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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 (ESI) 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 ESI, 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. [ABSTRACT FROM AUTHOR]

C-tail-anchored (TA) proteins often require a transmembrane domain of moderate hydrophobicity to maintain their endoplasmic reticulum residence, but the suggested role of protein-lipid interactions in this phenomenon has not been established. Here, we studied the interaction of TA proteins with lipids by differential scanning calorimetry by using a model system consisting of liposomes embedding either of two forms of cytochrome b5: the endoplasmic reticulum-resident wild-type (b5wt) and a mutant thereof (b5ext), that contains five extra nonpolar amino acids in its transmembrane domain and, therefore, reaches the plasma membrane. The proteins were incorporated into liposomes of palmitoyloleyl-phosphatidyicholine (POPC) or POPC mixed with either distearoyl-phosphatidylseri ne (DSPS), palmitoyl-oleyl-phosphatidylserine (POPS), distearoyl-phosphatidylcholine (DSPC), or C16- ceramide (CER). POPC liposomes displayed a single thermotropic transition centered at -3.4°C. When present the second lipid formed a domain within the POPC bilayer, as indicated by the appearance of an additional peak. This peak was centered at temperatures close to 0°C in the case of liposomes containing 10% CER, DSPS, and POPS and at 23°C in the case of DSPC, likely reflecting a higher degree of molecular packing for DSPC domains. In DSPS/POPC, POPS/POPC, or CER/POPC, but not in DSPC/POPC liposomes, the insertion of b5wt increased, whereas b5ext decreased, the relative contribution to the total enthalpy of the higher temperature, phase-separated component. These results were confirmed with fluorescence measurements by using pyrene-labeled phospholipids. The dissimilar interaction with lipids of these two differently localized TA proteins could have implications for their intracellular sorting. [ABSTRACT FROM AUTHOR]

Tail-anchored (TA) proteins are membrane proteins that contain an N-terminal domain exposed to the cytosol and a single transmembrane segment near the C-terminus followed by few or no polar residues. TA proteins with a mildly hydrophobic transmembrane domain, such as cytochrome b5 (b5), are able to insert post-translationally into pure lipid vesicles without assistance from membrane proteins. Here, we investigated whether any cytosolic proteins are needed to maintain b5 in a competent state for transmembrane integration. Using b5 constructs translated in vitro or produced in bacteria, we demonstrate that cytosolic proteins are neither necessary nor facilitatory for the unassisted translocation of b5. Furthermore, we demonstrate that no cytosolic protein is involved in the translocation of a C-terminal domain of 85 residues appended to the transmembrane domain of b5. Nevertheless, b5 does bind cytosolic proteins, and in their presence but not in their absence, its insertion into liposomes is inhibited by the thiol oxidant diamide and the alkylating agent N-ethylmaleimide. The effect of diamide is also observed in living cells. Thus, the specific in vivo targeting of b5 might be achieved by interaction with redox-sensitive targeting factors that hinder its nonspecific insertion into any permissive bilayer. [ABSTRACT FROM AUTHOR]