Chromalveolates are a diverse group of protists that include many ecologically and medically relevant organisms such as diatoms and apicomplexan parasites. They possess plastids generally surrounded by four membranes, which evolved by engulfment of a red alga. Today, most plastid proteins must be imported, but many aspects of protein import into complex plastids are still cryptic. In particular, how proteins cross the third outermost membrane has remained unexplained. We identified a protein in the third outermost membrane of the diatom Phaeodactylum tricornutum with properties comparable to those of the Omp85 family. We demonstrate that the targeting route of P. tricornutum Omp85 parallels that of the translocation channel of the outer envelope membrane of chloroplasts, Toc75. In addition, the electrophysiological properties are similar to those of the Omp85 proteins involved in protein translocation. This supports the hypothesis that P. tricornutum Omp85 is involved in precursor protein translocation, which would close a gap in the fundamental understanding of the evolutionary origin and function of protein import in secondary plastids.

Proteins of the Omp85 family are conserved in all kingdoms of life. They mediate protein transport across or protein insertion into membranes and reside in the outer membranes of Gramnegative bacteria, mitochondria, and chloroplasts. Omp85 proteins contain a C-terminal transmembrane ␤-barrel and a soluble N terminus with a varying number of polypeptide-transport-associated or POTRA domains. Here we investigate Omp85 from the cyanobacterium Anabaena sp. PCC 7120. The crystallographic three-dimensional structure of the N-terminal region shows three POTRA domains, here named P1 to P3 from the N terminus. Molecular dynamics simulations revealed a hinge between P1 and P2 but in contrast show that P2 and P3 are fixed in orientation. The P2-P3 arrangement is identical as seen for the POTRA domains from proteobacterial FhaC, suggesting this orientation is a conserved feature. Furthermore, we define interfaces for protein-protein interaction in P1 and P2. P3 possesses an extended loop unique to cyanobacteria and plantae, which influences pore properties as shown by deletion. It now becomes clear how variations in structure of individual POTRA domains, as well as the different number of POTRA domains with both rigid and flexible connections make the N termini of Omp85 proteins versatile adaptors for a plentitude of functions.Membrane proteins of the ␤-barrel type are pore proteins made up from a varying number of ␤-strands crossing the membrane. They are found exclusively in the outer membranes of bacteria, mitochondria, and chloroplasts (1). Specialized ␤-barrel proteins are involved in protein transport, called polypeptide-transporting ␤-barrel proteins (PTBs). 4 They can be divided into two classes according to their function (2-4). Class I PTBs are involved in transport of proteins from the periplasm to the extracellular space over the outer bacterial membrane. An example is the FhaC protein from Bordetella pertussis, belonging to the two-partner secretion system (4), which is required for transport of filamentous hemagglutinin. Class II proteins were initially discovered as surface-exposed antigens D15 and Oma87 from Haemophilus influenza (5, 6) and are now known to catalyze insertion of ␤-barrel proteins into the outer membrane (7-11). Analyzed members of this class include proteobacterial Omp85 proteins from Neisseria meningitidis (e.g. Refs. 12-14) and BamA from Escherichia coli (formerly named YaeT) (15-17), as well as Omp85 proteins from cyanobacteria like Synechocystis (18,19) and Anabaena (20 -22).All PTBs share a common structure with a C-terminal ␤-barrel domain, forming a membrane pore and a soluble N-terminal region. The N terminus contains a varying number of so-called polypeptide transport-associated or POTRA domains: class I PTBs have two, class II PTB one to six POTRA domains (Table 1) (15,23). The class I PTB FhaC from B. pertussis is known by crystallographic three-dimensional structure determination to comprise two POTRA domains and a 16-stranded ␤-barrel (24). For class II proteins, structural inf...

Gram-negative bacteria are surrounded by a cell wall including the outer membrane. The outer membrane is composed of two distinct monolayers where the outer layer contains lipopolysaccharides (LPS) with the non-phospholipid Lipid A as the core. The synthesis of Lipid A is initiated in the cytosol and thereby the molecule has to be transported across the inner and outer membranes. The β-barrel lipopolysaccharide-assembly protein D (LptD) was discovered to be involved in the transfer of Lipid A into the outer membrane of gram-negative bacteria. At present the molecular procedure of lipid transfer across the outer membrane remains unknown. Here we approached the functionality of the transfer system by an electrophysiological analysis of the outer membrane protein from Escherichia coli named ecLptD. In vitro the protein shows cation selectivity and has an estimated pore diameter of about 1.8 nm. Addition of Lipid A induces a transition of the open state to a sub-conductance state with two independent off-rates, which might suggest that LptD is able to bind and transport the molecule in vitro. To generalize our findings with respect to the Lipid A transport system of other gram-negative bacteria we have explored the existence of the proteins involved in this pathway by bioinformatic means. We were able to identify the membrane-inserted components of the Lipid A transport system in all gram-negative bacteria, whereas the periplasmic components appear to be species-specific. The LptD proteins of different bacteria are characterized by their periplasmic N-terminal domain and a C-terminal barrel region. The latter shows distinct sequence properties, particularly in LptD proteins of cyanobacteria, and this specific domain can be found in plant proteins as well. By electrophysiological experiments on LptD from Anabaena sp. PCC 7120 we are able to confirm the functional relation of anaLptD to Lipid A transport.

Proteins of the Omp85 family chaperone the membrane insertion of β-barrel-shaped outer membrane proteins in bacteria, mitochondria, and probably chloroplasts and facilitate the transfer of nuclear-encoded cytosolically synthesized preproteins across the outer envelope of chloroplasts. This protein family is characterized by N-terminal polypeptide transport-associated (POTRA) domains and a C-terminal membrane-embedded β-barrel. We have investigated a recently identified Omp85 family member of Arabidopsis thaliana annotated as P39. We show by in vitro and in vivo experiments that P39 is localized in chloroplasts. The electrophysiological properties of P39 are consistent with those of other Omp85 family members confirming the sequence based assignment of P39 to this family. Bioinformatic analysis showed that P39 lacks any POTRA domain, while a complete 16 stranded β-barrel including the highly conserved L6 loop is proposed. The electrophysiological properties are most comparable to Toc75-V, which is consistent with the phylogenetic clustering of P39 in the Toc75-V rather than the Toc75-III branch of the Omp85 family tree. Taken together P39 forms a pore with Omp85 family protein characteristics. The bioinformatic comparison of the pore region of Toc75-III, Toc75-V, and P39 shows distinctions of the barrel region most likely related to function. Proteins 2017; 85:1391-1401. © 2014 Wiley Periodicals, Inc.