Communication of mitochondria with the rest of the cell requires beta-barrel proteins of the outer membrane. All beta-barrel proteins are synthesized as precursors in the cytosol and imported into mitochondria by the general translocase TOM and the sorting machinery SAM. The SAM complex contains two proteins essential for cell viability, the channel-forming Sam50 and Sam35. We have identified the sorting signal of mitochondrial beta-barrel proteins that is universal in all eukaryotic kingdoms. The beta-signal initiates precursor insertion into a hydrophilic, proteinaceous membrane environment by forming a ternary complex with Sam35 and Sam50. Sam35 recognizes the beta-signal, inducing a major conductance increase of the Sam50 channel. Subsequent precursor release from SAM is coupled to integration into the lipid phase. We propose that a two-stage mechanism of signal-driven insertion into a membrane protein complex and subsequent integration into the lipid phase may represent a general mechanism for biogenesis of beta-barrel proteins.

The peroxisomal protein import machinery differs fundamentally from known translocons (endoplasmic reticulum, mitochondria, chloroplasts, bacteria) as it allows membrane passage of folded, even oligomerized proteins. However, the mechanistic principles of protein translocation across the peroxisomal membrane remain unknown. There are various models that consider membrane invagination events, vesicle fusion or the existence of large import pores. Current data show that a proteinaceous peroxisomal importomer enables docking of the cytosolic cargo-loaded receptors, cargo translocation and receptor recycling. Remarkably, the cycling import receptor Pex5p changes its topology from a soluble cytosolic form to an integral membrane-bound form. According to the transient pore hypothesis, the membrane-bound receptor is proposed to form the core component of the peroxisomal import pore. Here, we demonstrate that the membrane-associated import receptor Pex5p together with its docking partner Pex14p forms a gated ion-conducting channel which can be opened to a diameter of about 9 nm by the cytosolic receptor-cargo complex. The newly identified pore shows striking dynamics, as expected for an import machinery translocating proteins of variable sizes.

The Mim1 complex imports α-helical mitochondrial outer membrane proteins with multiple transmembrane segments.