The lumenal domain of Sec63p stimulates the ATPase activity of BiP and mediates BiP recruitment to the translocon in Saccharomyces cerevisiae.
The lumenal domain of Sec63p stimulates the ATPase activity of BiP and mediates BiP recruitment to the translocon in Saccharomyces cerevisiae.
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DOI：
10.1083/jcb.137.7.1483
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发表时间：
1997-06-30
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我们研究了完整膜蛋白Sec63p与管腔Hsp70Bip相互作用的分子本质，以阐明它们在前体转运到酿酒酵母内质网过程中的作用。Sec63p的管腔延伸与大肠杆菌蛋白DNAJ同源，是Sec63p与Bip之间可能的界面区域。该结构域被纯化为与谷胱甘肽S转移酶(GST)的融合蛋白(63Jp)，介导了63Jp与Bip之间稳定的依赖三磷酸腺苷的结合作用，并刺激了Bip的ATPase活性。当洗涤剂溶解的微粒与ATP和融合蛋白混合时，这种相互作用是高度选择性的，因为固定化的63Jp上只保留了Bip。在这些检测中，单独的GST是无效的。此外，在Sec63p的管腔区域含有点突变的GST融合不与Bip相互作用。最后，我们发现，可溶性的Sec63p腔结构域抑制了有效的前体进入重组的蛋白脂质体，从而整合了Bip和融合蛋白。我们的结论是，Sec63p的管腔结构域足以介导与Bip的酶相互作用，这种位于内质网腔面转位装置或转位的相互作用对于蛋白质转位到内质网是至关重要的。
We studied the molecular nature of the interaction between the integral membrane protein Sec63p and the lumenal Hsp70 BiP to elucidate their role in the process of precursor transit into the ER of Saccharomyces cerevisiae. A lumenal stretch of Sec63p with homology to the Escherichia coli protein DnaJ is the likely region of interface between Sec63p and BiP. This domain, purified as a fusion protein (63Jp) with glutathione S–transferase (GST), mediated a stable ATP-dependent binding interaction between 63Jp and BiP and stimulated the ATPase activity of BiP. The interaction was highly selective because only BiP was retained on immobilized 63Jp when detergent-solubilized microsomes were mixed with ATP and the fusion protein. GST alone was inactive in these assays. Additionally, a GST fusion containing a point mutation in the lumenal domain of Sec63p did not interact with BiP. Finally, we found that the soluble Sec63p lumenal domain inhibited efficient precursor import into proteoliposomes reconstituted so as to incorporate both BiP and the fusion protein. We conclude that the lumenal domain of Sec63p is sufficient to mediate enzymatic interaction with BiP and that this interaction positioned at the translocation apparatus or translocon at the lumenal face of the ER is vital for protein translocation into the ER.