We previously showed via electron microscopic immunocytochemistry that a 73 kDa polypeptide was an authentic peroxisomal membrane protein (PMP73) integrated exclusively into the boundary membrane of glyoxysomes in cucumber seedlings. In this paper we test the hypothesis that this PMP73 is a member of the heat-shock 70 protein (Hsp70) family by comparing amino acid sequences of cyanogen bromide (CNBr)-cleaved polypeptide fragments, immunoreactivities on protein blots, and microscopic immunofluorescence within suspension-cultured BY-2 tobacco cells. A sequence of eight amino acids (DAVGPEIQ) in PMP73 showed a high degree of similarity (up to 88%) with sequences in the same carboxy-terminal region of four plant Hsp70 proteins. IgGs affinity purified to PMP73 recognized on blots a membrane-bound Hsp72 (in pea cotyledon microsomes) and a cucumber PMP61, the latter shown by CNBr cleavage to be a distinct, but immunorelated polypeptide to PMP73. Conversely, IgGs specific for tomato Hsc70 (C-terminal half) recognized cucumber PMP73, and IgGs specific for cucumber DnaJ homologue (entire protein) recognized cucumber PMP61. In BY-2 cells, cucumber PMP73-specific IgGs localized only to peroxisomes. Antibodies raised against portions of tomato Hsc70 also localized to the BY-2 peroxisomes (as well as to cytosolic proteins). Collectively, the data show that authentic cucumber PMPs73 and 61 are immunorelated to each another, and that both exhibit selective immunoreactivity to IgGs from two classes of molecular chaperones, namely Hsp70 proteins and plant DnaJ homologues. They appear to be unique membrane-bound chaperones that likely function as part of the peroxisomal protein translocation machinery.

译文

我们先前通过电子显微镜免疫细胞化学显示,一个73 kDa的多肽是真正的过氧化物酶体膜蛋白 (PMP73),仅整合到黄瓜幼苗中乙醛酸体的边界膜中。在本文中,我们通过比较溴化氰 (CNBr) 切割的多肽片段的氨基酸序列,蛋白质印迹上的免疫反应性以及悬浮培养的by-2烟草细胞中的微观免疫荧光,检验了该PMP73是热休克70蛋白 (Hsp70) 家族成员的假设。PMP73中的八个氨基酸序列 (DAVGPEIQ) 与四种植物Hsp70蛋白的相同羧基末端区域中的序列显示出高度相似性 (高达88%)。纯化的与PMP73的IgGs亲和力在印迹上识别出与膜结合的Hsp72 (在豌豆子叶微粒体中) 和黄瓜PMP61,后者通过CNBr裂解显示为PMP73的独特但与免疫相关的多肽。相反,对番茄Hsc70 (C末端一半) 特异的igg识别黄瓜PMP73,对黄瓜DnaJ同源物 (整个蛋白质) 特异的igg识别黄瓜pmp61。在BY-2细胞中,黄瓜PMP73-specific igg仅定位于过氧化物酶体。针对番茄Hsc70部分产生的抗体也定位于BY-2过氧化物酶体 (以及胞质蛋白)。总的来说,数据表明,真实的黄瓜PMPs73和61彼此免疫相关,并且都对来自两类分子伴侣的igg表现出选择性免疫反应性,即Hsp70蛋白和植物DnaJ同源物。它们似乎是独特的膜结合伴侣,可能是过氧化物酶体蛋白易位机制的一部分。

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