Acetylcholine Receptor in a C2 Muscle Cell Variant Is Retained in the Endoplasmic Reticulum

We have examined the properties and intracellular localization of acetylcholine receptors in the C2 muscle cell line and in a variant (T-) that accumulates AChR intracellularly. On immunoblots, the subunit structures of the AChR from wild-type and Tcells were similar except that the 3' and t5 subunits of the variant AChR had altered mobilities. Digestion with endoglycosidases H and F demonstrated that this difference results from a failure of high-mannose N-linked oligosaccharides on AChR subunits to be processed to complex forms in the variant. N-linked glycosylation of other proteins in the variant was normal. When examined by immunocytochemistry, the distribution of internal AChR in wild-type cells was consistent with a location both in the endoplasmic reticulum and in the Golgi. Variant cells, however, showed no evidence of Golgi staining. Subcellular fractionation experiments also demonstrated AChR in the Golgi fractions of wild-type cells, but not in those derived from Tcells. We conclude that in Tmyotubes most of the AChR fails to be transported out of the endoplasmic reticulum. M EMBRANE proteins follow a common path of assembly and transport to the cell surface (reviewed in Burgess and Kelly, 1987; Pfeffer and Rothman, 1987). They are synthesized, glycosylated and assembled in the ER, transported to the Golgi apparatus where N-linked oligosaccharides are processed and other modifications made, and then transported to the cell surface. Proteins may deviate from this pathway under a variety of circumstances. Misfolding of the protein or failure to assemble can result in retention in the ER (Doyle et al., 1985, 1986; Copeland et al., 1986, 1988; Gething et al., 1986; Kreis and Lodish, 1986; Salter and Cresswell, 1986; Doms et al., 1988; Chen et al., 1988). Experimental manipulations such as change in pH, reduced temperature, or the addition of ionophores can also disrupt the movement of proteins to the surface, resulting in their intracellular retention (Matlin et al., 1988; Strous et al., 1985; Matlin, 1986; Griffiths et al., 1983; Matlin and Simons, 1983; Saraste and Kuismanen, 1984). These deviations from the normal pathway are useful for its dissection and for attempts to understand its regulation. The nicotinic acetylcholine receptor (AChR)' is an oligomeric membrane protein expressed in differentiated muscle cells that follows a pathway to the cell surface resembling that of other membrane proteins (Anderson and Blobel, 1981, Roy A. Black's present address is Immunex Corp., 51 University Street, Seattle, WA 98101. 1. Abbreviations used in this paper: AChR, acetylcholine receptor; t~-BuTx, ot-bungarotoxin; BiP, immunoglobulin heavy chain-binding protein; endo F, endoglycosidase F; endo H, endoglycosidase H; rho-c~-BuTx, rhodamine-conjugated a-bungarotoxin. 1983; Merlie et al., 1983; Carlin and Merlie, 1987). The receptor is assembled in the ER from four highly homologous glycosylated subunits to form the structure c~2fl3,6 (Smith et al., 1987). It is then transported to the Golgi apparatus (Fambrough and Devreotes, 1978), where some of the oligosaccharides on the 3' and 6 subunits are processed to complex forms (Gu and Hall, 1988b), and the fully processed AChR is then transported to the cell surface (Devreotes et al., 1977; Merlie et al., 1983). We have recently investigated a genetic variant of the C2 mouse muscle cell line, T-, that expresses a reduced amount of AChR on its surface (Black and Hall, 1985). Previous experiments (Gu et al., 1989) have shown that, although the Tvariant synthesizes a normal amount of receptor, only a small fraction of the assembled AChR reaches the cell surface. The remainder is accumulated in the cell interior. Here we investigate the molecular properties and intracellular location of the AChR in wild-type and variant C2 myotubes to determine the point in the intracellular pathway to the surface at which the AChR in variant cells has been retained or diverted. Materials and Methods