In vivo and in vitro processing of seed reserve protein in the endoplasmic reticulum: evidence for two glycosylation steps

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Abstract
Cotyledons of the common bean (Phaseolus vulgaris L.) synthesize large amounts of the reserve protein phaseolin. The polypeptides are synthesized on membrane-bound polysomes, pass through the endoplasmic reticulum (ER) and accumulate in protein bodies. For a study of the biosynthesis and processing of phaseolin, developing cotyledons were labeled with radioactive amino acids, glucosamine and mannose, and isolated fractions (polysomal RNA, polysomes, and rough ER) were used for in vitro protein synthesis. Newly synthesized phaseoline present in the ER of developing cotyledons can be fractionated into 4 glycopolypeptides by SDS PAGE [sodium dodecylsulfate polyacrylamide gel electrophoresis]. In vitro synthesis with polysomal RNA results in the formation of 2 polypeptides (or size-classes) while run-off synthesis with polysomes isolated from rough ER produces 2 unglycosylated and 2 glycosylated polypeptides. The formation of 2 glycosylated polypeptides by polysome run-off shows that glycosylation is a co-translational event. The 2 unglycosylated polypeptides formed by polysome run-off are slightly smaller than the 2 polypeptides formed by in vitro translation of isolated RNA, indicating that a signal peptide may be present on these polypeptides. Run-off synthesis with rough ER produces a pattern of 4 polypeptides similar to the one obtained by in vivo labeling. The 2 abundant glycopolypeptides formed in vivo or in vitro by rough ER are larger than the 2 glycosylated polypeptides formed by polysome run-off. This result indicates the existence of a 2nd glycosylation event for the abundant polypeptides. Inhibition of glycosylation by Triton X-100 during chain-completion with rough ER was used to show that these 2 glycosylation steps normally occur sequentially. Both glycosylation steps are inhibited by tunicamycin. Analysis of carbohydrate to protein ratios of the different polypeptides and of trypsin digests of polypeptides labeled with [3H]glucosamine confirmed the conclusion that some glycosylated polypeptides contain two oligosaccharide chains, while others contain only one. An analysis of tryptic peptide maps shows that each of the unglycosylated polypeptides is the precursor for 1 glycosylated polypeptide with 1 oligosaccharide chain and one with 2 oligosaccharide chains.