In vitro synthesis of the Ca2+ transport ATPase by ribosomes bound to sarcoplasmic reticulum membranes.
The calcium transport ATPase (M(r) 100,000) from sarcoplasmic reticulum membranes was synthesized in a cell-free translation system containing rough microsomes or detergent-treated bound polysomes from 14- to 16-day old chicken embryo muscles. Immunoprecipitates obtained from total translation mixtures treated with anti-ATPase antiserum contained 1.5% of the total radioactivity incorporated in vitro. A polypeptide with the electrophoretic mobility, isoelectric point, and [(35)S]methionine-labeled tryptic peptide pattern of the mature ATPase was a major component of these immunoprecipitates. By contrast, free polysomes from the same source, which were capable of high levels of in vitro protein synthesis, did not yield immunoprecipitable ATPase. ATPase synthesized in rough microsomes was not released by treatment with 10 mM EDTA in a high-salt medium (0.5 M KCl) which removes ribosomes and peripheral membrane proteins. Furthermore, labeled ATPase remained associated with the microsomes after these were treated with low concentrations of deoxycholate (0.1 mg/mg of protein in 0.3 ml) which release the luminal content of the vesicles. Only with higher deoxycholate concentrations (0.5 mg/mg of protein in 0.3 ml), which cause membrane dissolution, was the labeled ATPase found on the detergent extracts. These observations indicate that newly synthesized ATPase discharged from bound ribosomes is transferred directly to the sarcoplasmic reticulum membranes where it is incorporated as an integral membrane protein.
D000818:Animals; D000252:Calcium-Transporting ATPases / Q000096:biosynthesis*; D002645:Chickens; D007425:Intracellular Membranes / Q000201:enzymology; D008861:Microsomes / Q000201:enzymology; D008970:Molecular Weight; D009132:Muscles / Q000201:enzymology*; D010446:Peptide Fragments / Q000032:analysis; D011132:Polyribosomes / Q000201:enzymology; D014176:Protein Biosynthesis; D012270:Ribosomes / Q000201:enzymology*; D012519:Sarcoplasmic Reticulum / Q000201:enzymology*; D014357:Trypsin
D010446:Peptide Fragments; D014357:Trypsin; D000252:Calcium-Transporting ATPases