Early disulfide bond formation prevents heterotypic aggregation of membrane proteins in a cell-free translation system.
We previously demonstrated that a heterotypic complex of the two rat asialoglycoprotein receptor subunits was assembled during cell-free translation (Sawyer, J. T., and D. Doyle. 1990. Proc. Natl. Acad. Sci. USA. 87:4854-4858). We have characterized this system further by analyzing polypeptide interactions under both reducing and oxidizing translation conditions. This report shows that the complex represents a heterogeneous interaction between reduced membrane proteins rather than a specific oligomeric structure. In the reduced state membrane proteins interact in this system to form aggregates of diverse size and composition. The aggregated nascent polypeptides interact with the immunoglobulin heavy chain binding protein but this protein is not an integral component of the aggregate. Aggregation occurs via the exoplasmic domain, rather than the transmembrane domain, and the folding of this domain by the formation of intramolecular disulfides, prevents the interaction from occurring. Additionally, the folded molecules containing intramolecular disulfides lack high affinity binding activity and thus appear to resemble the earliest folding intermediates seen in vivo (Olson, J. T., and M. D. Lane. 198. FASEB (Fed. Am. Soc. Exp. Biol.) J. 3:1618-1624). These results lead us to suggest that the formation of intramolecular disulfides during early biogenesis serves to prevent nonspecific associations between nascent polypeptides.
Yilla
M
M
Department of Biological Sciences, State University of New York, Buffalo 14260.
Doyle
D
D
Sawyer
J T
JT
D000818:Animals; D037263:Asialoglycoprotein Receptor; D002474:Cell-Free System; D017630:Connexins; D018819:Dipeptidyl Peptidase 4; D004152:Dipeptidyl-Peptidases and Tripeptidyl-Peptidases / Q000235:genetics* / Q000378:metabolism; D004220:Disulfides / Q000378:metabolism*; D004229:Dithiothreitol / Q000494:pharmacology; D005978:Glutathione / Q000031:analogs & derivatives / Q000494:pharmacology; D019803:Glutathione Disulfide; D007535:Isomerases / Q000378:metabolism; D046911:Macromolecular Substances; D008565:Membrane Proteins / Q000235:genetics* / Q000378:metabolism; D010084:Oxidation-Reduction; D014176:Protein Biosynthesis; D019704:Protein Disulfide-Isomerases; D051381:Rats; D011971:Receptors, Immunologic / Q000235:genetics* / Q000378:metabolism; D015183:Restriction Mapping; D012156:Reticulocytes / Q000378:metabolism; D014158:Transcription, Genetic
D037263:Asialoglycoprotein Receptor; D017630:Connexins; D004220:Disulfides; D046911:Macromolecular Substances; D008565:Membrane Proteins; D011971:Receptors, Immunologic; D004152:Dipeptidyl-Peptidases and Tripeptidyl-Peptidases; D018819:Dipeptidyl Peptidase 4; D007535:Isomerases; D019704:Protein Disulfide-Isomerases; D005978:Glutathione; D004229:Dithiothreitol; D019803:Glutathione Disulfide