Discrimination between the endoplasmic reticulum and mitochondria by spontaneously inserting tail‐anchored proteins

Abstract

Tail-anchored (TA) proteins insert into their target organelles by incompletely elucidated posttranslational pathways. Some TA proteins spontaneously insert into protein-free liposomes, yet target a specific organelle in vivo. Two spontaneously inserting cytochrome b5 forms, b5-ER and b5-RR, which differ only in the charge of the C-terminal region, target the endoplasmic reticulum (ER) or the mitochondrial outer membrane (MOM), respectively. To bridge the gap between the cell-free and in cellula results, we analyzed targeting in digitonin-permeabilized adherent HeLa cells. In the absence of cytosol, the MOM was the destination of both b5 forms, whereas in cytosol the C-terminal negative charge of b5-ER determined targeting to the ER. Inhibition of the transmembrane recognition complex (TRC) pathway only partially reduced b5 targeting, while strongly affecting the classical TRC substrate synaptobrevin 2 (Syb2). To identify additional pathways, we tested a number of small inhibitors, and found that Eeyarestatin I (ES ) reduced insertion of b5-ER and of another spontaneously inserting TA protein, while not affecting Syb2. The effect was independent from the known targets of ES , Sec61 and p97/VCP. Our results demonstrate that the MOM is the preferred destination of spontaneously inserting TA proteins, regardless of their C-terminal charge, and reveal a novel, substrate-specific ER-targeting pathway.

A trap mutant reveals the physiological client spectrum of TRC40

“…S7" type="url"/>). Taken together, these results are consistent with the notion that mitochondrial TA proteins do not use the TRC pathway (Figueiredo Costa et al, 2018). Hence, the effect on Fis1 steady-state levels may relate to another function of TRC40.…”

“…Hence, it is necessary to carefully distinguish which TA proteins can use the TRC pathway from the precursors for which it is essential. While the existing tools such as in vitro assays and knockout cells or organisms have begun to provide answers to these questions (Casson et al, 2017; Figueiredo Costa et al, 2018; Guna et al, 2018; Haßdenteufel et al, 2017; Lin et al, 2016; Norlin et al, 2016, 2018; Pfaff et al, 2016; Rivera-Monroy et al, 2016; Vogl et al, 2016), an approach is still lacking that would reveal which TA proteins do in fact use the TRC pathway when all targeting options are operational (i.e. without the compensatory effects often provoked by knockout strategies).…”

Rare BANF1 Alleles and Relatively Frequent EMD Alleles Including ‘Healthy Lipid’ Emerin p.D149H in the ExAC Cohort

“…ExAC variants in this upstream region were rare (four unique variants between residues 158–193; Supplementary Table S2). Specific TRC40-dependent targeting to the ER/NE (rather than mitochondria or peroxisomes) requires negatively charged residues on the lumenal C-terminus (Costello et al, 2017; Figueiredo Costa et al, 2018). Emerin has two such residues, one of which is neutralized by variant p.E249G, identified in three individuals in ExAC (Figure 2).…”

Eeyarestatin Compounds Selectively Enhance Sec61-Mediated Ca2+ Leakage from the Endoplasmic Reticulum

“…Supporting this idea, it has been shown that ES1 is particularly cytotoxic against cancer cells when combined with proteasome inhibitors ( Wang et al., 2009 , Auner et al., 2013 ). Additional mechanisms that potentially contribute to the cytotoxic effects of ES1 are the impairment of both intracellular vesicular transport and membrane insertion of tail-anchored proteins ( Aletrari et al., 2011 , Naydenov et al., 2012 , Figueiredo Costa et al., 2018 ). Independently of targeting the ER-cytosol dislocation of proteins, ES1 also inhibits the co-translational translocation of nascent polypeptides into the ER, most likely by inhibiting Sec61 complexes both in cellula and in vitro ( Cross et al., 2009a ).…”