Quantitative proteomics and differential protein abundance analysis after the depletion of PEX3 from human cells identifies additional aspects of protein targeting to the ER

Protein import into the endoplasmic reticulum (ER) is the first step in the biogenesis of around 10,000 different soluble and membrane proteins in humans. It involves the co-or post-translational targeting of precursor polypeptides to the ER, and their subsequent membrane insertion or translocation. So far, three pathways for the ER targeting of precursor polypeptides and four pathways for the ER targeting of mRNAs have been described. Typically, these pathways deliver their substrates to the Sec61 polypeptide-conducting channel in the ER membrane. Next, the precursor polypeptides are inserted into the ER membrane or translocated into the ER lumen, which may involve auxiliary translocation components, such as the TRAP and Sec62/Sec63 complexes, or auxiliary membrane protein insertases, such as EMC and the TMCO1 complex. Recently, the PEX19/PEX3-dependent pathway, which has a well-known function in targeting and inserting vari-ous peroxisomal membrane proteins into pre-existent peroxisomal membranes, was also found to act in the targeting and, putatively, insertion of monotopic hairpin proteins into the ER. These either remain in the ER as resident ER membrane proteins, or are pinched off from the ER as components of new lipid droplets. Therefore, the question arose as to whether this pathway may play a more general role in ER protein targeting, i.e., whether it represents a fourth pathway for the ER targeting of precursor polypeptides. Thus, we addressed the client spectrum of the PEX19/PEX3-dependent pathway in both PEX3-depleted HeLa cells and PEX3-deficient Zellweger patient fibroblasts by an established approach which involved the label-free quantitative mass spectrometry of the total proteome of depleted or deficient cells, as well as differential protein abundance analysis. The negatively affected proteins included twelve peroxisomal proteins and two hairpin proteins of the ER, thus confirming two previously identified classes of putative PEX19/PEX3 clients in human cells. Interestingly, fourteen collagen-related proteins with signal peptides or N-terminal transmembrane helices belonging to the secretory pathway were also negatively affected by PEX3 deficiency, which may suggest compromised collagen biogenesis as a hitherto-unknown contributor to organ failures in the respective Zellweger patients.