Quantitative Proteomics and Differential Protein Abundance Analysis after the Depletion of PEX3 from Human Cells Identifies Additional Aspects of Protein Targeting to the ER

Abstract

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 various 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.

Proteomics identifies substrates and a novel component in hSnd2-dependent ER protein targeting

“…Therefore, the dual aim of this study was to gain further insights into the components and client spectrum of the SND pathway in human cells. For this, we used the previously successfully established experimental strategy of combining transient depletion of a central membrane component of the pathway, using two different HSND2 targeting siRNAs in parallel to a non-targeting siRNA from HeLa cells, with total cellular proteomic analysis by label-free quantitative MS analysis and differential protein abundance analysis (Nguyen et al, 2018; Schorr et al, 2020; Klein et al, 2020; Bhadra et al, 2021; Zimmermann et al, 2021). As an internal proof of principle, as well as for comparison, similar experiments were carried out for the SRP- and TRC-dependent targeting pathways for which data from different global approaches, including ribosome profiling and component trapping data are already available (Chartron et al, 2016; Costa et al, 2018; Coy-Vergara et al, 2019).…”

“…Interestingly, there was also hardly any overlap between the previously reported PEX3-dependent clients and the substrates of any of the other protein targeting routes SRP, SND, or TRC, with exception of the TA protein FAR1 (PEX3 and Wrb) and the type I membrane protein HLA-C (PEX3, SRα and Wrb) (Figure 2A). When human patient fibroblasts with a complete PEX3 deficiency were subjected to MS and differential protein abundance analysis (Zimmermann et al, 2021), the PEX3 knockout negatively affected clients with a SP (56%) to an even greater extent than the SRα knockdown employed in this study (44%) and, likewise, gave an even higher enrichment of C-terminal targeting signals (32%) in clients with TMH as compared to hSnd2 or Wrb knockdown (21% and 27%, respectively) (Figures 2B and 2G). In contrast to hSnd2 and Wrb, PEX3 did not show any preferences for multi-spanning membrane proteins and had a lower preference for membrane proteins with central targeting signals (Figures 2D-2G).…”

“…In contrast to hSnd2 and Wrb, PEX3 did not show any preferences for multi-spanning membrane proteins and had a lower preference for membrane proteins with central targeting signals (Figures 2D-2G). Notably, however, unlike the other three ER membrane receptors, PEX3 appears to be restricted to certain areas of the ER or ER subdomains, where lipid droplets are formed and peroxisomal precursors and large cargo secretory vesicles are budding off (Schrul and Kopito, 2016; Jansen and van der Klei, 2019; Zimmermann et al, 2021). This restricted localization may explain, by an as of yet unknown mechanism, its different client spectrum (Figure 2A and 7B).…”

“…The aim of this study was to gain further insights into the components and client spectrum of the SND pathway in human cells under physiological conditions, including physiological translation rates and competing precursor polypeptides. For this, we used the established experimental strategy of combining transient depletion of a central component of the pathway, here hSnd2, combined with total cellular proteomic analysis by label-free quantitative mass spectrometry/MS and differential protein abundance analysis in HeLa cells (Nguyen et al, 2018; Schorr et al, 2020; Klein et al, 2020; Bhadra et al, 2021; Zimmermann et al, 2021). For comparison, similar experiments were carried out for the SRP and TRC targeting pathways, for which different global approaches, such as ribosome profiling or component trapping data, are also available for human cells (Chartron et al, 2016; Costa et al, 2018; Coy-Vergara et al, 2019).…”

Mechanisms of ER Protein Import

“…As further reading on this subject, we suggest recent original articles by Jomaa et al [3] as well as Tirincsi et al [4]. J. Herrmann and colleagues [5] and B. Schrul and colleagues [6] round up the section on protein targeting to the ER and provide a state of the art view of the ER-SURF pathway and recent insights into the client spectrum of the PEX3/PEX19 pathway to the ER, respectively. Here, we suggest as further reading an article on the targeting of mRNAs and ribosome-nascent-chain complexes to the ER [7].…”