Coupled cell-free synthesis, segregation, and core glycosylation of a secretory protein.
mRNA from rat mammary glands 13-15 days post partum was translated in a wheat germ cell-free system either in the absence or in the presence of ribosome-denuded membranes prepared from isolated rough microsomes of dog pancreas. Newly synthesized alpha-lactalbumin was identified by immunoprecipitation with a monospecific rabbit antiserum against rat alpha-lactalbumin and was characterized by partial amino-terminal sequence determination and by lectin affinity chromatography. In the absence of membranes a presumably unglycosylated form of alpha-lactalbumin was synthesized that bound neither to concanavalin A-Sepharose nor to Ricinus communis lectin-agarose and that contained an amino-terminal signal peptide region comprising 19 amino acid residues. In the presence of membranes a processed form was synthesized that lacked the signal peptide portion and that had an amino-terminal sequence identical to that of mature alpha-lactalbumin. Furthermore, this processed form was found to be segregated, presumably within the microsomal vesicles, because it was resistant to post-translational proteolysis. It was also found to be glycosylated, and because it bound to concanavalin A-Sepharose, from which it could be eluted specifically by alpha-methyl mannoside, but not to R. communis lectin-agarose, it was presumably core-glycosylated. Processing, segregation, and core glycosylation were observed to proceed only when membranes were present during translation and not when they were added after translation.
D000595:Amino Acid Sequence; D000818:Animals; D050260:Carbohydrate Metabolism; D002474:Cell-Free System; D005260:Female; D006023:Glycoproteins / Q000096:biosynthesis* / Q000378:metabolism; D007768:Lactalbumin / Q000096:biosynthesis* / Q000378:metabolism; D008321:Mammary Glands, Animal / Q000378:metabolism / Q000648:ultrastructure; D008861:Microsomes / Q000378:metabolism*; D011498:Protein Precursors / Q000096:biosynthesis*; D051381:Rats
D006023:Glycoproteins; D011498:Protein Precursors; D007768:Lactalbumin