Tripathi Arati, Mandon Elisabet C, Gilmore Reid, Rapoport Tom A
From the Howard Hughes Medical Institute and the Department of Cell Biology, Harvard Medical School, Boston, Massachusetts 02115 and
the Department of Biochemistry and Molecular Pharmacology, University of Massachusetts Medical School, Worcester, Massachusetts 01605.
J Biol Chem. 2017 May 12;292(19):8007-8018. doi: 10.1074/jbc.M116.761122. Epub 2017 Mar 12.
The biosynthesis of many eukaryotic proteins requires accurate targeting to and translocation across the endoplasmic reticulum membrane. Post-translational protein translocation in yeast requires both the Sec61 translocation channel, and a complex of four additional proteins: Sec63, Sec62, Sec71, and Sec72. The structure and function of these proteins are largely unknown. This pathway also requires the cytosolic Hsp70 protein Ssa1, but whether Ssa1 associates with the translocation machinery to target protein substrates to the membrane is unclear. Here, we use a combined structural and biochemical approach to explore the role of Sec71-Sec72 subcomplex in post-translational protein translocation. To this end, we report a crystal structure of the Sec71-Sec72 complex, which revealed that Sec72 contains a tetratricopeptide repeat (TPR) domain that is anchored to the endoplasmic reticulum membrane by Sec71. We also determined the crystal structure of this TPR domain with a C-terminal peptide derived from Ssa1, which suggests how Sec72 interacts with full-length Ssa1. Surprisingly, Ssb1, a cytoplasmic Hsp70 that binds ribosome-associated nascent polypeptide chains, also binds to the TPR domain of Sec72, even though it lacks the TPR-binding C-terminal residues of Ssa1. We demonstrate that Ssb1 binds through its ATPase domain to the TPR domain, an interaction that leads to inhibition of nucleotide exchange. Taken together, our results suggest that translocation substrates can be recruited to the Sec71-Sec72 complex either post-translationally through Ssa1 or co-translationally through Ssb1.
许多真核生物蛋白质的生物合成需要精确靶向并跨内质网膜转运。酵母中的翻译后蛋白质转运既需要Sec61转运通道，也需要另外四种蛋白质组成的复合物：Sec63、Sec62、Sec71和Sec72。这些蛋白质的结构和功能在很大程度上尚不清楚。该途径还需要胞质Hsp70蛋白Ssa1，但尚不清楚Ssa1是否与转运机制结合以将蛋白质底物靶向到膜上。在这里，我们使用结构和生化相结合的方法来探索Sec71-Sec72亚复合物在翻译后蛋白质转运中的作用。为此，我们报道了Sec71-Sec72复合物的晶体结构，该结构表明Sec72包含一个四肽重复（TPR）结构域，该结构域通过Sec71锚定在内质网膜上。我们还确定了该TPR结构域与源自Ssa1的C末端肽的晶体结构，这表明了Sec72与全长Ssa1的相互作用方式。令人惊讶的是，Ssb1是一种结合核糖体相关新生多肽链的胞质Hsp70，它也与Sec72的TPR结构域结合，尽管它缺乏Ssa1的TPR结合C末端残基。我们证明Ssb1通过其ATP酶结构域与TPR结构域结合，这种相互作用导致核苷酸交换的抑制。综上所述，我们的结果表明转运底物可以在翻译后通过Ssa1或在翻译过程中通过Ssb1被招募到Sec71-Sec72复合物中。
