B.Bölter and R.Hemmler contributed equally to this workPhosphorylated carbohydrates are the main photoassimilated export products from chloroplasts that support the energy household and metabolism of the plant cell. Channels formed by the chloroplastic outer envelope protein OEP21 selectively facilitate the translocation of triosephosphate, 3-phosphoglycerate and phosphate, central intermediates in the source-sink relationship between the chloroplast and the cytosol. The anion selectivity and asymmetric transport properties of OEP21 are modulated by the ratio between ATP and triosephosphates, 3-phosphoglycerate and phosphate in the intermembrane space. Conditions that lead to export of triosephosphate from chloroplasts, i.e. photosynthesis, result in outward-rectifying OEP21 channels, while a high ATP to triosephosphate ratio, e.g. dark metabolism, leads to inward-rectifying OEP21 channels with a less pronounced anion selectivity. We conclude that solute exchange between plastids and cytosol can already be regulated at the level of the organellar outer membrane.

The preprotein translocation at the inner envelope membrane of chloroplasts so far involves ®ve proteins: Tic110, Tic55, Tic40, Tic22 and Tic20. The molecular function of these proteins has not yet been established. Here, we demonstrate that Tic110 constitutes a central part of the preprotein translocation pore. Dependent on the presence of intact Tic110, radiolabelled preprotein speci®cally interacts with isolated inner envelope vesicles as well as with puri®ed, recombinant Tic110 reconstituted into liposomes. Circular dichroism analysis reveals that Tic110 consists mainly of b-sheets, a structure typically found in pore proteins. In planar lipid bilayers, recombinant Tic110 forms a cation-selective high-conductance channel with a calculated inner pore opening of 1.7 nm. Puri®ed transit peptide causes strong¯ickering and a voltage-dependent block of the channel. Moreover, at the inner envelope membrane, a peptide-sensitive channel is described that shows properties basically identical to the channel formed by recombinant Tic110. We conclude that Tic110 has a distinct preprotein binding site and functions as a preprotein translocation pore at the inner envelope membrane.

For the analysis of membrane transport processes two single molecule methods are available that differ profoundly in data acquisition principle, achievable information, and application range: the widely employed electrical single channel recording and the more recently established optical single transporter recording. In this study dense arrays of microscopic horizontal bilayer membranes between 0.8 microm and 50 microm in diameter were created in transparent foils containing either microholes or microcavities. Prototypic protein nanopores were formed in bilayer membranes by addition of Staphylococcus aureus alpha-hemolysin (alpha-HL). Microhole arrays were used to monitor the formation of bilayer membranes and single alpha-HL pores by confocal microscopy and electrical recording. Microcavity arrays were used to characterize the formation of bilayer membranes and the flux of fluorescent substrates and inorganic ions through single transporters by confocal microscopy. Thus, the unitary permeability of the alpha-HL pore was determined for calcein and Ca(2+) ions. The study paves the way for an amalgamation of electrical and optical single transporter recording. Electro-optical single transporter recording could provide so far unresolved kinetic data of a large number of cellular transporters, leading to an extension of the nanopore sensor approach to the single molecule analysis of peptide transport by translocases.

The flux of phosphorylated carbohydrates, the major export products of chloroplasts, is regulated at the level of the inner and presumably also at the level of the outer membrane. This is achieved through modulation of the outer membrane Oep21 channel currents and tuning of its ion selectivity. Refined analysis of the Oep21 channel properties by biochemical and electrophysiological methods revealed a channel formed by eight ␤-strands with a wider pore vestibule of d vest ϳ2.4 nm at the intermembrane site and a narrower filter pore of d restr ϳ1 nm. The Oep21 pore contains two high affinity sites for ATP, one located at a relative transmembrane electrical distance ␦ ‫؍‬ 0.56 and the second close to the vestibule at the intermembrane site. The ATP-dependent current block and reduction in anion selectivity of the Oep21 channel is relieved by the competitive binding of phosphorylated metabolic intermediates like 3-phosphoglycerate and glycerinaldehyde 3-phosphate. Deletion of a C-terminal putative FX 4 K binding motif in Oep21 decreased the capability of the channel to tune its ion selectivity by about 50%, whereas current block remained unchanged.Plastid organelles perform vital biosynthetic functions in every plant organ. They are surrounded by double membranes, the inner and the outer envelope, which delimit spatially and temporally the plastid compartment from the cytoplasm. Both envelope membranes are distinguishable by their structure, function, and biochemical properties but also cooperate, for example, in the synthesis of lipids or in protein translocation. Chloroplasts are the site of carbon dioxide reduction and its assimilation into carbohydrates, amino acids, fatty acids, and terpenoid compounds. The manifold biosynthetic functions of chloroplasts require the existence of different selective transport mechanisms across the envelope membranes to provide the cell with carbohydrates, organic nitrogen, and sulfur compounds (1, 2 Until recently, the outer envelope membrane was considered to be freely permeable for most small molecular mass solutes up to 10 kDa (1).Correspondingly, it was believed that the osmotic barrier against the cytosol is formed exclusively by the inner envelope membrane, containing specific carrier proteins, some of which have been identified on the functional and also on the molecular level (2). However, our recent reports reveal the presence of several specific solute pores in the outer envelope, indicating that the intermembrane space is not freely accessible to low molecular weight solutes (3-6).Whereas the inner envelope carrier proteins (e.g. the triose phosphate-phosphate translocator, the dicarboxylic acid translocator, and the hexose phosphate carrier) show a distinct substrate selectivity and specificity, it remains elusive to what extent the transport through these outer membrane channels is regulated (7). The ancestral relation between plastids and Gram-negative bacteria suggests the presence of different solute channel proteins in the organellar outer membrane. In pea chlor...

HepaChip-MP: a 24-culture-chamber, automated microfluidic in vitro model of the liver sinusoid in multiwellplate format.