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In vitro synthesis, glycosylation, and membrane insertion of the four subunits of Torpedo acetylcholine receptor

Anderson, David J. and Blobel, Gunter (1981) In vitro synthesis, glycosylation, and membrane insertion of the four subunits of Torpedo acetylcholine receptor. Proceedings of the National Academy of Sciences of the United States of America, 78 (9). pp. 5598-5602. ISSN 0027-8424. http://resolver.caltech.edu/CaltechAUTHORS:ANDpnas81

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Abstract

We have characterized the early biosynthetic forms of the Torpedo electroplax acetylcholine receptor by using a cell-free protein synthesizing system. We obtained primary translation products of ≈38, 50, 49, and 60 kilodaltons for the α, ß, γ, and δ polypeptides, respectively, by using immunoprecipitation with subunit-specific antisera. These chains could each be labeled by the formylated initiator [35S]Met-tRNA. On cotranslational incubation with pancreatic rough microsomes, glycosylated forms of each subunit were obtained that had molecular weights close to those of their mature authentic counterparts. Extensive trypsinization reduced the glycosylated forms of the receptor subunits to glycosylated membrane-protected fragments of ≈35(α), 37(ß), 45(γ), and 44(δ) kilodaltons. In this system, then, each receptor chain spans the membrane at least once. This in vitro-synthesized material apparently exhibited neither oligomeric assembly nor -bungarotoxin binding.


Item Type:Article
Additional Information:© 1981 by the National Academy of Sciences. Communicated by Jerard Hurwitz, April 20, 1981. D.J.A. is supported in part by a National Science Foundation Predoctoral Fellowship. The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U. S. C. §1734 solely to indicate this fact.
Subject Keywords:cell-free translation of electroplax mRNA; immunoprecipitation by subunit-specific antisera; transmembrane orientation of acetylcholine receptor subunits; biosynthesis of multisubunit integral membrane proteins
Record Number:CaltechAUTHORS:ANDpnas81
Persistent URL:http://resolver.caltech.edu/CaltechAUTHORS:ANDpnas81
Alternative URL:http://dx.doi.org/10.1073/pnas.78.9.5598
Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:9278
Collection:CaltechAUTHORS
Deposited By: Tony Diaz
Deposited On:06 Dec 2007
Last Modified:26 Dec 2012 09:47

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