Unassembled Subunits Research Articles

Endoplasmic Reticulum Retention and Associated Degradation of a GABAA Receptor Epilepsy Mutation That Inserts an Aspartate in the M3 Transmembrane Segment of the α1 Subunit

A GABA(A) receptor alpha1 subunit epilepsy mutation (alpha1(A322D)) introduces a negatively charged aspartate residue into the hydrophobic M3 transmembrane domain of the alpha1 subunit. We reported previously that heterologous expression of alpha1(A322D)beta2gamma2 receptors in mammalian cells resulted in reduced total and surface alpha1 subunit protein. Here we demonstrate the mechanism of this reduction. Total alpha1(A322D) subunit protein was reduced relative to wild type protein by a similar amount when expressed alone (86 +/- 6%) or when coexpressed with beta2 and gamma2S subunits (78 +/- 6%), indicating an expression reduction prior to subunit oligomerization. In alpha1beta2gamma2S receptors, endoglycosidase H deglycosylated only 26 +/- 5% of alpha1 subunits, consistent with substantial protein maturation, but in alpha1(A322D)beta2gamma2S receptors, endoglycosidase H deglycosylated 91 +/- 4% of alpha1(A322D) subunits, consistent with failure of protein maturation. To determine the cellular localization of wild type and mutant subunits, the alpha1 subunit was tagged with yellow (alpha1-YFP) or cyan (alpha1-CFP) fluorescent protein. Confocal microscopic imaging demonstrated that 36 +/- 4% of alpha1-YFPbeta2gamma2 but only 5 +/- 1% alpha1(A322D)-YFPbeta2gamma2 colocalized with the plasma membrane, whereas the majority of the remaining receptors colocalized with the endoplasmic reticulum (55 +/- 4% alpha1-YFPbeta2gamma2S, 86 +/- 3% alpha1(A322D)-YFP). Heterozygous expression of alpha1-CFPbeta2gamma2S and alpha1(A322D)-YFPbeta2gamma2S or alpha1-YFPbeta2gamma2S and alpha1(A322D)-CFPbeta2gamma2S receptors showed that membrane GABA(A) receptors contained primarily wild type alpha1 subunits. These data demonstrate that the A322D mutation reduces alpha1 subunit expression after translation, but before assembly, resulting in endoplasmic reticulum-associated degradation and membrane alpha1 subunits that are almost exclusively wild type subunits.

Ubiquilin-1 Regulates Nicotine-induced Up-regulation of Neuronal Nicotinic Acetylcholine Receptors

Chronic exposure to nicotine, as in tobacco smoking, up-regulates nicotinic acetylcholine receptor surface expression in neurons. This up-regulation has been proposed to play a role in nicotine addiction and withdrawal. The regulatory mechanisms behind nicotine-induced up-regulation of surface nicotinic acetylcholine receptors remain to be determined. It has recently been suggested that nicotine stimulation acts through increased assembly and maturation of receptor subunits into functional pentameric receptors. Studies of muscle nicotinic acetylcholine receptors suggest that the availability of unassembled subunits in the endoplasmic reticulum can be regulated by the ubiquitin-proteosome pathway, resulting in altered surface expression. Here, we describe a role for ubiquilin-1, a ubiquitin-like protein with the capacity to interact with both the proteosome and ubiquitin ligases, in regulating nicotine-induced up-regulation of neuronal nicotinic acetylcholine receptors. Ubiquilin-1 interacts with unassembled alpha3 and alpha4 subunits when coexpressed in heterologous cells and interacts with endogenous nicotinic acetylcholine receptors in neurons. Coexpression of ubiquilin-1 and neuronal nicotinic acetylcholine receptors in heterologous cells dramatically reduces the expression of the receptors on the cell surface. In cultured superior cervical ganglion neurons, expression of ubiquilin-1 abolishes nicotine-induced up-regulation of nicotinic acetylcholine receptors but has no effect on the basal level of surface receptors. Coimmunostaining shows that the interaction of ubiquilin-1 with the alpha3 subunit draws the receptor subunit and proteosome into a complex. These data suggest that ubiquilin-1 limits the availability of unassembled nicotinic acetylcholine receptor subunits in neurons by drawing them to the proteosome, thus regulating nicotine-induced up-regulation.

