Intracellular targeting of tail-anchored proteins

Tail-anchored (TA) proteins are integral membrane proteins that carry out important and diverse functions and that are targeted to their destination by unique post-translational pathways. Although the main pathway for targeting to the endoplasmic reticulum (ER) of TA proteins is represented by the TRC40/Get3 pathway, many TA proteins, i.e., those targeted in vivo to the mitochondrial outer membrane (MOM) and some ER targeted ones, can insert in vitro into pure phospholipid bilayers without assistance from any chaperone. The mechanism of precise in vivo targeting of these TA proteins is unclear. Cytochrome b5 is a spontaneously inserted TA protein, of which two forms are known, targeting the ER (b5-ER) or the MOM (b5-RR). The recombinant proteins microinjected into cultured cells are faithfully targeted, indicating that the targeting information is present in the protein and not in the mRNA. Using digitonin semi-permeabilized cells and in the presence of rabbit reticulocyte lysate as the source of cytosol, we have obtained faithful targeting for both forms of cytochrome b5 that approaches the in cellula situation. In contrast, in the absence of cytosol both forms target the mitochondria. We tested also the effects of energy depletion of the RRL and we observed an effect on b5-ER, confirmed by the reduction of ER localization. Thus, energy-dependent chaperones are required for b5-ER’s avoidance of the MOM and its specific targeting to the ER. Taking this into consideration, we investigated the role of the principal TA protein target TRC40 system on b5-ER targeting, by using two different approaches in our system: the coiled-coil domain of WRB (ER membrane receptor of TRC40), which act as a decoy receptor, and WRB silencing. These experiments demonstrated that TCR40 plays only a modest role in the post-translational delivery of b5-ER to the ER membrane, suggesting the contribution of redundant pathways to b5 biogenesis.