The Journal of Cell Biology, Vol 96, 633-638, Copyright © 1983 by The Rockefeller University Press
In vitro synthesis and membrane insertion of bovine MP26, an integral protein from lens fiber plasma membrane
DL Paul and DA Goodenough
Synthesis of MP26, the principal protein of lens fiber plasma membranes,
was directed in the reticulocyte lysate system by poly A mRNA enriched from
whole bovine lens RNA using oligo (dt)-cellulose chromatography.
Synthesized MP26 was enriched by immune precipitation. The in
vitro-synthesized MP26 had an electrophoretic mobility indistinguishable
from that of the native molecule. MP26 showed a cotranslational requirement
for dog pancreas microsomes in order for membrane association to occur.
Microsome-associated in vitro- synthesized MP26 showed a sensitivity to
digestion with chymotrypsin which was similar to the sensitivity of native
MP26 in isolated lens fiber plasma membranes, indicating correct insertion
of the MP26 into the microsome. Synthesis and membrane insertion of MP26
using N-formyl- [35S]methionyl tRNA as label demonstrated that no
proteolytic processing or significant glycosylation accompanied membrane
insertion. Chymotryptic cleavage of membrane-inserted,
N-formyl-[35S]methionine- labeled MP26 resulted in loss of label,
suggesting that the N-terminal of the in vitro-synthesized MP26 faces the
cytoplasm.
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