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.