Nicotine Acts as a Pharmacological Chaperone to Up-Regulate Human α4β2 Acetylcholine Receptors

Human neuronal nicotinic acetylcholine receptor (AChR) alpha4 subunits and an alpha4 mutant (S247Falpha4) found in autosomal-dominant nocturnal frontal lobe epilepsy (ADNFLE) were expressed along with beta2 in permanently transfected tsA201 human embryonic kidney cell lines. Their sensitivity to activation, desensitization, and up-regulation by cholinergic ligands was investigated. Up-regulation after 3 to 24 h resulted primarily from an increase in assembly of AChRs from large pools of unassembled subunits, but up-regulation also resulted from a 5-fold increase in the lifetime of AChRs in the surface membrane. Up-regulation does not require current flow through surface membrane AChRs, because up-regulation occurs in the presence of the channel blocker mecamylamine and with the alpha4 mutant, which prevents nearly all AChR function. Both membrane-permeable ligands like nicotine and much less permeable quaternary amine cholinergic ligands can act as pharmacological chaperones within the endoplasmatic reticulum to promote the assembly of AChRs. Agonists are more potent pharmacological chaperones than antagonists, presumably because activated or desensitized conformations assemble more efficiently. Assembly intermediates are disrupted by solubilization in Triton X-100, but chemical cross-linking stabilizes a putative assembly intermediate approximately the size of an alpha4beta2alpha4beta2 tetramer.

Analysis of COX2 mutants reveals cytochrome oxidase subassemblies in yeast

Cytochrome oxidase catalyses the reduction of oxygen to water. The mitochondrial enzyme contains up to 13 subunits, 11 in yeast, of which three, Cox1p, Cox2p and Cox3p, are mitochondrially encoded. The assembly pathway of this complex is still poorly understood. Its study in yeast has been so far impeded by the rapid turnover of unassembled subunits of the enzyme. In the present study, immunoblot analysis of blue native gels of yeast wild-type and Cox2p mutants revealed five cytochrome oxidase complexes or subcomplexes: a, b, c, d and f; a is likely to be the fully assembled enzyme; b lacks Cox6ap; d contains Cox7p and/or Cox7ap; f represents unassembled Cox1p; and c, observed only in the Cox2p mutants, contains Cox1p, Cox3p, Cox5p and Cox6p and lacks the other subunits. The identification of these novel cytochrome oxidase subcomplexes should encourage the reexamination of other yeast mutants.

RIC-3 Enhances Functional Expression of Multiple Nicotinic Acetylcholine Receptor Subtypes in Mammalian Cells

Recent studies have shown that RIC-3, originally identified in Caenorhabditis elegans as the protein encoded by the gene resistance to inhibitors of cholinesterase (ric-3), can enhance functional expression of alpha7 nicotinic acetylcholine receptors (nAChRs). In the present study, the influence of C. elegans and human RIC-3 upon multiple homomeric (alpha7, alpha8, and alpha9) and heteromeric (alpha3beta2, alpha3beta4, alpha4beta2, alpha4beta4, and alpha9alpha10) nAChR subtypes has been examined in transfected mammalian cells by radioligand binding and functional characterization. Coexpression of RIC-3 facilitates a dramatic enhancement of the ability of alpha7 (and the closely related alpha8 subunit) to generate functional nAChRs in otherwise nonpermissive mammalian cells. In contrast, coexpression of RIC-3 did not facilitate functional expression of either homomeric alpha9 or heteromeric alpha9alpha10 nAChRs in mammalian cell lines. It is noteworthy that whereas RIC-3 has been reported to cause a marked functional inhibition of heteromeric nAChRs such as alpha3beta4 and alpha4beta2 expressed in Xenopus laevis oocytes, RIC-3 significantly enhances levels of functional expression of these and other (alpha3beta2 and alpha4beta4) heteromeric nAChRs when expressed in mammalian cell lines. In addition, the interaction of multiple nAChR subunits (alpha3, alpha4, alpha7, beta2, and beta4) with RIC-3 has been demonstrated by coimmunoprecipitation from metabolically labeled transfected cells. It is significant that coimmunoprecipitation experiments have provided evidence that RIC-3 associates with unassembled nAChR subunits, a finding that is consistent with previous suggestions that RIC-3 may act by enhancing the maturation (subunit folding and assembly) of nAChRs. We conclude that RIC-3 is an nAChR-associated protein that can enhance functional expression of multiple nAChR subtypes in transfected mammalian cells.

