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 mitochondrial outer membrane contains machinery for the import of preproteins encoded by nuclear genes. Eight different Tom (translocase of outer membrane) proteins have been identified that function as receptors and/or are related to a hypothetical general import pore. Many mitochondrial membrane channel activities have been described, including one related to Tim23 of the inner-membrane protein-import system; however, the pore-forming subunit(s) of the Tom machinery have not been identified until now. Here we describe the expression and functional reconstitution of Tom40, an integral membrane protein with mainly beta-sheet structure. Tom40 forms a cation-selective high-conductance channel that specifically binds to and transports mitochondrial-targeting sequences added to the cis side of the membrane. We conclude that Tom40 is the pore-forming subunit of the mitochondrial general import pore and that it constitutes a hydrophilic, approximately 22 A wide channel for the import of preproteins.

We report the identification, functional expression, purification, reconstitution and electrophysiological characterization of an up to now unique prokaryotic potassium ion channel (KcsA

Mitochondria import hundreds of different precursor proteins from the cytosol. More than 50% of mitochondrial proteins do not use the classical import pathway that is guided by amino-terminal presequences, but instead contain different types of internal targeting signals. Recent studies have revealed an unexpected complexity of the mitochondrial protein import machinery and have led to the discovery of new transport pathways. Here, we review the versatility of mitochondrial protein import and its connection to mitochondrial morphology, redox regulation and energetics.

The mitochondrial inner membrane imports numerous proteins that span it multiple times using the membrane potential Deltapsi as the only external energy source. We purified the protein insertion complex (TIM22 complex), a twin-pore translocase that mediated the insertion of precursor proteins in a three-step process. After the precursor is tethered to the translocase without losing energy from the Deltapsi, two energy-requiring steps were needed. First, Deltapsi acted on the precursor protein and promoted its docking in the translocase complex. Then, Deltapsi and an internal signal peptide together induced rapid gating transitions in one pore and closing of the other pore and drove membrane insertion to completion. Thus, protein insertion was driven by the coordinated action of a twin-pore complex in two voltage-dependent steps.

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.

Aggregation of ␣-synuclein is a key event in several neurodegenerative diseases, including Parkinson disease. Recent findings suggest that oligomers represent the principal toxic aggregate species. Using confocal single-molecule fluorescence techniques, such as scanning for intensely fluorescent targets (SIFT) and atomic force microscopy, we monitored ␣-synuclein oligomer formation at the single particle level. Organic solvents were used to trigger aggregation, which resulted in small oligomers ("intermediate I"). Under these conditions, Fe 3؉ at low micromolar concentrations dramatically increased aggregation and induced formation of larger oligomers ("intermediate II"). Both oligomer species were on-pathway to amyloid fibrils and could seed amyloid formation. Notably, only Fe 3؉ -induced oligomers were SDS-resistant and could form ion-permeable pores in a planar lipid bilayer, which were inhibited by the oligomerspecific A11 antibody. Moreover, baicalein and N-benzylidenebenzohydrazide derivatives inhibited oligomer formation. Baicalein also inhibited ␣-synuclein-dependent toxicity in neuronal cells. Our results may provide a potential disease mechanism regarding the role of ferric iron and of toxic oligomer species in Parkinson diseases. Moreover, scanning for intensely fluorescent targets allows high throughput screening for aggregation inhibitors and may provide new approaches for drug development and therapy.

The chloroplastic outer envelope protein OEP75 with a molecular weight of 75 kDa probably forms the central pore of the protein import machinery of the outer chloroplastic membrane. Patch-clamp analysis shows that heterologously expressed, purified and reconstituted OEP75 constitutes a voltage-gated ion channel with a unit conductance of Λ ϭ 145pS. Activation of the OEP75 channel in vitro is completely dependent on the magnitude and direction of the voltage gradient. Therefore, movements of protein charges of parts of OEP75 in the membrane electric field are required either for pore formation or its opening. In the presence of precursor protein from only one side of the bilayer, strong flickering and partial closing of the channel was observed, indicating a specific interaction of the precursor with OEP75. The comparatively low ionic conductance of OEP75 is compatible with a rather narrow aqueous pore (d pore ≅ 8-9 Å). Provided that protein and ion translocation occur through the same pore, this implies that the environment of the polypeptide during the transit is mainly hydrophilic and that protein translocation requires almost complete unfolding of the precursor.