Identification of a complex between centrin and heat shock proteins in CSF-arrested Xenopus oocytes and dissociation of the complex following oocyte activation.
Identification of a complex between centrin and heat shock proteins in CSF-arrested Xenopus oocytes and dissociation of the complex following oocyte activation.
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DOI：
10.1006/dbio.1995.1259
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发表时间：
1995-09
影响因子：
2.7
通讯作者：
Marie Uzawa;J. Grams;B. Madden;D. Toft;J. Salisbury
中科院分区：
文献类型：
--
作者：
Marie Uzawa;J. Grams;B. Madden;D. Toft;J. Salisbury
使用单克隆抗中心蛋白抗体（20 H5）和细胞生长抑制因子（CSF）逮捕爪蟾卵母细胞提取物的免疫共沉淀实验特异性沉淀卵母细胞中心蛋白（20 kDa）和两个相关的蛋白质70和90 kDa。微序列分析的胰蛋白酶肽片段的70 kDa的蛋白质揭示了100%的同一性与13个氨基酸的肽序列从非洲爪蟾热休克蛋白HSP-70。使用抗hsp单克隆抗体（N27和AC-88）对免疫沉淀物进行Western印迹分析，确认了70-kDa蛋白为hsp-70的身份，并鉴定了90-kDa蛋白为hsp-90。当在免疫沉淀过程中使用抗hsp-70或抗hsp-90单克隆抗体（分别为BB 70和4F 3）作为一抗时，中心蛋白/hsp复合物也被免疫沉淀。centrin/hsp复合物对pH和Ca ~（2+）浓度敏感。该复合物显示在各种条件下HSP-70和HSP-90的差异解离，表明每个HSP可以独立地与中心蛋白结合。当卵母细胞首先通过电击或离子载体处理激活，然后使用抗中心蛋白单克隆抗体20 H5进行免疫沉淀时，中心蛋白沉淀物中HSP-70的水平显着降低，并且这些复合物不含明显的HSP-90。我们的结论是，在CSF逮捕的卵母细胞，中心体蛋白，中心蛋白，是作为一个复杂的热休克蛋白，HSP-70和HSP-90，这种复杂的解离激活后的卵母细胞。中心蛋白和这些热休克蛋白之间形成复合物的功能后果是未知的。然而，根据已定义的热休克蛋白在其他系统中的作用，提出了几种可能性。
Coimmunoprecipitation experiments using a monoclonal anti-centrin antibody (20H5) and cytostatic factor (CSF)-arrested Xenopus oocyte extracts specifically precipitates oocyte centrin (20-kDa) and two associated proteins of 70- and 90-kDa. Microsequence analysis of a tryptic peptide fragment of the 70-kDa protein reveals 100% identity with a 13-amino-acid peptide sequence from Xenopus heat shock protein hsp-70. Western blot analysis of immunoprecipitates using anti-hsp monoclonal antibodies (N27 and AC-88) confirms the identity of the 70-kDa protein as hsp-70 and identifies the 90-kDa protein as hsp-90. The centrin/hsp complex is also immunoprecipitated when anti-hsp-70 or anti-hsp-90 monoclonal antibodies (BB70 and 4F3, respectively) are used as primary antibodies during immunoprecipitation. The centrin/hsp complex is sensitive to pH and Ca2+ concentration. The complex shows differential dissociation of hsp-70 and hsp-90 under a variety of conditions, suggesting that each hsp can bind to centrin independently of the other. When oocytes are first activated by electric shock or ionophore treatment, followed by immunoprecipitation using anti-centrin monoclonal antibody 20H5, centrin precipitates with significantly reduced levels of hsp-70 in the complex, and these complexes contain no apparent hsp-90. We conclude that, in CSF-arrested oocytes, the centrosomal protein, centrin, is associated as a complex with the heat shock proteins, hsp-70 and hsp-90, and that this complex dissociates upon activation of the oocyte. The functional consequences of the formation of complexes between centrin and these hsps are unknown. However, based on the roles that have been defined for heat shock proteins in other systems, several possibilities are suggested.