Distinct Domains of Small Tims Involved in Subunit Interaction and Substrate Recognition

Tim9 and Tim10 belong to the small Tim family of mitochondrial ATP-independent chaperones. They are organised in a specific hetero-oligomeric complex (TIM10) that escorts polytopic proteins into the mitochondrial inner membrane. The contributions of the individual subunits to the assembly and function of the TIM10 complex remain poorly understood. Here, we show that substrate recognition and assembly of the complex are mediated by distinct domains of the subunits. These are unrelated to the characteristic "twin CX3C" motif that is present in all small Tims and ensures proper folding of the unassembled subunits. Specifically, we show that substrate recognition is achieved by the Tim10 subunit, whilst Tim9 serves a more structural role. The N-terminal domain of Tim10 is a substrate sensor whilst its C-terminal part is essential for complex formation. By contrast, both N and C-terminal domains of Tim9 are involved in the stability of the complex.

Loss of zolpidem efficacy in the hippocampus of mice with the GABAA receptor γ2 F77I point mutation

Zolpidem is a hypnotic benzodiazepine site agonist with some gamma-aminobutyric acid (GABA)(A) receptor subtype selectivity. Here, we have tested the effects of zolpidem on the hippocampus of gamma2 subunit (gamma2F77I) point mutant mice. Analysis of forebrain GABA(A) receptor expression with immunocytochemistry, quantitative [(3)H]muscimol and [(35)S] t-butylbicyclophosphorothionate (TBPS) autoradiography, membrane binding with [(3)H]flunitrazepam and [(3)H]muscimol, and comparison of miniature inhibitory postsynaptic current (mIPSC) parameters did not reveal any differences between homozygous gamma2I77/I77 and gamma2F77/F77 mice. However, quantitative immunoblot analysis of gamma2I77/I77 hippocampi showed some increased levels of gamma2, alpha1, alpha4 and delta subunits, suggesting that differences between strains may exist in unassembled subunit levels, but not in assembled receptors. Zolpidem (1 microm) enhanced the decay of mIPSCs in CA1 pyramidal cells of control (C57BL/6J, gamma2F77/F77) mice by approximately 60%, and peak amplitude by approximately 20% at 33-34 degrees C in vitro. The actions of zolpidem (100 nm or 1 microm) were substantially reduced in gamma2I77/I77 mice, although residual effects included a 9% increase in decay and 5% decrease in peak amplitude. Similar results were observed in CA1 stratum oriens/alveus interneurons. At network level, the effect of zolpidem (10 microm) on carbachol-induced oscillations in the CA3 area of gamma2I77/I77 mice was significantly different compared with controls. Thus, the gamma2F77I point mutation virtually abolished the actions of zolpidem on GABA(A) receptors in the hippocampus. However, some residual effects of zolpidem may involve receptors that do not contain the gamma2 subunit.

Les cholinestérases : des enzymes ancrées dans les membranes et les lames basales

Two proteins, ColQ and PRiMA, organize tetramers of acetylcholinesterase (AChE) and of butyrylcholinesterase (BChE) through peptide interactions. A short proline rich sequence in the N-terminal domain of ColQ or PRiMA associates four C-terminal extension of AChE or BChE. ColQ targets the enzymes in the basal lamina, PRiMA targets the enzymes at the plasma membrane. These complexes represent the mature proteins. The unassembled C-terminal extention of AChE is the key determinant recognized during the "quality control" of protein synthesis. Unassembled catalytic subunits are then degraded by the proteasome pathway. At the neuromuscular junction, ColQ/AChE represents the concentrated enzyme. The clusterisation of AChE depends upon ColQ through three sites of interactions: two different heparin binding domains in the collagen domain interact with heparan sulfate proteoglycan particularly the perlecan and the C-terminal non collagenic domain interacts with MuSK, the tyrosine kinase receptor organiser of the neuromuscular junction. The absence of ColQ and AChE has revealed that the excess of Ach stimulates more nicotinic receptors but probably not until their desensitization. Several morphological modifications may help the clearance of Ach. Conversely the synapse transmission fails during high frequency nerve stimulation.

