Mechanism of precise intracellular targeting of tail-anchored proteins

Tail-anchored (TA) proteins are a subclass of type II integral membrane proteins that carry out important and diverse functions within cells. Many TA proteins, i.e., those targeted in vivo to the mitochondrial outer membrane (MOM) and a certain proportion of endoplasmic reticulum (ER) targeted ones, can insert into pure phospholipid bilayers without assistance from any chaperone. Cytochrome b5 is a spontaneously inserted TA protein, of which two forms are known: one targeting the ER (b5-ER) and other targeting the MOM (b5-RR). Microinjection of the recombinant proteins results in faithful targeting of each of the two proteins, indicating that the targeting information is present in the protein and not in the mRNA. Now, using digitonin semi-permeabilized cells and in presence of rabbit reticulocyte lysate (RRL) 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 b5ER, confirmed by the reduction of endoplasmic reticulum colocalization. Thus, energy-dependent chaperones are required for b5ER’s avoidance of the MOM and its specific targeting to the ER. Taking this into consideration, we have started now testing the role of the energy-dependent TRC40 system on b5ER targeting, by adding the coiled-coil domain of WRB (ER membrane receptor of TRC40). So far, our results tell us that WRBcc causes a minor shift in the insertion from ER to MOM, suggesting a modest role of this chaperone in the post-translational delivery of b5-ER to the ER membrane. We are presently investigating the role of other factors in b5 targeting, including chaperones of the Hsp70 family. Although this study is being carried out on model proteins b5-ER and b5-RR, it could be relevant for other physiologically crucial proteins, such as those of the Bcl2 family. Thus, one could speculate that under different conditions, or in different tissues, the availability of different targeting factors could result in different subcellular distribution of these proteins, with different effects on cell physiology.