Corsi A K, Schekman R
Department of Molecular and Cell Biology, Howard Hughes Medical Institute, University of California, Berkeley, California 94720, USA.
J Cell Biol. 1997 Jun 30;137(7):1483-93. doi: 10.1083/jcb.137.7.1483.
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.
我们研究了整合膜蛋白Sec63p与内质网腔热休克蛋白70（Hsp70）BiP之间相互作用的分子本质，以阐明它们在酿酒酵母前体转运到内质网过程中的作用。Sec63p的一段与大肠杆菌蛋白DnaJ具有同源性的内质网腔序列，可能是Sec63p与BiP之间的界面区域。该结构域作为与谷胱甘肽S-转移酶（GST）的融合蛋白（63Jp）进行纯化，介导了63Jp与BiP之间稳定的ATP依赖性结合相互作用，并刺激了BiP的ATP酶活性。这种相互作用具有高度选择性，因为当用去污剂溶解的微粒体与ATP和融合蛋白混合时，只有BiP保留在固定化的63Jp上。单独的GST在这些试验中无活性。此外，含有Sec63p内质网腔结构域点突变的GST融合蛋白不与BiP相互作用。最后，我们发现可溶性Sec63p内质网腔结构域抑制前体有效导入重组的蛋白脂质体，该重组蛋白脂质体同时包含BiP和融合蛋白。我们得出结论，Sec63p的内质网腔结构域足以介导与BiP的酶促相互作用，并且这种位于内质网腔面易位装置或易位子处的相互作用对于蛋白质向内质网的易位至关重要。
