Chrispeels M J, Higgins T J, Craig S, Spencer D
J Cell Biol. 1982 Apr;93(1):5-14. doi: 10.1083/jcb.93.1.5.
Developing pea (Pisum sativum L.) cotyledons were labeled with radioactive amino acids, glucosamine, and mannose in pulse an pulse-chase experiments to study the synthesis, glycosylation, and transport of the reserve proteins vicilin and legumin to the protein bodies. Tissue extracts were fractionated on sucrose gradients to isolate either the endoplasmic reticulum (ER) or the protein bodies. Immunoaffinity gels were used to determine radioactivity in the reserve proteins (legumin and vicilin). After pulse-labeling for 45 min with amino acids, about half the total incorporated radioactivity coincided closely with the position of the ER marker enzyme NADH-cytochrome c reductase at a density of 1.13 g . cm-3 on the sucrose gradient. Both radioactivity and enzyme activity shifted to a density of 1.18 g . cm-3 in the presence of 3 mM MgCl2 indicating that the radioactive proteins were associated with the rough ER. Approximately half of the incorporated radioactivity associated with the rough ER was in newly synthesized reserve protein and this accounted for 80% of the reserve protein synthesized in 45 min. Trypsin digestion experiments indicated that these proteins were sequestered within the ER. In pulse-chase experiments, the reserve proteins in the ER became radioactive without appreciable lag and radioactivity chased out of the ER with a half-life of 90 min. Radioactive reserve proteins became associated with a protein body-rich fraction 20-30 min after their synthesis and sequestration by the ER. Pulse-chase experiments with radioactive glucosamine and mannose in the presence and absence of tunicamycin indicated that glycosylation of vicilin occurs in the ER. However, glycosylation is not a prerequisite for transport of vicilin from ER to protein bodies. Examination of the reserve protein polypeptides by SDS PAGE followed by fluorography showed that isolated ER contained legumin precursors (Mr 60,000-65,000) but not the polypeptides present in mature legumin (Mr 40,000 and 19,000) as well as the higher molecular weight polypeptides of vicilin (Mr 75,000, 70,000, 50,000, and 49,000). The smaller polypeptides of vicilin present in vicilin extracted from protein bodies (Mr 12,000-34,000) were absent from the ER. The results show that newly synthesized reserve proteins are preferentially and transiently sequestered within the ER before they move to the protein bodies, and that the ER is the site of storage protein glycosylation.
在脉冲和脉冲追踪实验中，用放射性氨基酸、葡糖胺和甘露糖标记发育中的豌豆（Pisum sativum L.）子叶，以研究贮藏蛋白豌豆球蛋白和豆球蛋白的合成、糖基化以及向蛋白体的转运。将组织提取物在蔗糖梯度上进行分级分离，以分离内质网（ER）或蛋白体。使用免疫亲和凝胶来测定贮藏蛋白（豆球蛋白和豌豆球蛋白）中的放射性。用氨基酸脉冲标记45分钟后，大约一半的总掺入放射性与内质网标记酶NADH - 细胞色素c还原酶在蔗糖梯度上密度为1.13 g·cm⁻³处的位置紧密重合。在3 mM MgCl₂存在下，放射性和酶活性均转移至密度为1.18 g·cm⁻³处，表明放射性蛋白与粗面内质网相关。与粗面内质网相关的掺入放射性中约一半存在于新合成的贮藏蛋白中，这占45分钟内合成的贮藏蛋白的80%。胰蛋白酶消化实验表明这些蛋白被隔离在内质网内。在脉冲追踪实验中，内质网中的贮藏蛋白迅速变得具有放射性，且放射性以90分钟的半衰期从内质网中被追踪出来。放射性贮藏蛋白在被内质网合成和隔离后20 - 30分钟与富含蛋白体的部分相关联。在有和没有衣霉素存在的情况下用放射性葡糖胺和甘露糖进行脉冲追踪实验表明，豌豆球蛋白的糖基化发生在内质网中。然而，糖基化不是豌豆球蛋白从内质网转运到蛋白体的先决条件。通过SDS - PAGE随后进行荧光自显影检查贮藏蛋白多肽表明，分离的内质网含有豌豆球蛋白前体（Mr 60,000 - 65,000），但不含有成熟豌豆球蛋白中存在的多肽（Mr 40,000和19,000）以及豌豆球蛋白的高分子量多肽（Mr 75,000、70,000、50,000和49,000）。从蛋白体中提取的豌豆球蛋白中存在的较小的豌豆球蛋白多肽（Mr 12,000 - 34,000）在内质网中不存在。结果表明，新合成的贮藏蛋白在转移到蛋白体之前优先且短暂地被隔离在内质网中，并且内质网是贮藏蛋白糖基化的场所。
