Precursor protein targeting toward organellar surfaces is assisted by different cytosolic chaperones. We demonstrate that the chloroplast protein translocon subunit Toc64 is the docking site for Hsp90 affiliated preproteins. Thereby, Hsp90 is recognised by the clamp type TPR domain of Toc64. The subsequent transfer of the preprotein from Toc64 to the major receptor of the Toc complex, namely Toc34, is affinity driven and nucleotide dependent. We propose that Toc64 acts as an initial docking site for Hsp90 associated precursor proteins. We outline a mechanism in which chaperones are recruited for a specific targeting event by a membrane-inserted receptor.

In the postgenomic era, the transformation of genetic information into biochemical meaning is required. We have analyzed the proteome of the chloroplast outer envelope membrane by an in silico and a proteomic approach. Based on its evolutionary relation to the outer membrane of Gram-negative bacteria, the outer envelope membrane should contain a large number of ␤-barrel proteins. We therefore calculated the probability for the existence of ␤-sheet, ␤-barrel, and hairpin structures among all proteins of the Arabidopsis thaliana genome. According to the existence of these structures, a number of candidates were selected. This protein pool was analyzed by TargetP to discard sequences with signals that would direct the protein to other organelles different from chloroplasts. In addition, the pool was manually controlled for the presence of proteins known to function outside of the chloroplast envelope. The approach developed here can be used to predict the topology of ␤-barrel proteins. For the proteomic approach, proteins of highly purified outer envelope membranes of chloroplasts from Pisum sativum were analyzed by ESI-MS/MS mass spectrometry. In addition to the known components, four new proteins of the outer envelope membranes were identified in this study.

Several ␤-barrel-type channels are involved in the translocation or assembly of outer membrane proteins of bacteria or endosymbiotically derived organelles. Here we analyzed the functional units of the ␤-barrel polypeptide transporter Toc75 (translocon in outer envelope of chloroplasts) of the outer envelope of chloroplasts and of a protein, alr2269, from Nostoc PCC7120 with homology to Toc75, both proteins having a similar domain organization. We demonstrated that the N-terminal region functions as a recognition and complex assembly unit, whereas the C terminus forms the ␤-barrel-type pore. The pore region is, in turn, modulated by the N terminus of the proteins. The protein from Nostoc PCC7120, which shares a common ancestor with Toc75, is able to recognize precursor proteins destined for chloroplasts. In contrast, the recognition of peripheral translocon subunits by Toc75 is a novel feature acquired through evolution.␤-barrel-type channels are involved in the translocation of polypeptides (1), the assembly of proteins in the outer membrane of endosymbiotic organelles (2-4), or in the assembly of proteins in the outer membrane of bacteria (5, 6). These proteins belong to one class, which can be termed polypeptide-transporting ␤-barrel channels (2, 4, 7). Four proteins are in the focus of recent investigation, namely the bacterial outer membrane proteins Omp85 and ShlB, the mitochondrial outer membrane protein Tob55/Sam50, and the chloroplast outer envelope protein Toc75.ShlB is an outer membrane protein involved in the secretion of hemolysins or adhesins in various Gram-negative pathogens (8, 9). Omp85 is an essential component for outer membrane biogenesis in Neisseria meningitidis that might have two functions: the assembly of outer membrane proteins (5) and the translocation of lipids (10). Recently, it was discussed that the effect on lipid transfer by Omp85 depletion might be indirect and explained by an assembly defect of the required outer membrane protein, suggesting a function of Omp85 in outer membrane protein assembly only (11). As for ShlB, a ␤-barrel transmembrane structure was suggested for Omp85 (5). Recently, a new polypeptide-transporting protein was identified in the outer membrane of mitochondria and termed Sam50 (3), Tob55 (2), or mitochondrial Omp85 homologue (4). This protein facilitates the assembly of proteins into the outer membrane of mitochondria. Tob55/Sam50 is found in a larger complex with Mas37 (3, 12) and Tob38/Sam35 (13-15).The fourth investigated ␤-barrel-type polypeptide transporter is the 75-kDa subunit of the translocon of the outer envelope of chloroplasts, Toc75. Toc75 forms a complex with Toc34, Toc64, and Toc159 (16). In contrast to the other identified polypeptide transporters, such as Omp85, the translocation of proteins through Toc75 requires the action of assisting proteins, such as Toc159 (17), but still Toc75 seems to contain a preprotein-binding site as determined by electrophysiological measurements (1). Topological modeling of Toc75 from Pisum sativum (18) or T...

Background: Different iron transport systems evolved in Gram-negative bacteria during evolution. Most of the transport systems depend on outer membrane localized TonB-dependent transporters (TBDTs), a periplasma-facing TonB protein and a plasma membrane localized machinery (ExbBD). So far, iron chelators (siderophores), oligosaccharides and polypeptides have been identified as substrates of TBDTs. For iron transport, three uptake systems are defined: the lactoferrin/transferrin binding proteins, the porphyrin-dependent transporters and the siderophore-dependent transporters. However, for cyanobacteria almost nothing is known about possible TonB-dependent uptake systems for iron or other substrates.

Anabaena is a model to analyze the evolutionary development of plastids, cell differentiation, and the regulation of nitrogen fixation. Thereby, the outer membrane proteome is the place of sensing environmental differences and during plastid development, systems for intracellular communication had to be added to the proteome of this membrane. We present a protocol for the isolation of the outer membrane from Anabaena and the analysis of the proteome using different tools. 55 proteins were identified.