Kalinski A, Melroy D L, Dwivedi R S, Herman E M
Plant Molecular Biology Laboratory, United States Department of Agriculture, Beltsville, Maryland 20705.
J Biol Chem. 1992 Jun 15;267(17):12068-76.
We have examined the synthesis, posttranslational processing, and localization of soybean P34, a member of the papain superfamily. P34 has been identified as a constituent of oil storage organelles or oil bodies isolated from seed lysates and has been assumed to be one of the oil body proteins. Electron microscopic immunocytochemistry with a monoclonal antibody demonstrated that P34 is localized in the protein storage vacuoles but not in the oil bodies. Immunocytochemical observations of partially disrupted seed cells showed that the association of P34 with oil bodies appears to occur as a consequence of cell lysis. In vitro synthesis of P34 results in the formation of a 46-kDa polypeptide that increases to 47 kDa due to core glycosylation by canine microsomes. In vivo synthesis studies in the presence and absence of tunicamycin, an inhibitor of N-linked glycosylation, indicate that pro-P34 is 47 kDa. Since the cDNA sequence of prepro-P34 contains a single putative glycosylation site in the precursor domain, we conclude that P34, like a few other vacuolar proteins, is synthesized as a glycoprotein precursor. Pulse-chase experiments showed that the processing of pro-P34 to mature P34 occurs in a single step and that this posttranslational cleavage occurs on the carboxyl side of an Asn, which is typical of seed vacuolar proteins. Pro-P34 (47 kDa) is detected in immunoblots of maturing seeds. Analysis of RNA indicates that the P34 genes are expressed only during seed maturation and that the P34 mRNA is related to other thiol protease mRNAs detectable in other organs and plants. Unlike other seed thiol proteases that are synthesized only after seed germination, P34 accumulates during seed maturation.
我们研究了木瓜蛋白酶超家族成员大豆P34的合成、翻译后加工及定位。P34已被鉴定为从种子裂解物中分离出的油贮藏细胞器或油体的一种成分，并被认为是油体蛋白之一。用单克隆抗体进行的电子显微镜免疫细胞化学分析表明，P34定位于蛋白质贮藏液泡而非油体中。对部分破裂的种子细胞进行免疫细胞化学观察显示，P34与油体的结合似乎是细胞裂解的结果。P34的体外合成产生一条46 kDa的多肽，由于犬微粒体的核心糖基化作用，该多肽增加到47 kDa。在有无衣霉素（一种N-连接糖基化抑制剂）存在的情况下进行的体内合成研究表明，前体P34为47 kDa。由于前原P34的cDNA序列在前体结构域中含有一个推定的糖基化位点，我们得出结论，P34与其他一些液泡蛋白一样，是以糖蛋白前体的形式合成的。脉冲追踪实验表明，前体P34加工成成熟P34是一步完成的，这种翻译后切割发生在Asn的羧基侧，这是种子液泡蛋白的典型特征。在成熟种子的免疫印迹中可检测到前体P34（47 kDa）。RNA分析表明，P34基因仅在种子成熟期间表达，且P34 mRNA与在其他器官和植物中可检测到的其他硫醇蛋白酶mRNA相关。与其他仅在种子萌发后才合成的种子硫醇蛋白酶不同，P34在种子成熟期间积累。
