The transmembrane topology of the amino terminus of the alpha subunit of the nicotinic acetylcholine receptor

R.A Chavez,Z.W Hall

doi:10.1016/s0021-9258(18)98649-7

Abstract

We have investigated the transmembrane topology of the amino-terminal domain of the alpha subunit of the mouse muscle nicotinic acetylcholine receptor synthesized in vitro and in vivo. Using oligonucleotide-directed mutagenesis we introduced new glycosylation consensus sequences at alpha 154 and at alpha 200. For each novel site, additional constructs were made in which the original site at alpha N141 was eliminated. Glycosylation at the new sites, as exhibited in a rabbit reticulocyte cell-free translation system supplemented with canine pancreatic microsomes and in a transient transfection system with COS cells, was taken as evidence of the transmembrane translocation of the new site. Each of the new sites was glycosylated in both systems. In separate experiments we found that an alpha subunit fragment terminating at alpha M207 could be extracted from microsomal membranes with sodium carbonate after in vitro translation, indicating that this fragment is not an integral membrane protein. Our results, taken together with previous experiments, indicate that the amino terminus of the alpha subunit up to at least residue alpha 207 is translocated across the membrane of the endoplasmic reticulum. This topology probably represents the orientation of the amino terminus of the alpha subunit in the assembled receptor.

Highlights

We have investigated the transmembrane topology of the amino-terminal domain of the a subunit of the mouse muscle nicotinic acetylcholine receptor synthesized in vitro and in vivo
An alternative model of the amino-terminal domain (Fig. 1B) arises from experiments with monoclonal antibodies raised againstasynthetic peptide whose sequence corresponds to residues a152-167 of the a subunit of the Torpedo acetylcholine receptor (AChR)
Taken bind the AChR in Torpedo vesicles only after treatments that together with previous experiments, indicate that the permeabilize the membrane, Criado and co-workers have proamino terminus of the a subunit up to at least residue posed that residues a152-159 are cytoplasmic and connect a207 is translocated across the membrane of the endoplasmic reticulum

Summary

RESULTS

NovelGlycosylation Sites Can Be Processed inin Vitro Translations-Initial experiments were carried out to characterize the translation products of the normal mammalian muscle a subunit. mRNA was synthesized in vitro from a subunit cDNA using SP6 RNA polymerase and was used t o prime a rabbit reticulocyte in vitro translation system. To visualize glycosylation at the new site alone, we tested a construct containing the new site at a154 but with the old site eliminated (ax g154) In this case, after translation in the presence of microsomes, a single glycosylated species that the extreme amino terminus and amino acid residues appeared, which co-migrated with the primary translation (~141-143of the a subunit are transported across the mem- product as seen witha g154 (Fig. 3, lune k ) .This glycosylation brane of the microsomal vesicle. Treattion from COS cells transfected with normala subunit cDNA ment of microsomal vesicles with sodium carbonate at pH (Fig. 4, lane A ) revealed three bands: a major band of the 11.5 opens thevesicles into phospholipid sheets and strips the same mobility as the processed and glycosylated a subunit membrane of peripheral proteins. HCI (7.5) (SUC)or 0.1 M sodium carbonate (11.5) (CARB).After incubation and centrifugation as described (see "Materialsand Methods") supernatants ( S )and pellets (P)were separated, trichloroacetic acid-precipitated, and electrophoresed as described

Tr: RM

Findings

DISCUSSION