IDENTIFICATION AND SUBSEQUENT PHOSPHORYLATION OF SEQUESTERED PARTIALLY PROCESSED CASEINS IN THE LACTATING GUINEA-PIG MAMMARY-GLAND

Golgi and endoplasmic-reticulum fractions were prepared from the lactating guinea-pig mammary gland. The endoplasmic-reticulum fraction was highly active in the processing and sequestration of milk-protein primary translation products. Explants from the lactating gland in organ culture were used to identify milk-protein intermediates present in the secretory pathway and the timing of the events leading to their post-translational modification. With [35S]methionine, the milk proteins labeled after a short pulse (3 min) were represented by the partially processed (but not phosphorylated) caseins and .alpha.-lactalbumin sequestered within membrane-bound vesicles. After a 30 in labeling period, higher-MW caseins with electrophoretic mobilities identical with those of the phosphorylated caseins isolated from milk were identified in the incubation medium, and sequestered within membrane-bound vesicles. Pulse-chase experiments established a precursor-product relationship between these forms. Secretion is apparent .apprx. 30 min after sequestration. Caseins are highly phosphorylated; removal of the phosphate residues with acid phosphatase results in proteins with increased electrophoretic mobility, similar to those of the partially processed early casein intermediates found sequestered in explants after a 3 min pulse with [35S]methionine, and those sequestered within microsomal membranes after mRNA-directed cell-free protein synthesis. A comparison of the proteins labeled during both short (5 min) and long (30 min) pulses with [35S]methionine and [32P]Pi shows that, in contrast with the 35S-labeled caseins, those labeled with [32P]Pi exhibit only electrophoretic mobilities identical with those of the mature caseins isolated from milk and those identified after long labeling periods with [35S]methionine. No phosphorylated early intermediate forms of casins were identified. The synthesis and post-translational modification of guinea-pig caseins occurs in 2 stages, an early event involving synthesis and sequestration within the endoplasmic reticulum, an event that involves signal-peptide removal, followed 10-20 min later by phosphorylation at a different point in the secretory pathway, probably in the Golgi complex. Secretion of the phosphorylated caseins occurs 10-20 min later.