Physiological effects of unassembled chaperonin Cct subunits in the yeastSaccharomyces cerevisiae

Eukaryotic chaperonins, the Cct complexes, are assembled into two rings, each of which is composed of a stoichiometric array of eight different subunits, which are denoted Cct1p-Cct8p. Overexpression of a single CCT gene in Saccharomyces cerevisiae causes an increase of the corresponding Cct subunit, but not of the Cct complex. Nevertheless, overexpression of certain Cct subunits, especially CCT6, suppresses a wide range of abnormal phenotypes, including those caused by the diverse types of conditional mutations tor2-21, lst8-2 and rsp5-9 and those caused by the concomitant overexpression of Sit4p and Sap155p. The examination of 73 altered forms of Cct6p revealed that the cct6-24 mutation, containing GDGTT --> AAAAA replacements of the conserved ATP-binding motif, was unable to suppress any of these traits, although the cct6-24 allele was completely functional for growth. These results provide evidence for functional differences among Cct subunits and for physiological properties of unassembled subunits. We suggest that the suppression is due to the competition of specific Cct subunits for activities that normally modify various cellular components. Furthermore, we also suggest that the Cct subunits can act as suppressors only in certain states, such as when associated with ATP.

Regulation of nicotinic receptor expression by the ubiquitin-proteasome system.

Control of ligand-gated ion channel (LGIC) expression is essential for the formation, maintenance and plasticity of synapses. Treatment of mouse myotubes with proteasome inhibitors increased the number of surface nicotinic acetylcholine receptors (AChRs), indicating LGIC expression is regulated by the ubiquitin-proteasome system (UPS). Elevated surface expression resulted from increased AChR delivery to the plasma membrane and not from decreased turnover from the surface. The rise in AChR trafficking was the direct result of increased assembly of subunits in the endoplasmic reticulum (ER). Because proteasome inhibitors also blocked ER-associated degradation (ERAD) of unassembled AChR subunits, the data indicate that the additional AChRs were assembled from subunits normally targeted for ERAD. Our data show that AChR surface expression is regulated by the UPS through ERAD, whose activity determines oligomeric receptor assembly efficiency.

Export from the Endoplasmic Reticulum of Assembled N-Methyl-D-aspartic Acid Receptors Is Controlled by a Motif in the C Terminus of the NR2 Subunit

Functional N-methyl-d-aspartic acid (NMDA) receptors are formed from the assembly of NR1 and NR2 subunits. When expressed alone, the major NR1 splice variant and the NR2 subunits are retained in the endoplasmic reticulum (ER), reflecting a quality control mechanism found in many complex multisubunit proteins to ensure that only fully assembled and properly folded complexes reach the cell surface. Recent studies have identified an RRR motif in the C terminus of the NR1 subunit, which controls the ER retention of the unassembled subunit. Here we investigated the mechanisms controlling the ER retention of the NR2 subunit and the export of the assembled complex from the ER. We found that Tac chimeras of the C terminus of the NR2B subunit show that an ER retention signal is also present in the NR2B subunit. In assembled complexes, ER retention signals on the individual subunits must be overcome to allow the complex to leave the ER. One common mechanism involves mutual masking of the signals on the individual subunits. Our data do not support such a mechanism for regulating the release of assembled NMDA receptors from the ER. We found that the motif, HLFY, immediately following transmembrane domain 4 of the NR2 subunit, is required for the assembled complex to exit from the ER. Mutation of this motif allowed the assembly of NR1 and NR2 subunits into a complex that was functional, based on MK-801 binding, but it is retained in the ER. These results are consistent with HLFY functioning as a signal that is necessary for the release of the assembled functional NMDA receptor complex from the ER.

