NUOVE STRATEGIE DI STUDIO DELLA BIOGENESI DELLE MEMBRANE: IL PROBLEMA DEL CORRETTO 'TARGETING' POST-TRADUZIONALE

Tail-anchored (TA) proteins have a transmembrane domain near the C terminus and an N-terminal cytosolic moiety; because of this topology they do not have access to the SRP-mediated co-translational pathway for insertion into the ER, and can be integrated in their target membranes only posttranslationally. The TRC40/Get3 system is known to play an important role in post-translational targeting to the ER, but some TA proteins can integrate into lipid bilayers independently from this system, yet undergo precise targeting in vivo. The best studied example of this phenomenon is given by cytochrome b5 (b5), of which two isoforms, one targeting the ER and the other the outer mitochondrial membrane (MOM) have been characterized (b5 ER and b5 RR). Surprisingly, the in vivo targeting specificity of the two isoforms is not reproduced in vitro, inasmuch as both proteins are able to insert into protein-free liposomes in the absence of any chaperone, and neither of them shows a preference for ER or MOM-derived vesicles even when presented with both these membranes together. In this thesis I have set up new in vivo and in vitro systems to investigate the molecular basis of this mode of post-translational targeting. (i) The in vivo system consists in the microinjection into cultured cells of the two b5 recombinant proteins, under control or perturbed conditions, and the subsequent determination of their localization by immunofluorescence. At 15 minutes after the microinjection, each of the two isoforms is localized correctly to its respective organelle, indicating that no ribosomal factor is required for the targeting. RNAi and the use of dominant negative proteins confirmed the absence of involvement of TRC40/Get3. Instead, pharmacological inhibition demonstrated that other chaperones , such as Hsp90 , Hsp70, also calmodulin are involved, and that the two isoforms are affected differently by these cytosolic proteins. In addition, the physico-chemical properties of the membrane target are involved, since the cholesterol overload inhibits insertion. (ii) The in vitro system consists of cell semi- permeabilized by digitonin treatment. Surprsingly, in this system, in the absence of cytosol, or in the presence of a cytosolic fraction of reticulocytes, both proteins are inserted exclusively into the MOM; instead, in the presence of a cytosolic fraction derived from rat liver, b5 ER is partially localized also to the ER. My results indicate that one or more limiting chaperones are required for the correct targeting of b5 ER and that in the absence of these factors, the MOM is the preferred destination of both isoforms. These results have important implications for the Bcl-2 family of TA proteins, involved in the regulation of apoptosis, some of which have a dual ER-MOM localization.