Biosynthesis of ionotropic acetylcholine receptors requires the evolutionarily conserved ER membrane complex

Abstract

The number of nicotinic acetylcholine receptors (AChRs) present in the plasma membrane of muscle and neuronal cells is limited by the assembly of individual subunits into mature pentameric receptors. This process is usually inefficient, and a large number of the synthesized subunits are degraded by endoplasmic reticulum (ER)-associated degradation. To identify cellular factors required for the synthesis of AChRs, we performed a genetic screen in the nematode Caenorhabditis elegans for mutants with decreased sensitivity to the cholinergic agonist levamisole. We isolated a partial loss-of-function allele of ER membrane protein complex-6 (emc-6), a previously uncharacterized gene in C. elegans. emc-6 encodes an evolutionarily conserved 111-aa protein with two predicted transmembrane domains. EMC-6 is ubiquitously expressed and localizes to the ER. Partial inhibition of EMC-6 caused decreased expression of heteromeric levamisole-sensitive AChRs by destabilizing unassembled subunits in the ER. Inhibition of emc-6 also reduced the expression of homomeric nicotine-sensitive AChRs and GABAA receptors in C. elegans muscle cells. emc-6 is orthologous to the yeast and human EMC6 genes that code for a component of the recently identified ER membrane complex (EMC). Our data suggest this complex is required for protein folding and is connected to ER-associated degradation. We demonstrated that inactivation of additional EMC members in C. elegans also impaired AChR synthesis and induced the unfolded protein response. These results suggest that the EMC is a component of the ER folding machinery. AChRs might provide a valuable proxy to decipher the function of the EMC further.

Fig. 1.

emc-6 encodes a conserved transmembrane protein. (A) Structure of the emc-6 genomic locus. emc-6 is predicted to be the upstream gene of an operon (www.wormbase.org). Black boxes represent the coding regions, and gray boxes represent the 5′ untranslated region. The black triangle illustrates the site of Mos1 insertion in kr150. SL1, SL1 transspliced leader; SL2, SL2 transspliced leader. (B) ClustalX alignment of C. elegans EMC-6 with orthologs from nematodes, fly, and vertebrates. Predicted transmembrane regions (TM1 and TM2) are boxed. Residues conserved in all species are highlighted in dark gray, residues with strongly similar properties (scoring >0.5) are highlighted in medium gray, and residues with weakly similar properties (scoring ≤0.5) are highlighted in light gray. The position of the kr150 mutation is indicated by a triangle. (C) EMC-6 topology model based on TMHMM and Topo2 predictions. Residues conserved in all species are labeled in gray. Cter, C-terminus; CYT, cytoplasm; LUM, lumen; Nter, N-terminus. (D) Expression of an emc-6 genomic fragment rescues levamisole resistance in kr150 mutants. Gray bars illustrate the percentage of dead animals after overnight exposure to 0.6 mM levamisole, and black bars illustrate the percentage of surviving animals. Three independent transgenic lines were tested. n, number of animals tested. ***P < 0.001, Fisher exact probability test.

Fig. 2.

emc-6 is required in muscle cells for L-AChR function and localizes to the ER. (A) Expression of an emc-6 cDNA and GFP::Cb-EMC-6 or HA::Ce-EMC-6 translational fusion in muscle (Pmyo-3 promoter) but not in neurons (Prab-3 promoter) rescues levamisole resistance of emc-6(kr150) mutants. n, number of tested animals. n.s. (nonsignificant), P > 0.05; ***P < 0.001, Fisher exact probability test. (B) GFP::Cb-EMC-6 fusion proteins localize to the ER. (i) GFP::Cb-EMC-6 and tagRFP::KDEL translational fusions used for expression in BWM (Pmyo-3 promoter). (ii) Live images of GFP::Cb-EMC-6 and tagRFP::KDEL displaying a reticular staining pattern throughout the cytoplasm surrounding the nucleus. (Scale bars, 5 μm.) (iii) Magnified view of the reticulum network. (Scale bars, 2.5 μm.) (C) HA::Ce-EMC-6 fusion does not colocalize with a Golgi-resident tagRFP-tagged Mannosidase II protein (MANS::tagRFP). (i) HA::Ce-EMC-6 and MANS::tagRFP used for expression in BWMs (Pmyo-3 promoter). (ii) Immunostaining with anti-HA and anti-tagRFP antibodies. DAPI staining of cell nuclei. (Scale bars, 5 μm.)

Fig. 3.

