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 labelled 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 min labelling period, higher-Mr 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 approx. 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 labelled during both short (5 min) and long (30 min) pulses with [35S]methionine and [32P]Pi shows that, in contrast with the 35S-labelled caseins, those labelled with [32P]Pi exhibit only electrophoretic mobilities identical with those of the mature caseins isolated from milk and those identified after long labelling periods with [35S]methionine. No phosphorylated early intermediate forms of caseins were identified. We conclude that the synthesis and post-translational modification of guinea-pig caseins occurs in two stages, (i) an early event involving synthesis and sequestration within the endoplasmic reticulum, an event that involves signal-peptide removal, followed (ii) 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.
D000818:Animals; D002364:Caseins / Q000378:metabolism*; D002499:Centrifugation, Density Gradient; D004591:Electrophoresis, Polyacrylamide Gel; D005260:Female; D006168:Guinea Pigs; D007425:Intracellular Membranes / Q000378:metabolism; D007700:Kinetics; D007768:Lactalbumin / Q000378:metabolism; D007774:Lactation; D008321:Mammary Glands, Animal / Q000378:metabolism*; D008861:Microsomes / Q000378:metabolism; D009924:Organ Culture Techniques; D010766:Phosphorylation; D011247:Pregnancy; D014176:Protein Biosynthesis
D002364:Caseins; D007768:Lactalbumin