Synthesis of spectrin in avian erythroid cells: association of nascent polypeptide chains with the cytoskeleton
The site of synthesis of spectrin was investigated in erythroid cells from 10-day chicken embryos. After various periods of [35S]methionine incorporation the cells were lysed in a Triton X-100 (TX-100)-containing buffer and were separated into a TX-100-soluble and -insoluble (cytoskeletal) fraction. Analysis of these two fractions by two-dimensional gel electrophoresis after a short pulse-labeling period reveals that alpha-spectrin nascent polypeptides are present predominantly in the TX-100-insoluble fraction. These polypeptides can be immunoprecipitated with alpha-spectrin antisera and the [35S]methionine incorporated into them during a short pulse can be chased into mature alpha-spectrin molecules. The alpha-spectrin nascent polypeptide chains are released quantitatively from the TX-100 cytoskeleton by treatment of lysed cells with puromycin, suggesting that they themselves are not associated with the cytoskeleton. A small fraction of the newly synthesized mature alpha-spectrin molecules is rapidly incorporated into the cytoskeleton, as shown by the fact that they are not released by the puromycin treatment; the rest are recovered in the soluble fraction. These results suggest that alpha-spectrin is synthesized in association with the cytoskeleton during chicken erythropoiesis and assembles onto the membrane-cytoskeleton posttranslationally.
Additional InformationCopyright ©1983 by the National Academy of Sciences. Communicated by Clifford Grobstein, February 7, 1983. We thank Dr. W. James Nelson for performing the immunoprecipitations and Drs. W. James Nelson, Lars Carlsson, and Randall T. Moon for their many helpful comments on the manuscript. This work was supported by grants from the National Institutes of Health, the National Science Foundation, and the Muscular Dystrophy Association of America. I.B. was also supported by a postdoctoral fellowship from the Swedish Natural Science Research Council. E.L. is a recipient of a Research Career Development Award from the National Institutes of Health. 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.
Published - BLIpnas83.pdf