Proteomics explains client specificity of the human Sec62/Sec63 complex in ER protein import

In mammalian cells, one-third of all polypeptides are transported into/across the ER-membrane via the Sec61-channel. While the Sec61-complex facilitates translocation of all polypeptides with amino-terminal signal peptides (SP) or transmembrane helices, the Sec61-associated Sec62/Sec63-complex supports translocation of only a subset, i.e. in a substrate-specific manner. To characterize Sec62- and Sec63-dependent precursors, we combined siRNA-mediated Sec62- or Sec63-depletion in human cells, label-free quantitative proteomics, and differential expression analysis. Alternatively, their CRISPR/Cas9-mediated knock-out in HEK 293 cells was carried out. The results were validated by western blotting under similar steady-state conditions and after short-term over-expression and simultaneous proteasome inhibition, respectively. SP analysis of Sec62- and Sec63-clients revealed the distinguishing feature. Thus, Sec62/Sec63-complex acts as an sp-receptor on the ER-membrane’s cytosolic face for certain precursor polypeptides and triggers substrate-specific (and regulated) opening of the Sec61-channel by its interactions with Sec61α.

在哺乳动物细胞中，三分之一的多肽通过Sec61通道（Sec61-channel）被转运至内质网膜（ER-membrane）内或跨内质网膜。尽管Sec61复合物（Sec61-complex）可介导所有带有氨基端信号肽（SP）或跨膜螺旋的多肽的跨膜转运，但与Sec61结合的Sec62/Sec63复合物（Sec61-associated Sec62/Sec63-complex）仅能辅助部分底物的转运，即遵循底物特异性模式。为了鉴定依赖Sec62与Sec63的前体多肽，我们将人类细胞中经小干扰RNA（siRNA）介导的Sec62或Sec63敲低、无标记定量蛋白质组学以及差异表达分析相结合。此外，我们还在HEK 293细胞中开展了Sec62和Sec63基因的CRISPR/Cas9介导敲除实验。我们分别在相似的稳态条件下，以及经过短期过表达并同时抑制蛋白酶体的条件下，通过蛋白质印迹（western blotting）对实验结果进行了验证。对Sec62与Sec63的作用底物进行SP分析后，我们明确了其区别于其他转运复合物的核心特征。综上，Sec62/Sec63复合物（Sec62/Sec63-complex）可作为特定前体多肽的信号肽受体，定位于内质网膜的胞质侧，并通过与Sec61α亚基的相互作用，触发Sec61通道以底物特异性（并受调控）的方式开放。