Atp10p Assists Assembly of Atp6p into the F0 Unit of the Yeast Mitochondrial ATPase

The F(0)F(1)-ATPase complex of yeast mitochondria contains three mitochondrial and at least 17 nuclear gene products. The coordinate assembly of mitochondrial and cytosolic translation products relies on chaperones and specific factors that stabilize the pools of some unassembled subunits. Atp10p was identified as a mitochondrial inner membrane component necessary for the biogenesis of the hydrophobic F(0) sector of the ATPase. Here we show that, following its synthesis on mitochondrial ribosomes, subunit 6 of the ATPase (Atp6p) can be cross-linked to Atp10p. This interaction is required for the integration of Atp6p into a partially assembled subcomplex of the ATPase. Pulse labeling and chase of mitochondrial translation products in vivo indicate that Atp6p is less stable and more rapidly degraded in an atp10 null mutant than in wild type. Based on these observations, we propose Atp10p to be an Atp6p-specific chaperone that facilitates the incorporation of Atp6p into an intermediate subcomplex of ATPase subunits.

Alpha bungarotoxin-1.4 nm gold: a novel conjugate for visualising the precise subcellular distribution of alpha 7* nicotinic acetylcholine receptors

Alpha 7 subunit-containing nicotinic acetylcholine receptors (α7* nAChR) are involved in a variety of functions in the mammalian brain, including modulating neurotransmitter release and synaptic plasticity. Identifying the precise cellular distribution of α7* nAChRs with respect to the local neurochemical environment is crucial to understanding these biological roles. Current strategies for localising α7* nAChRs at the subcellular level have limitations. Anti-α7 subunit antibodies detect both assembled and unassembled subunits whereas biotinylated αbungarotoxin (αBgt) only binds to assembled α7* nAChRs, but interpretation of labelling is marred by co-detection of endogenous tissue biotin. To overcome these problems, we have characterised a novel 1.4 nm gold αBgt conjugate used to directly localise α7* nAChR. Gold conjugation does not significantly decrease binding affinity, and gold αBgt specifically labels α7* nAChR in both unfixed and aldehyde-fixed tissue at the light and electron microscope levels, labelling being abolished in the presence of excess competing toxin. At the ultrastructural level, gold αBgt is associated with neuronal membranes and located at axon-dendritic synapses in the rat hippocampus CA1 stratum radiatum. These results reveal gold αBgt to be a valuable new tool in elucidating the functional neuroanatomy of α7* nAChR in the central nervous system.

Endoplasmic reticulum quality control of unassembled iron transporter depends on Rer1p-mediated retrieval from the golgi.

Endoplasmic reticulum (ER) quality control is a conserved process by which misfolded or unassembled proteins are selectively retained in the endoplasmic reticulum (ER). Failure in oligomerization of multisubunit membrane proteins is one of the events that triggers ER quality control. The transmembrane domains (TMDs) of unassembled subunits are determinants of ER retention in many cases, although the mechanism of the TMD-mediated sorting of unassembled subunits remains elusive. We studied a yeast iron transporter complex on the cell surface as a new model system for ER quality control. When Fet3p, a transmembrane subunit, is not assembled with the other membrane subunit, Ftr1p, unassembled Fet3p is exclusively localized to the ER at steady state. The TMD of Fet3p contains a determinant for this process. However, pulse-chase analysis and in vitro budding assays indicate that unassembled Fet3p rapidly escapes from the ER. Furthermore, Rer1p, a retrieval receptor for ER-resident membrane proteins in the Golgi, is responsible for the TMD-dependent ER retrieval of unassembled Fet3p. These findings provide clear evidence that the ER quality control of unassembled membrane proteins can be achieved by retrieval from the Golgi and that Rer1p serves as a specific sorting receptor in this process.

