The Evolutionarily Related β-Barrel Polypeptide Transporters from Pisum sativum and Nostoc PCC7120 Contain Two Distinct Functional Domains

Abstract

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...

Chloroplast Omp85 proteins change orientation during evolution

“…Interestingly, an interaction between the POTRA domains of the pea Toc75-III and the in vitro translated pea Toc33 G domain was observed (30). This could indicate either a Toc33-dependent import pathway of Toc75-III or an interaction within the TOC complex, which would then require a reversed topology, contrary to what would be predicted.…”

“…Thus, the available data are contradictory concerning the topology of the chloroplast Omp85 proteins. Because the POTRA domains provide a specific binding site for chloroplast preproteins (30) and display a flexible interface for protein-protein interaction as shown by MD simulations based on the crystal structure of the POTRA domains of alr2269, the cyanobacterial ancestor of Toc75-III (6), both possible topologies are reasonable. The importance of solving this question for the understanding of TOC function becomes apparent when the potential function of the POTRA domains during the translocation event is considered.…”

Relative Orientation of POTRA Domains from Cyanobacterial Omp85 Studied by Pulsed EPR Spectroscopy

“…PCC 7120 (hereafter, Anabaena sp. (15)). They are considered to be ancestors of the translocation pore of the translocase of the outer envelope membrane of the chloroplast (TOC (16)).…”

“…contains three open reading frames coding for distinct Omp85 proteins (17), the one encoded by alr2269 (hereafter referred to as anaOmp85) is considered the most abundant Omp85 protein in the outer membrane (18) and is the subject of this study. anaOmp85 contains three POTRA domains (anaP1, anaP2, and anaP3), which were found to regulate the pore gating of the b-barrel (5,15,19). In contrast to BamA, anaOmp85 contains an N-terminal proline-rich region of~200 amino acids that precedes the POTRA domains and for which structural information does not exist (19).…”

“…A mutant of the Omp85 protein in the sorting and assembly machinery (SAM, also referred to as topogenesis of mitochondrial outer membrane b-barrel proteins (20)), Sam50, which lacks the POTRA domain, is viable (21), although it is said that this domain is involved in substrate release from the SAM complex (22). Similarly, the POTRA domain of Omp85 in the TOC complex, Toc75, recognizes the transit peptide of the chloroplast proteins that have to be translocated across the outer envelope and also interacts with another complex component, the receptor Toc34 (15). However, in Arabidopsis thaliana, two Omp85 homologs without the POTRA domain have been described experimentally (23,24), which is in line with the notion that Omp85 with either alternative or without soluble domains has evolved to perform alternative functions (25).…”