Biosynthesis of calcium-binding protein of chick embryonic chorioallantoic membrane: In vitro organ culture and cell-free translation

Abstract

Biosynthesis of the calcium-binding protein (CaBP) of the chick embryonic chorioallantoic membrane (CAM) was studied using in vitro organ culture and cell-free translation. The organ culture studies showed: 1) The CaBP is a relatively stable protein ( t 1 2 = 60 h ); 2) Biosynthesis of the CaBP involves microsomes and includes two posttranslational modifications, glycosylation and γ-glutamyl carboxylation; and 3) During embryonic development, a single species of the CaBP is expressed in the CAM. Cell-free translation of total CAM mRNA, including CaBP mRNA, was achieved in a rabbit reticulocyte lysate system using [ 35S]Met as a tracer. Based on the properties of the nascent CaBP polypeptide translated in the presence or absence of microsomal membranes, the early stages of CaBP synthesis appear to be: translation of CaBP mRNA on membrane-bound polysomes, insertion and translocation of the nascent polypeptide across microsomal membranes, and co-translational cleavage of a signal sequence.