The mechanism underlying perinuclear calcium spiking induced during legume root endosymbioses is largely unknown. Lotus japonicus symbiosis-defective castor and pollux mutants are impaired in perinuclear calcium spiking. Homology modeling suggested that the related proteins CASTOR and POLLUX might be ion channels. Here, we show that CASTOR and POLLUX form two independent homocomplexes in planta. CASTOR reconstituted in planar lipid bilayers exhibited ion channel activity, and the channel characteristics were altered in a symbiosis-defective mutant carrying an amino acid replacement close to the selectivity filter. Permeability ratio determination and competition experiments reveled a weak preference of CASTOR for cations such as potassium over anions. POLLUX has an identical selectivity filter region and complemented a potassium transport-deficient yeast mutant, suggesting that POLLUX is also a potassium-permeable channel. Immunogold labeling localized the endogenous CASTOR protein to the nuclear envelope of Lotus root cells. Our data are consistent with a role of CASTOR and POLLUX in modulating the nuclear envelope membrane potential. They could either trigger the opening of calcium release channels or compensate the charge release during the calcium efflux as counter ion channels.

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...

␤-Barrel-shaped channels of the Omp85 family are involved in the translocation or assembly of proteins of bacterial, mitochondrial, and plastidic outer membranes. We have compared these proteins to understand the evolutionary development of the translocators. We have demonstrated that the proteins from proteobacteria and mitochondria have a pore diameter that is at least five times smaller than found for the Omp85 in cyanobacteria and plastids. This finding can explain why Omp85 from cyanobacteria (but not the homologous protein from proteobacteria) was remodeled to become the protein translocation pore after endosymbiosis. Further, the pore-forming region of the Omp85 proteins is restricted to the C terminus. Based on a phylogenetic analysis we have shown that the pore-forming domain displays a different evolutionary relationship than the N-terminal domain. In line with this, the affinity of the N-terminal domain to the C-terminal region of the Omp85 from plastids and cyanobacteria differs, even though the N-terminal domain is involved in gating of the pore in both groups. We have further shown that the N-terminal domain of nOmp85 takes part in homo-oligomerization. Thereby, the differences in the phylogeny of the two domains are explained by different functional constraints acting on the regions. The poreforming domain, however, is further divided into two functional regions, where the distal C terminus itself forms a dimeric pore. Based on functional and phylogenetic analysis, we suggest an evolutionary scenario that explains the origin of the contemporary translocon.Polypeptide transport and assembly of proteins into or across the outer membrane of endosymbiotic organelles or Gram-negative bacteria depend on ␤-barrel-shaped channels (1-4). One class of these proteins is composed of polypeptide-transporting ␤-barrel (PTB) 6 channels, whose topology was determined by modeling (5-7). PTBs of recent interest are, e.g. outer membrane proteins (which secrete adhesins such as hemagglutinin) (8, 9) and bacterial (1, 7, 10 -12), mitochondrial (Tob55/Sam50) (5, 13, 14), and chloroplast outer membrane proteins (Toc75) (15) of the Omp85 family. The PTBs are partitioned into two functional categories, namely in translocation of precursor proteins across the membrane and in the assembly of outer membrane proteins (3). Furthermore, comparison between chloroplastic, mitochondrial, and bacterial Omp85 protein sequences revealed a high similarity of these PTBs (14,16,17).The PTB Toc75 forms a complex with Toc34, Toc64, and Toc159 (3). A precursor protein-binding site at Toc75 (15, 18), together with the action of Toc159 (19), facilitates the translocation of precursor proteins across the membrane. In contrast, the Omp85 proteins from Neisseria meningitidis, Escherichia coli, and mitochondria are involved in the assembly of outer membrane proteins (1,5,7,(11)(12)(13)(14). As found for Toc75, the mitochondrial PTB is a component of a larger complex with Mas37 (20, 13) and Tob38/Sam35 (21,22).Recently, it was demonstrated that t...

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.

Transport of solutes or macromolecules such as proteins across membranes requires a proteinaceous channel or transporter. Besides their way of action, these proteins can be divided according to their substrates or to their secondary structure of the membrane domain. In terms of secondary structure a-helical or b-sheet channels can be differentiated [1]. Both types of channels show a high neighbourhood correlation according to the fold [2] suggesting similar folds of the membrane inserted domains. In the past, much attention was given to the a-helical channels [3-5]. However, recently ion channels formed by the b-sheets moved into the focus of interest [6,7]. While analyzing these channels it became obvious that they emerged from outer membrane proteins of prokaryotic endo-symbionts, as these proteins were the only b-barrel type membrane proteins found in bacteria [6]. This class of proteins is present in organellar membranes of eukaryotic organisms, like in the outer mitochondrial membrane [8] emerged from a-proteobacteria [9], in the outer envelope of chloroplasts [10,11] emerged from cyanobacteria [9] and maybe even in the peroxi-somal membrane [12]. The peroxisomal b-barrel protein might be an indication either of the discussed endosymbiotic origin (for example [13]) or of a redistribution of proteins within the cell as a result of the gene transfer of the other two endosymbiotic events [14]. Most of the b-barrel type channels of eukaryotes belong to the porin type family. Recent research revealed that b-barrel type channels are also involved in the translocation of polypeptides [15], in the assembly of proteins in the outer membrane of endosymbio-tic organelles [16-19] or in the assembly of proteins in the outer membrane of bacteria [7,20,21]. One polypeptide-transporter that was found in Bordetella pertussis is FhaC, which secretes the main Transport of solutes and polypeptides across membranes is an essential process for every cell. In the past, much focus has been placed on helical transporters. Recently, the b-barrel-shaped transporters have also attracted some attention. The members of this family are found in the outer bacterial membrane and the outer membrane of endosymbiotically derived organ-elles. Here we analyze the features and the evolutionary development of a specified translocator family, namely the b-barrel-shaped polypeptide-transporters. We identified sequence motifs, which characterize all transporters of this family, as well as motifs specific for a certain subgroup of proteins of this class. The general motifs are related to the structural composition of the pores. Further analysis revealed a defined distance of two motifs to the C-terminal portion of the proteins. Furthermore, the evolutionary relationship of the proteins and the motifs are discussed. Abbreviation EBS, exact b-sheet.