Assembly of the sarcoplasmic reticulum. Cell-free synthesis of te Ca2+ + Mg2+-adenosine triphosphatase and calsequestrin.
Polyadenylated RNA prepared from neonatal rat muscle was translated in a rabbit reticulocyte cell-free system. Two sarcoplasmic reticulum proteins, the Ca2+ + Mg2+-dependent adenosine triphosphatase (ATPase) and calsequestrin, were isolated from the translation mixture by immunoprecipitation, followed by electrophoresis in sodium dodecyl sulfate-polyacrylamide gels. The [35S]methionine-labeled translation products were characterized by molecular weight, peptide mapping, and NH2-terminal sequence analysis. The ATPase synthesized in the cell-free system was found to have the same molecular weight (Mr = 100,000) and [35S]-methionine-labeled peptide map as the mature ATPase. The methionine residue present at the NH2 terminus of the mature ATPase was donated by initiator methionyl-tRNArMet and it became acetylated during translation. These results suggest that the ATPase was synthesized without an NH2-terminal signal sequence. Calsequestrin (Mr - 63,000) was synthesized as a higher molecular weight precursor (Mr = 66,000) that contained an additional [35S]methionine-labeled peptide when compared to mature calsequestrin. The NH2-terminal sequence of the precursor was different from the mature protein. The precursor was processed to a polypeptide with a molecular weight identical with mature calsequestrin when microsomal membranes prepared from canine pancreas were included during translation. These results show that calsequestrin is synthesized with an NH2-terminal signal sequence that is removed during translation. These data add to the evidence that the ATPase and calsequestrin follow distinctly different biosynthetic pathways, even though, ultimately, they are both located in the same membrane.
D000107:Acetylation; D000596:Amino Acids / Q000032:analysis; D000818:Animals; D000831:Animals, Newborn; D017301:Ca(2+) Mg(2+)-ATPase; D000252:Calcium-Transporting ATPases / Q000096:biosynthesis*; D002155:Calsequestrin / Q000096:biosynthesis*; D007700:Kinetics; D009124:Muscle Proteins / Q000096:biosynthesis*; D010446:Peptide Fragments / Q000032:analysis; D011061:Poly A / Q000378:metabolism; D011132:Polyribosomes / Q000378:metabolism; D014176:Protein Biosynthesis; D012313:RNA / Q000378:metabolism; D012333:RNA, Messenger / Q000378:metabolism; D011817:Rabbits; D051381:Rats; D012156:Reticulocytes / Q000378:metabolism; D012519:Sarcoplasmic Reticulum / Q000378:metabolism*; D014357:Trypsin
D000596:Amino Acids; D002155:Calsequestrin; D009124:Muscle Proteins; D010446:Peptide Fragments; D012333:RNA, Messenger; D011061:Poly A; D012313:RNA; D014357:Trypsin; D017301:Ca(2+) Mg(2+)-ATPase; D000252:Calcium-Transporting ATPases