Biogenesi di membrane : un nuovo meccanismo di inserimento di proteine nel doppio strato lipidico

Membrane proteins are generally inserted into biological membranes with the assistance of dedicated complexes. We demonstrated the ability of unassisted integration for the eukaryotic protein cytochrome b5 (b5), which inserts into protein-free liposomes, provided that these have low cholesterol content. b5 belongs to the functionally important group of Tail-Anchored (TA) proteins, that are anchored to intracellular membranes by a single transmembrane domain (TMD) close to the C-terminus. We also investigated the limits of unassisted translocation, and report that surprisingly long (up to 85 residues) polar domains, placed downstream to b(5)'s TMD, can efficiently translocate across protein-free bilayers in the near absence of energy.Unassisted translocation was not a unique property of b(5),but shared with another TA protein (PTP1B) which, like b(5), has a moderately hydrophobic TMD. In contrast, more hydrophobic TMDs, like the one of the TA protein synaptobrevin-2 (Syb2), were unable to support translocation of even a short sequence across protein-free liposomes;these TMD-bearing constructs showed the same requirements for energy and for an ER trypsin-sensitive component as reported for native Syb2. Our data solve long-standing discrepancies on TA protein biogenesis and show that unexpectedly large lumenal domains, downstream to a suitable TMD, can translocate across a lipid bilayer without assistance from ER proteins.