Inositol 1,4,5-Trisphosphate Receptor Ubiquitination Is Mediated by Mammalian Ubc7, a Component of the Endoplasmic Reticulum-associated Degradation Pathway, and Is Inhibited by Chelation of Intracellular Zn2+

In response to activation of certain cell surface receptors, inositol 1,4,5-trisphosphate receptors (InsP3Rs), which are located in the endoplasmic reticulum, can be rapidly ubiquitinated and then degraded by the proteasome. Ubiquitination is mediated by the concerted action of ubiquitin-conjugating enzymes (Ubcs or E2s) and ubiquitin-protein ligases (E3s). In the present study we have examined the enzymology of ubiquitination of endogenous InsP3Rs in muscarinic agonist-stimulated SH-SY5Y human neuroblastoma cells, focusing our attention on two mammalian E2s, MmUbc6 and MmUbc7, that have been implicated in endoplasmic reticulum-associated degradation (ERAD) and are homologous to the yeast ERAD E2s, Ubc6p and Ubc7p. Analysis of SH-SY5Y cells stably expressing these enzymes and their dominant-negative mutants revealed that MmUbc7 mediates InsP3R ubiquitination and down-regulation, but that MmUbc6 does not. These data indicate that InsP3Rs are processed by a component of the ERAD pathway and suggest that MmUbc7 may be employed selectively to ubiquitinate proteins, like InsP3Rs, that are subject to regulated ERAD. Additional studies showed that the Zn2+ chelator N,N,N',N'-tetrakis(2-pyridylmethyl)ethylenediamine blocked InsP3R ubiquitination, suggesting that a RING finger domain-containing E3 is also involved in this process. Finally, muscarinic agonist-induced InsP3R ubiquitination was seen in rat brain slices, indicating that the results obtained from SH-SY5Y cells reflect a physiological process.

Regulation of nicotinic acetylcholine receptor assembly.

The four muscle-type nicotinic acetylcholine receptor (AChR) subunits, alpha, beta, gamma, and delta, assemble into functional alpha(2)betagammadelta pentamers in the endoplasmic reticulum (ER) through a series of interdependent folding and oligomerization events. The first stable assembly intermediate is a trimer composed of alpha, beta, and gamma subunits. The formation of alphabetagamma trimers initiates a series of subunit folding and processing events that allow addition of delta subunits to form alphabetagammadelta tetramers. Subunit folding and processing continue with formation of the ligand-binding sites on the alpha subunit of alphabetagammadelta tetramers and the second alpha subunit added to assemble alpha(2)betagammadelta pentamers. AChR assembly is inefficient. Only 20-30% of synthesized subunits assemble into mature receptors in the ER, while the remaining unassembled subunits are degraded. However, the efficiency of subunit assembly can be regulated under certain conditions leading to higher AChR expression. Increased intracellular cAMP levels cause a 2- to 3-fold increase in AChR assembly efficiency and a comparable increase in surface expression. Additionally, block of ubiquitin-proteasome degradation appears to enhance AChR assembly and expression. Thus, the regulation of AChR assembly through posttranslational mechanisms is a potential therapeutic target for increasing AChR expression in diseases in which expression is compromised.

Cell Surface Expression of 5-Hydroxytryptamine Type 3 Receptors Is Controlled by an Endoplasmic Reticulum Retention Signal

Two subunits of the 5-hydroxytryptamine type 3 (5-HT3) have been identified (5-HT3A and 5-HT3B) that assemble into homomeric (5-HT3A) and heteromeric (5-HT3A+5-HT3B) complexes. Unassembled 5-HT3B subunits are efficiently retained within the cell. In this study, we address the mechanism controlling the release of 5-HT3B from the endoplasmic reticulum (ER). An analysis of chimeric 5-HT3A receptor(R).5-HT3BR constructs suggests the presence of elements downstream of the first transmembrane domain of 5-HT3B subunits that inhibit cell surface expression. To investigate this possibility, truncated 5-HT3B subunits were constructed and assessed for their ability to access the cell surface in COS-7 and ts201 cells. Using this approach, we have identified the presence of an ER retention signal located within the first cytoplasmic loop between transmembrane domains I and II of 5-HT3B. Transplantation of this signal (CRAR) into the homologous region of 5-HT3A results in the ER retention of this subunit until rescued by co-assembly with wild-type 5-HT3A. The mutation of this ER retention signal in 5-HT3B (5-HT3BSGER) does not lead to cell surface expression, suggesting the presence of other signals or mechanisms to control the surface expression of 5-HT3BRs. The generation of truncated 5-HT3BSGER constructs confirmed that the CRAR signal does play an important role in the ER retention of 5-HT3B.

