and by treatment of pulse chase labeled protein with endoglycosidase H, A23187 and ionomycin cause newly made secretory proteins to remain within the rough endoplasmic reticulum (ER). Experiments in which A23187 is added at different times during a pulse or chase show that secretion of newly made alpha 1-antitrypsin becomes resistant to the ionophore, on average, 15 min after synthesis; this is about 20 min before it reaches the trans-Golgi, and while it is still within the rough ER. We speculate that a high concentration of Ca2+ within the ER may be essential for certain secretory proteins to fold properly, that folding is inhibited when ER Ca2+ levels are lowered by ionophore treatment, and that unfolded proteins, particularly alpha 1-antitrypsin, cannot exit the rough ER. Treatment of murine 3T3 fibroblasts or human hepatoma HepG2 cells with the Ca2+ ionophores A23187 or ionomycin also induces a severalfold accumulation of the ER lumenal protein Bip (Grp78). These findings disagree with a recent report that Ca2+ ionophores cause secretion of Bip and other resident ER proteins, but is consistent with other reports that A23187 causes accumulation of mRNAs for Bip and other ER lumenal proteins.     

cTAGE5 mediates collagen secretion through interaction with TANGO1 at endoplasmic reticulum exit sites

Cutaneous T-cell lymphoma-associated antigen 5 (cTAGE5), an originally identified tumor antigen, is overexpressed in various cancer cell lines. The cDNA encodes an integral membrane protein containing two coiled-coil motifs and a proline-rich domain. We show that cTAGE5 specifically localizes to the endoplasmic reticulum (ER) exit sites. In addition, cTAGE5 forms a complex with TANGO1 (MIA3), a previously characterized cargo receptor for collagen VII, by the interaction of their coiled-coil motifs. Of interest, cTAGE5, as well as TANGO1, is capable of interacting with the inner-layer coatomer of COPII Sec23/24 complex through their C-terminal proline-rich domains and required for collagen VII secretion. We propose that cTAGE5 acts as a coreceptor of TANGO1 for collagen VII export from the ER.     

Phosphatidylinositol synthesis at the endoplasmic reticulum

Phosphatidylinositol (PI) is a minor phospholipid with a characteristic fatty acid profile; it is highly enriched in stearic acid at the sn-1 position and arachidonic acid at the sn-2 position. PI is phosphorylated into seven specific derivatives, and individual species are involved in a vast array of cellular functions including signalling, membrane traffic, ion channel regulation and actin dynamics. De novo PI synthesis takes place at the endoplasmic reticulum where phosphatidic acid (PA) is converted to PI in two enzymatic steps. PA is also produced at the plasma membrane during phospholipase C signalling, where hydrolysis of phosphatidylinositol (4,5) bisphosphate (PI(4,5)P₂) leads to the production of diacylglycerol which is rapidly phosphorylated to PA. This PA is transferred to the ER to be also recycled back to PI. For the synthesis of PI, CDP-diacylglycerol synthase (CDS) converts PA to the intermediate, CDP-DG, which is then used by PI synthase to make PI. The de novo synthesised PI undergoes remodelling to acquire its characteristic fatty acid profile, which is altered in p53-mutated cancer cells. In mammals, there are two CDS enzymes at the ER, CDS1 and CDS2. In this review, we summarise the de novo synthesis of PI at the ER and the enzymes involved in its subsequent remodelling to acquire its characteristic acyl chains. We discuss how CDS, the rate limiting enzymes in PI synthesis are regulated by different mechanisms. During phospholipase C signalling, the CDS1 enzyme is specifically upregulated by cFos via protein kinase C.     

Regulation of ApoB secretion by the low density lipoprotein receptor requires exit from the endoplasmic reticulum and interaction with ApoE or ApoB

Apolipoprotein B (apoB) is required for the hepatic assembly and secretion of very low density lipoprotein (VLDL). The LDL receptor (LDLR) promotes post-translational degradation of apoB and thereby reduces VLDL particle secretion. We investigated the trafficking pathways and ligand requirements for the LDLR to promote degradation of apoB. We first tested whether the LDLR drives apoB degradation in an endoplasmic reticulum (ER)-associated pathway. Primary mouse hepatocytes harboring an ethyl-nitrosourea-induced, ER-retained mutant LDLR secreted comparable levels of apoB with LDLR-null hepatocytes, despite reduced secretion from cells expressing the wild-type LDLR. Additionally, treatment of cells with brefeldin A inhibited LDLR-dependent degradation. However, this rescue was reversible, and degradation of apoB occurred upon removal of brefeldin A. To characterize the lipoprotein reuptake pathway of degradation, we employed an LDLR mutant defective in constitutive endocytosis and internalization of apoB. This mutant was as effective in reducing apoB secretion as the wild-type LDLR. However, the effect was dependent on apolipoprotein E (apoE) as only the wild-type LDLR, and not the endocytic mutant, reduced apoB secretion in apoE-null cells. Treatment with heparin rescued a pool of apoB in cells expressing the endocytic mutant, indicating that reuptake of VLDL via apoE still occurs with this mutant. Finally, an LDLR mutant defective in binding apoB but not apoE reduced apoB secretion in an apoE-dependent manner. Together, these data suggest that the LDLR directs apoB to degradation in a post-ER compartment. Furthermore, the reuptake mechanism of degradation occurs via internalization of apoB through a constitutive endocytic pathway and apoE through a ligand-dependent pathway.     

Unscrambling exit site patterns on the endoplasmic reticulum as a quenched demixing process

The endoplasmic reticulum (ER) is a vital organelle in mammalian cells with a complex morphology. Consisting of sheet-like cisternae in the cell center, the peripheral ER forms a vast tubular network on which a dispersed pattern of a few hundred specialized domains (ER exit sites (ERESs)) is maintained. Molecular details of cargo sorting and vesicle formation at individual ERESs, fueling the early secretory pathway, have been studied in some detail. The emergence of spatially extended ERES patterns, however, has remained poorly understood. Here, we show that these patterns are determined by the underlying ER morphology, suggesting ERESs to emerge from a demixing process that is quenched by the ER network topology. In particular, we observed fewer but larger ERESs when transforming the ER network to more sheet-like morphologies. In contrast, little to no changes with respect to native ERES patterns were observed when fragmenting the ER, indicating that hampering the diffusion-mediated coarse graining of domains is key for native ERES patterns. Model simulations support the notion of effective diffusion barriers impeding the coarse graining and maturation of ERES patterns. We speculate that tuning a simple demixing mechanism by the ER topology allows for a robust but flexible adaption of ERES patterns, ensuring a properly working early secretory pathway in a variety of conditions.