In vitro biosynthesis of the β-subunit of the Na +/K +-ATPase in developing brine shrimp: glycosylation and membrane insertion

Abstract

We demonstrate here translation, glycosylation, and membrane insertion of the β-subunit of the Na +/K +-ATPase of the developing brine shrimp, Artemia, in a reticulocyte lysate translation system. The apparent molecular weight of the primary translation product as determined by SDS-PAGE is 33 000 ± 1000 ( n = 7). When microsomal membranes are present during the entire translation period, a new band with an apparent molecular weight of 37 000 ± 1000 ( n = 7) appears. This change in apparent molecular weight is due to the addition of about two N-linked oligosaccharides. The temporal relationship between protein synthesis and glycosylation have also been examined. Glycosylation and membrane insertion could be achieved if membranes were added after completion of about 70% of the peptide chain. However, glycosylation did not occur if membranes were added after the completion of translation of the β-subunit. The β-subunit was synthesized on membrane-bound polysomes, where about two N-linked oligosaccharides were added to the growing polypeptide chain. These studies demonstrate that in vitro translation systems will be useful for studying the biosynthesis of the β-subunit of the brine shrimp, which is a good model system to examine the developmental regulation of the Na +/K +-ATPase.