Human rhinovirus type 16: mutant V1210A requires capsid-binding drug for assembly of pentamers to form virions during morphogenesis.

Our laboratory has previously reported isolation of human rhinovirus type 16 (HRV16) mutants which depend on WIN 52035 to grow. A rapid rise of progeny virus infectivity occurred when drug was added late in growth cycles, suggesting that the drug-dependence lesion was at the step of virus assembly (W. Wang et al., J. Virol. 72:1210-1218, 1998). Here, we report that capsid subunits, 5S protomers and 14S pentamers, of a drug-dependent mutant were produced normally in the absence of drug, but mutant 80S empty capsids and 150S provirions were not formed, maturation cleavage of provirions (VP0 --> VP2 + VP4) did not occur, and the unassembled mutant capsid subunits were degraded with a half-life of 15 min. Drug was not required by mutant virus for attachment, uncoating, RNA synthesis and protein synthesis, and polyprotein processing except maturation cleavage. The requirement of drug for assembly of mutant pentamers to form provirions and the rapid assembly of preformed subunits (synthesized in the absence of drug) after drug addition suggested that after native pentamers (P5) have been formed they must be converted to an assembly active state (P5(*)), possibly by a conformational change induced by the binding of drug. We propose that pocket factor plays the same role in wild-type virus. In addition, we also report the construction and the properties of a full-length cDNA clone of HRV16, pR16.11, which produces in vitro transcripts with infectivity similar to that of virion RNA. This cDNA clone is available at the American Type Culture Collection.

14-3-3 Dimers Probe the Assembly Status of Multimeric Membrane Proteins

Background: Arginine-based endoplasmic reticulum (ER) localization signals are involved in the heteromultimeric assembly of membrane protein complexes like ATP-sensitive potassium channels (KATP) or GABAB G protein-coupled receptors. They constitute a trafficking checkpoint that prevents ER exit of unassembled subunits or partially assembled complexes. For KATP channels, the mechanism that leads to masking of the ER localization signals in the fully assembled octameric complex is unknown.Results: By employing a tetrameric affinity construct of the C terminus of the KATP channel α subunit, Kir6.2, we found that 14-3-3 isoforms epsilon and zeta specifically recognize the arginine-based ER localization signal present in this cytosolic tail. The interaction was reconstituted by using purified 14-3-3 proteins. Competition with a nonphosphorylated 14-3-3 high-affinity binding peptide implies that the canonical substrate binding groove of 14-3-3 is involved. Comparison of monomeric CD4, dimeric CD8, and artificially tetramerized CD4 fusions correlates the copy number of the tail containing the arginine-based signal with 14-3-3 binding, resulting in the surface expression of the membrane protein. Binding experiments revealed that the COPI vesicle coat can specifically recognize the arginine-based ER localization signal and competes with 14-3-3 for the binding site.Conclusions: The COPI vesicle coat and proteins of the 14-3-3 family recognize arginine-based ER localization signals on multimeric membrane proteins. The equilibrium between these two competing reactions depends on the valency and spatial arrangement of the signal-containing tails. We propose a mechanism in which 14-3-3 bound to the correctly assembled multimer mediates release of the complex from the ER.

Two birds with one stone: a novel motif for ACh receptor assembly quality control

Assembly of ACh receptors is under tight quality control. Only functional ACh receptors are expressed on the cell surface; unassembled subunits are retained in the endoplasmic reticulum (ER). In a recent elegant study, Wang and colleagues have identified a novel motif in the M1 domain of the ACh-receptor subunit that is responsible for ER retention and degradation of unassembled subunits. This signal appears to play an important role in regulating surface trafficking of functional ACh receptors.