Regulation of nicotinic receptor trafficking by the transmembrane Golgi protein UNC-50

Abstract

Nicotinic acetylcholine receptors (AChRs) are pentameric ligand-gated ion channels that mediate fast synaptic transmission at the neuromuscular junction (NMJ). After assembly in the endoplasmic reticulum (ER), AChRs must be transported to the plasma membrane through the secretory apparatus. Little is known about specific molecules that mediate this transport. Here we identify a gene that is required for subtype-specific trafficking of assembled nicotinic AChRs in Caenorhabditis elegans. unc-50 encodes an evolutionarily conserved integral membrane protein that localizes to the Golgi apparatus. In the absence of UNC-50, a subset of AChRs present in body-wall muscle are sorted to the lysosomal system and degraded. However, the trafficking of a second AChR type and of GABA ionotropic receptors expressed in the same muscle cells is not affected in unc-50 mutants. These results suggest that, in addition to ER quality control, assembled AChRs are sorted within the Golgi system by a mechanism that controls the amount of cell-surface AChRs in a subtype-specific way.

Figure 1

Body-wall muscles of unc-50 mutants do not respond to levamisole, however the response to nicotine and GABA are unaffected. The electrophysiological responses of wild type (N2) and unc-50 mutant (alleles x515 and x47) body-wall muscles to pressure ejection of 500 μM levamisole (A), 500 μM nicotine (B), and 100 μM GABA (C) are shown. The arrow marks drug application. Results are presented as the means of independent experiments. Error bars represent the standard error of the mean (s.e.m.).

Figure 2

unc-50 mutants do not display the Lev-AChR at the cell surface. Wild-type, unc-50(e306) heterozygote and unc-50(e306) mutant animals were engineered to express the C-terminally tagged LEV-1-4xHA Lev-AChR subunit (A, B). Similarly wild-type and unc-50(e306) mutant animals were engineered to express the UNC-49-3xmyc GABA_A receptor subunit (C, D), and subsequently, fluorescently labeled antibodies against the respective epitopes were injected into the body cavity. After a recovery period of 6 h, in which the body cavity was cleared of unbound antibodies, the signal resulting from antibodies bound to the epitope-tagged receptors at the cell surface was measured and quantified. The signals originating from the ventral cord NMJs are shown in panels A and C, and quantified in panels B and D, respectively. The number of animals counted is indicated and the error bars represent the standard error of the mean (s.e.m.). ^**P<0.01, t-test. The scale bars correspond to 10 μm.

Figure 3

No Lev-AChR can be detected in unc-50 mutants by immunostaining and Western blot analysis. (A) Wild-type (N2) and unc-50(x47) mutant young adult animals were freeze fractured, fixed in methanol/acetone and immunostained with polyclonal antibodies against the Lev-AChR subunit UNC-29. Antibodies to the ACh vesicular transporter UNC-17 were used to detect presynaptic terminals along the nerve cord. The positions of the nerve ring (NR), dorsal-, and ventral cord (DC and VC) NMJs are indicated. The scale bar represents 20 μm. (B) Total membrane fractions of staged L4 wild-type worms, unc-29, and unc-50 mutants were size fractionated by SDS gel electrophoresis and transferred onto a nitrocellulose membrane. The membrane was sequentially probed with antibodies against UNC-29 and VHA-5. The TM subunit of the vacuolar ATPase VHA-5 was used to normalize the membrane fractions.

Figure 4

Total membrane fractions from staged L4 hermaphrodites of the indicated genotypes were size separated, blotted, and probed with antibodies against the Lev-AChR subunit UNC-29 and against the vacuolar ATPase subunit VHA-5 as a loading control. (A) The unassembled Lev-AChR is stable and can be detected in the mutants unc-38(x20), unc-63(x37), and lev-1(kr6) that lack one of the receptor subunits. While membrane extracts of unc-29 mutants exhibit no detectable UNC-29 staining (1.1±0.6% (n=4)), wild-type levels of staining are seen in unc-38 (115±18% (n=3)), unc-63 (101±11% (n=2)), and lev-1 (107±25% (n=2)) mutants. (B) UNC-50 acts after Lev-AChR assembly. The unassembled receptor can be detected in unc-50; unc-63 double mutants and not in unc-50 mutants. (C) The Lev-AChR is degraded by the lysosomal system. UNC-29 levels in cup-5 mutants (110±6% (n=2)) are comparable to wild-type N2 animals (100% (n=2)), but mutations in cup-5 stabilize the Lev-AChR in an unc-50 mutant background.

Figure 5

unc-50 encodes an evolutionarily conserved integral membrane protein. (A) Genomic organization of T07A5.2/unc-50. unc-50 is part of an operon, but all unc-50 alleles carry mutations in the gene T07A5.2. The black regions represent coding regions, while white parts represent the untranslated regions. Splice leaders are indicated. (B) ClustalX alignment of UNC-50 with its orthologs from yeast to humans. The locations of the predicted TM regions are indicated. Residues conserved between all species are highlighted in black and conserved residues between most species in gray. The positions of the mutations detected in unc-50 alleles are indicated above the protein sequence, asterisks correspond to stop mutations while the triangles mark the two point mutations found in the allele x56. The GenBank accession numbers for the sequences used can be found in the Supplementary Figure 3. The C-terminus of UNC-50 is truncated in this panel, but is shown in panel C. (C) Proposed membrane topology of UNC-50. The residues conserved between all species are underlined in gray.

Figure 6

Muscle-specific expression of unc-50 cDNA is sufficient to rescue the resistance to levamisole (A) and the uncoordinated movement phenotype (B) of unc-50 mutant animals. Transgenic unc-50 hermaphrodites express the unc-50 cDNA under the control of the muscle-specific myo-3 promoter from an extrachromosomal array. (C) Conditional expression of the unc-50 cDNA under the control of the heat-shock promoter is able to rescue the levamisole resistance after induction by heat shock in adult animals carrying the transgene.

Figure 7

UNC-50 localizes to the Golgi system. (A–C) mRFP-UNC-50 fusion proteins display a vesicular staining throughout the cytoplasm of a body-wall muscle cell, as shown. This largely overlaps the staining of the Golgi-resident Mannosidase II-GFP fusion protein. Representative Golgi structures are marked by arrowheads. (D–E) The mRFP-UNC-50 fusion protein does not overlap with an ER-localized GFP-cb5 protein. (The scale bar=10 μm.)

Figure 8

UNC-50 interacts with the SEC7 domain-containing Arf GEF Gea1p. Yeast two-hybrid interaction test of a yeast DB-GEA1 fusion protein with either the yeast GMH1, the human Gmh1, or C. elegans UNC-50 fused to the GAL4 activation domain (AD). A positive interaction allows yeast cells to grow on selective media lacking histidine.