Protein secretion and outer membrane assembly in Alphaproteobacteria

Abstract

The assembly of beta-barrel proteins into membranes is a fundamental process that is essential in Gram-negative bacteria, mitochondria and plastids. Our understanding of the mechanism of beta-barrel assembly is progressing from studies carried out in Escherichia coli and Neisseria meningitidis. Comparative sequence analysis suggests that while many components mediating beta-barrel protein assembly are conserved in all groups of bacteria with outer membranes, some components are notably absent. The Alphaproteobacteria in particular seem prone to gene loss and show the presence or absence of specific components mediating the assembly of beta-barrels: some components of the pathway appear to be missing from whole groups of bacteria (e.g. Skp, YfgL and NlpB), other proteins are conserved but are missing characteristic domains (e.g. SurA). This comparative analysis is also revealing important structural signatures that are vague unless multiple members from a protein family are considered as a group (e.g. tetratricopeptide repeat (TPR) motifs in YfiO, beta-propeller signatures in YfgL). Given that the process of the beta-barrel assembly is conserved, analysis of outer membrane biogenesis in Alphaproteobacteria, the bacterial group that gave rise to mitochondria, also promises insight into the assembly of beta-barrel proteins in eukaryotes.

Fig. 1

What is a β-barrel? (a) The polypeptide backbone of LamB from Escherichia coli (Wang et al., 1997) is traced to show the residues contributing to each β-strand (in squares) aligned to based on the hydrogen bond interactions between each strand. The unpaired hydrogen bond donors and acceptors in the first and last strand are highlighted by the dotted lines. (b) The structure of the assembled β-barrel membrane protein, represented as if in three dimensions, shows sequential β-strands (shown as colored arrows) form an antiparallel sheet that wraps into a cylinder: the final β-strand hydrogen bonds to the first strand to complete the barrel. Loops of polypeptide between the strands tend to be short on the periplasmic rim of the barrel, while longer loops are exposed to the extracellular face of the membrane. These longer loops are structured and can be folded back into the barrel lumen.

Fig. 2

In Escherichia coli, outer membrane proteins are synthesized in the cytoplasm as precursors with a signal sequence where they are recognized by the chaperones SecB and SecA (1). SecA assists translocation through the SecYEG complex in the inner membrane (2), and the signal sequence is processed by SP I (3). The substrate proteins are assisted across the periplasm by the chaperone SurA (4), and delivered to the BAM complex (5), to catalyse insertion into the outer membrane (6). Other chaperones, Skp and DegP, might cooperate to help ensure transfer to the outer membrane; DegP can also function as a protease to degrade misfolded outer membrane proteins in situations of environmental stress (Young & Hartl, 2003; Krojer et al., 2008).

Fig. 3

The BAM complexes in Escherichia coli and Brucella melitensis. The interactions between subunits of the BAM complex in E. coli are depicted. The POTRA domains are labelled numerically 1–5: POTRA domains 2, 3 and 4 are required to mediate interactions with YfgL (BamB=‘B’), while the fifth POTRA domain is crucial for interactions with YfiO (‘D’) and SmpA (‘E’), with BamD serving as the docking point for NlpB (‘C’) (Malinverni et al., 2006; Kim et al., 2007; Sklar et al., 2007b). In Brucella melitensis, the SurA chaperone is diminished lacking the PPIase domains, and BamB (YfgL) and BamC (NlpB) are lacking from the BAM complex.

Fig. 4

Sequence analyses of the Alphaproteobacteria suggest BamD (YfiO) has at least five TPR motifs and BamB (YfgL) could have a β-propeller structure. (a) BamD (YfiO) sequences from Alphaproteobacteria were analysed with three independent TPR prediction strategies (SMART, http://smart.embl-heidelberg.de/; HHpred, http://toolkit.tuebingen.mpg.de/hhpred; and TPRpred, http://toolkit.tuebingen.mpg.de/tprpred), revealing the presence of five TPR motifs in various Alphaproteobacteria, including Rickettsia, Caulobacter and Mesorhizobium. The TPR is a degenerate motif with very few strictly conserved positions – the position of the consensus residues G–Y–A–F–P are shown. Although TPR signatures are not clear in all BamD sequences (such as the one from Escherichia coli), clustalw aligns the BamD homologs readily and at least some of the key residues found in TPR motifs are evident in all species. The alignment corresponding to the first TPR motif (‘TPR1’) is shown. (b) BamB (YfgL) sequences from Alphaproteobacteria were analysed with SMART to determine the presence of seven or eight β-propeller motifs. Homology searching using HHpred suggests BamB from E. coli is most similar to proteins with an eight-bladed β-propeller fold. Each of the blades would interact via hydrophobic contacts (heavy black line) and the outer β-strand can make additional hydrogen bond contacts (red lines). BamB from E. coli can readily be modelled using six of the β-propeller structures in the Protein Data Bank (2AD6, 1YIQ, 1KB0, 1W6S, 1FLG and 1KV9) as template structures. The model structure of BamB is shown from two views. In this structural model the three mutations in BamB (L173, L175, and R176; the Cα atoms of each are highlighted as orange spheres), that cause defects for docking to the POTRA domains of BamA (Vuong et al., 2008), come together in one of the β-propeller motifs.

Fig. 5

Assembly pathways for β-barrel proteins in mitochondria and Alphaproteobacteria. In eukaryotes, β-barrel proteins are translated in the cytosol, transported across the mitochondrial outer membrane by the TOM complex and passed to the inner surface of the SAM complex for assembly. Passage through the intermembrane space depends on tiny TIM chaperones, such as the Tim9/10 complex (Pfanner et al., 2004; Paschen et al., 2005; Bolender et al., 2008). The final steps of assembly depend on the metaxins, Sam35 and Sam37 (‘35’ and ‘37’), on the outer face of the membrane (Pfanner et al., 2004; Paschen et al., 2005; Bolender et al., 2008; Chan & Lithgow, 2008). The Rickettsia are considered the closest living relatives to the progenitor of mitochondria, and use a BAM complex mechanism largely equivalent to that found in other bacteria.