Assembly of AKAP82, a protein kinase A anchor protein, into the fibrous sheath of mouse sperm
Assembly of AKAP82, a protein kinase A anchor protein, into the fibrous sheath of mouse sperm
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DOI：
10.1006/dbio.1997.8767
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发表时间：
1997-12-15
影响因子：
2.7
通讯作者：
Gerton, GL
中科院分区：
文献类型：
--
作者：
Johnson, LR;Foster, JA;Gerton, GL
哺乳动物精子鞭毛的组装是一个复杂的发育过程，在单倍体精子分化过程中，需要编码组成部分的基因的顺序激活和这些蛋白质的协调组装。在这项研究中，研究了轴突周围纤维鞘组装的机制。该研究的对象是小鼠精子鞭毛的主要纤维鞘蛋白AKAP82，它是a激酶锚定蛋白（AKAP）家族的一员，该多肽结合蛋白激酶a （PK-A）的调控（RII）亚基。免疫电镜显示，AKAP82遍布于纤维鞘的横肋和纵柱。由于AKAP82最初是在精子发生过程中作为前体（pro-AKAP82）合成的，因此针对pro-AKAP82加工区域的肽（M-r 97,000）产生了抗血清。在免疫印迹实验中，抗体在凝集精子中检测到pro-AKAP82，而在附睾精子中检测不到。此外，附睾精子中还存在另外两个m - r109000 （p109）和m - r26000 （p26，代表前体的“pro”结构域）的免疫反应蛋白。对附睾精子蛋白的碱性磷酸酶处理表明，p109是保留在精子中的前akap82的磷酸化形式。通过免疫荧光检测，在睾丸精子中，pro-AKAP82定位于主片段的整个长度，而在附睾精子中，p109和p26仅存在于主片段的近端。用Triton X-100提取生殖细胞时，将Pro-AKAP82溶解。然而，在精子中，AKAP82和p109几乎完全抵抗这些提取条件，即使用Triton和二硫代苏糖醇提取后，它们仍保留在颗粒部分。与pro-AKAP82类似，PK-A的RII亚基存在于发育中的生殖细胞的Triton x -100可溶性部分中。在精子中，大部分RII也变成了微粒，这与AKAP82将RII固定在鞭毛中的假设一致。这些数据表明，前AKAP82在细胞体中合成，沿着轴突转运到其在纤维鞘中的组装位点，然后通过蛋白水解剪切形成成熟的AKAP82。(C) 1997学术出版社。
The assembly of the mammalian sperm flagellum is a complex developmental event requiring the sequential activation of genes encoding the component parts and the coordinated assembly of these proteins during the differentiation of the haploid spermatid. In this study, the mechanism underlying the assembly of the fibrous sheath surrounding the axoneme was examined. The subject of the study was the major fibrous sheath protein of the mouse sperm flagellum, AKAP82, a member of the A Kinase Anchor Protein (AKAP) family of polypeptides that bind the regulatory (RII) subunit of protein kinase A (PK-A). Immunoelectron microscopy demonstrated that AKAP82 is present throughout the transverse ribs and longitudinal columns of the fibrous sheath. Since AKAP82 is initially synthesized as a precursor (pro-AKAP82) during spermiogenesis, an antiserum was raised against a peptide from the processed region of pro-AKAP82 (M-r 97,000). In immunoblotting experiments, the antibody detected pro-AKAP82 in condensing spermatids but not in epididymal sperm. In addition, two other immunoreactive proteins of M-r 109,000 (p109) and M-r 26,000 (p26, representing the "pro" domain of the precursor) were present in epididymal sperm. Alkaline phosphatase treatment of epididymal sperm proteins demonstrated that p109 was a phosphorylated form of pro-AKAP82 that remained in sperm. By immunofluorescence, pro-AKAP82 was localized to the entire length of the principal piece in testicular sperm, while in epididymal sperm p109 and p26 were present only in the proximal portion of the principal piece. Pro-AKAP82 was solubilized when germ cells were extracted with Triton X-100. However, in sperm, both AKAP82 and p109 were almost totally resistant to these extraction conditions and remained in the particulate fraction even after extraction with Triton and dithiothreitol. Similar to pro-AKAP82, the RII subunit of PK-A was present in the Triton X-100-soluble fraction of developing germ cells. In sperm, much of the RII also became particulate, consistent with the hypothesis that AKAP82 anchors RII in the flagellum. These data indicate that pro-AKAP82 is synthesized in the cell body, transported down the axoneme to its site of assembly in the fibrous sheath, and then proteolytically clipped to form mature AKAP82. (C) 1997 Academic Press.