In vivo and in vitro processing of seed reserve protein in the endoplasmic reticulum: evidence for two glycosylation steps.
In vivo and in vitro processing of seed reserve protein in the endoplasmic reticulum: evidence for two glycosylation steps.
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DOI：
10.1083/jcb.96.4.999
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发表时间：
1983-04
影响因子：
7.8
通讯作者：
Chrispeels, M J
中科院分区：
文献类型：
--
作者：
Bollini, R;Vitale, A;Chrispeels, M J
普通菜豆（Phaseolus vulgaris L.）合成大量的储备蛋白菜豆蛋白。多肽在膜结合的多聚核糖体上合成，通过内质网（ER）并在蛋白体中积累。为了研究菜豆蛋白的生物合成和加工，用放射性氨基酸、葡糖胺和甘露糖标记发育中的子叶，并将分离的组分（多核糖体RNA、多核糖体和粗糙ER）用于体外蛋白质合成。新合成的菜豆蛋白存在于发育中的子叶的内质网中，可以通过SDS PAGE分离成四种糖多肽。在体外合成与多聚核糖体RNA的结果，在形成两个多肽的多聚核糖体径流表明，糖基化是一个共翻译事件。由多核糖体流出形成的两个未糖基化的多肽略小于由分离的RNA的体外翻译形成的两个多肽，表明信号肽可能存在于这些多肽上。径流合成与粗糙ER产生的四个多肽的模式类似于在体内标记获得的一个。多聚体流出形成的两个丰富的糖多肽。该结果表明对于丰富的多肽存在第二糖基化事件。使用Triton X-100在粗ER链完成期间抑制糖基化，以表明这两个糖基化步骤通常顺序发生。两个糖基化步骤均被衣霉素抑制。对不同多肽的碳水化合物与蛋白质的比率以及用[（3）H]葡糖胺标记的多肽的胰蛋白酶的分析证实了以下结论：一些糖基化多肽含有两个寡糖链，而另一些仅含有一个。胰蛋白酶肽图谱分析表明，每个非糖基化多肽是一个糖基化多肽的前体，其中一个糖基化多肽具有一个寡糖链，另一个糖基化多肽具有两个寡糖链。
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 phaseolin present in the ER of developing cotyledons can be fractioned into four glycopolypeptides by SDS PAGE. In vitro synthesis with polysomal RNA results in the formation of two polypeptides by polysome run-off shows that glycosylation is a co-translational event. The two unglycosylated polypeptides formed by polysome run-off are slightly smaller than the two 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 four polypeptides similar to the one obtained by in vivo labeling. The two abundant glycopolypeptides formed by polysome run-off. This result indicates the existence of a second 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 two glycosylation steps normally occur sequentially. Both glycosylation steps are inhibited by tunicamycin. Analysis of carhohydrate to protein ratios of the different polypeptides and of trypsin digests of polypeptides labeled with [(3)H]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 one glycosylated polypeptide with one oligosaccharide chain and one with two oligosaccharide chains.