Quantitative Proteomics and Differential Protein Abundance Analysis after Depletion of Putative mRNA Receptors in the ER Membrane of Human Cells Identifies Novel Aspects of mRNA Targeting to the ER

Abstract

In human cells, one-third of all polypeptides enter the secretory pathway at the endoplasmic reticulum (ER). The specificity and efficiency of this process are guaranteed by targeting of mRNAs and/or polypeptides to the ER membrane. Cytosolic SRP and its receptor in the ER membrane facilitate the cotranslational targeting of most ribosome-nascent precursor polypeptide chain (RNC) complexes together with the respective mRNAs to the Sec61 complex in the ER membrane. Alternatively, fully synthesized precursor polypeptides are targeted to the ER membrane post-translationally by either the TRC, SND, or PEX19/3 pathway. Furthermore, there is targeting of mRNAs to the ER membrane, which does not involve SRP but involves mRNA- or RNC-binding proteins on the ER surface, such as RRBP1 or KTN1. Traditionally, the targeting reactions were studied in cell-free or cellular assays, which focus on a single precursor polypeptide and allow the conclusion of whether a certain precursor can use a certain pathway. Recently, cellular approaches such as proximity-based ribosome profiling or quantitative proteomics were employed to address the question of which precursors use certain pathways under physiological conditions. Here, we combined siRNA-mediated depletion of putative mRNA receptors in HeLa cells with label-free quantitative proteomics and differential protein abundance analysis to characterize RRBP1- or KTN1-involving precursors and to identify possible genetic interactions between the various targeting pathways. Furthermore, we discuss the possible implications on the so-called TIGER domains and critically discuss the pros and cons of this experimental approach.

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

“…Applying the established statistical analysis, we found that PEX3 deficiency significantly affected the steady-state levels of 238 proteins: 141 negatively and 97 positively (permutation false discovery rate-adjusted p value < 0.05). Of the negatively affected proteins, GO terms assigned 39.2% to organelles of the endocytic and exocytic pathways (Figure 2c, large pies), which corresponds to a 1.36-fold enrichment (Figure 2c, large pies, 39.2% divided by 28.91% = 1.36) and is below the average values of 1.46 and 1.94 observed after depletion of the mRNA targeting components KTN1 (1.55) plus RRBP1 (1.37) and the translocation components Sec61 (2.37) plus TRAP (1.5), respectively (Table 2) [33,46]. In contrast to the PEX3-knock-down cells (Figure S1c), we also detected enrichment of proteins with SP (2.65-fold), N-glycosylated proteins (2.2-fold), and membrane proteins (1.36-fold) (Figure 2c, small pies), which was lower as compared to the Sec61-(6.51, 2.83, 2.51) or TRAP-experiment (3.3, 2.7, 2.1) but higher as compared to the KTN1-(1, 2.09, 1,76) and RRBP1-experiment (2.44, 2.12, 1.46) [33,46].…”

“…Interestingly, there were 14 precursors of mitochondrial proteins negatively affected by PEX3-deficiency (Table S4), a phenomenon previously observed after depletion of RRBP1 from HeLa cells and attributed to their physiological trafficking from the ER to mitochondria via the newly identified ER-SURF pathway [11,33]. Among these negatively affected mitochondrial proteins were three outer membrane proteins (AIFM2, RHOT1, VAT1), one inner membrane protein (NDUFV3), and ten matrix proteins, two of which have a dual localization in mitochondria and peroxisomes (ACAD11, SCP2).…”

“…Our approach to characterize the client spectrum of PEX3 in ER protein targeting involves gene silencing of the putative receptor PEX3 in HeLa Kyoto cells with two different targeting siRNAs in parallel to a non-targeting or control siRNA and differential proteomic analysis by label-free quantitative MS analysis and differential protein abundance analysis (Figure 1b). This protocol was developed and previously used to characterize the client spectrum and client SP features of ER protein translocation components including Sec61 complex (as a proof on concept), TRAP complex, Sec62/Sec63 complex, TRAM1-protein, ERj1, plus BiP, and the mRNA targeting components KTN1 and RRBP1 [33,[46][47][48]. The approach is based on the assumption that polypeptide precursors, which have to be imported into the ER, are degraded by the proteasome in the cytosol upon interference with their ER targeting or translocation because their SPs or TMHs are not easily compatible with the aqueous character of the cytosol.…”

“…Therefore, their cellular levels are decreased as compared to control cells, and this change is detected by quantitative MS and subsequent differential protein abundance analysis [46]. Typically, the decrease was observed to be accompanied by an increase of ubiquitin-conjugating enzymes [33,[46][47][48]. Furthermore, a simultaneous increase in other ER import components was detected, which is consistent with the overlap of pathways and, additionally, may indicate a genetic interaction between different pathways and cellular compensation.…”

“…In addition, there is targeting of mRNAs to the ER membrane, which involves mRNA receptors (such as KTN1), or receptors for ribosome nascent chain complexes with nascent chains, which are not yet long enough to be able to interact with SRP (such as RRBP1) [27][28][29][30][31][32][33]. However, one general lesson from the analysis of all these pathways is that they are not strictly separated from each other and that there are at least some precursor polypeptides, which can be targeted to the ER by more than one pathway (such as some small presecretory proteins and some tail-anchored membrane proteins) [23,25,26].…”