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.
D000818:Animals; D002642:Chick Embryo; D003599:Cytoskeleton / Q000187:drug effects / Q000378:metabolism*; D004900:Erythroblasts / Q000187:drug effects / Q000378:metabolism*; D004912:Erythrocytes / Q000378:metabolism*; D008565:Membrane Proteins / Q000096:biosynthesis*; D008715:Methionine / Q000378:metabolism; D017830:Octoxynol; D011092:Polyethylene Glycols; D011691:Puromycin / Q000494:pharmacology; D013049:Spectrin / Q000096:biosynthesis*
D008565:Membrane Proteins; D013049:Spectrin; D011092:Polyethylene Glycols; D011691:Puromycin; D017830:Octoxynol; D008715:Methionine