EMC-6 is required for surface expression of ionotropic receptors. (A) Response to pressure ejection of levamisole in voltage-clamped ventral BWMs is reduced in emc-6(kr150) [mean ± SEM (WT: 367 ± 37 pA, n = 7; emc-6(kr150): 87 ± 8 pA, n = 6); P = 0.0012]. Arrowheads mark application onset. (B) L-AChR–dependent evoked currents recorded from BWMs after ventral nerve cord stimulation are decreased in emc-6(kr150). Electrically evoked responses were obtained in a unc-49(e407); acr-16(ok789) genetic background to eliminate GABA_A receptor and N-AChR contributions (WT: 553 ± 50 pA, n = 6; emc-6(kr150): 210 ± 31 pA, n = 6; P = 0.0022). (C) L-AChR expression is decreased at NMJs of emc-6(kr150) mutants (i, iii, iv, and vi), whereas presynaptic differentiation is unaffected (ii, iii, v, and vi). L-AChRs are labeled using anti–UNC-38. Cholinergic boutons are labeled using an anti-vesicular acetylcholine transporter UNC-17 (VAChT) antibody. (Scale bars, 10 μm.) (D) Visualization of L-AChRs in living worms using a knock-in UNC-29–tagRFP L-AChR subunit engineered by MosTIC homologous recombination. DC, dorsal cord; NR, nerve ring. Arrowheads indicate L-AChR clusters at the dorsal nerve cord. Intestinal autofluorescence is indicated with an asterisk. (Scale bars, 10 μm.) (E) UNC-29–tagRFP fluorescence is reduced in emc-6(kr150) mutants (WT: 18.1 ± 2.5 arbitrary units, n = 14; emc-6(kr150): 2.9 ± 0.3 arbitrary units, n = 13; ***P < 0.001, Mann–Whitney test). (F) EMC-6 is required for N-AChR function. Response to pressure ejection of nicotine (WT: 931 ± 78 pA, n = 8; emc-6(kr150): 633 ± 94 pA, n = 12; P = 0.023). Arrowheads mark application onset. (G) EMC-6 is required for GABA_A receptor function. Response to pressure ejection of GABA (WT: 1552 ± 118 pA, n = 6; emc-6(kr150): 710 ± 172 pA, n = 5; P = 0.0043). Arrowheads mark application onset.

Fig. 4.

emc-6 is required before or during L-AChR assembly. (A) L-AChR expression is reduced in emc-6(kr150) mutants. Levels of unassembled UNC-29 L-AChR subunits detected in unc-63(kr13) are further decreased in unc-63(0); emc-6(kr150) double mutants. UNC-29 levels are quantified by Western blot using anti–UNC-29 antibodies and normalized to tubulin levels. The migration profile of UNC-29 subunit appears to be slightly different in emc-6(kr150) compared with other genotypes, which might reflect slight changes of UNC-29 glycosylation. Seven independent experiments were quantified (mean ± SEM). n.s. (not significant), P > 0.05; *P < 0.05; **P < 0.01 after Holm correction, Mann–Whitney–Wilcoxon test. (B) Remaining L-AChRs exit the ER in emc-6(kr150). Treatments with EndoH or N-Glycosidase F (PNGase) were performed on protein extracts of mixed-stage animals before SDS/PAGE analysis. Black arrowheads indicate glycosylated forms resistant to EndoH, gray arrowheads indicate partially glycosylated forms partially resistant to EndoH, and white arrowheads indicate unglycosylated forms sensitive to EndoH.

Fig. 5.

EMC is required for L-AChR expression and ER homeostasis. (A) EMC inactivation by RNAi leads to resistance to levamisole. Bars represent the percentage of resistant animals after overnight exposure to 0.4 mM levamisole following preembryonic RNAi in eri-1(mg366). Four to 13 independent experiments were performed (20–45 animals per condition per experiment) (mean ± SEM). n.s. (not significant), P > 0.05; ***P < 0.001 after Holm correction, Mann–Whitney–Wilcoxon test. (B) EMC inactivation triggers the UPR. (i) Representative pictures of young adults expressing GFP under the control of the hsp-4 promoter reporting the extent of the UPR (intensity: +, white; ++, gray; +++ black). (ii) Bar graph represents UPR in worms exposed to control (empty vector), emc, or unc-50 RNAi. RNAi was performed preembryonically by feeding the parent WT worms with bacteria expressing dsRNA. n, number of animals tested. Four to eight independent experiments were pooled. n.s. (not significant), P > 0.05; ***P < 0.001 after Bonferroni correction, Fisher exact test.

Fig. P1.

Under physiological conditions, EMC-6 ensures the expression of L-AChRs as well as two other multimeric ionotropic receptors. EMC-6 likely functions within the recently identified EMC to stabilize unassembled AChR subunits and promote their assembly into mature receptors. Inactivation of the EMC triggers unassembled L-AChR subunit degradation via ERAD and causes activation of the unfolded protein response (UPR). The EMC appears as a component of the ER folding machinery and seems essential for maintaining ER homeostasis in vivo.