Mitochondrial-bacterial hybrids of BamA/Tob55 suggest variable requirements for the membrane integration of β-barrel proteins

Abstract

β-Barrel proteins are found in the outer membrane (OM) of Gram-negative bacteria, chloroplasts and mitochondria. The assembly of these proteins into the corresponding OM is facilitated by a dedicated protein complex that contains a central conserved β-barrel protein termed BamA in bacteria and Tob55/Sam50 in mitochondria. BamA and Tob55 consist of a membrane-integral C-terminal domain that forms a β-barrel pore and a soluble N-terminal portion comprised of one (in Tob55) or five (in BamA) polypeptide transport-associated (POTRA) domains. Currently the functional significance of this difference and whether the homology between BamA and Tob55 can allow them to replace each other are unclear. To address these issues we constructed hybrid Tob55/BamA proteins with differently configured N-terminal POTRA domains. We observed that constructs harboring a heterologous C-terminal domain could not functionally replace the bacterial BamA or the mitochondrial Tob55 demonstrating species-specific requirements. Interestingly, the various hybrid proteins in combination with the bacterial chaperones Skp or SurA supported to a variable extent the assembly of bacterial β-barrel proteins into the mitochondrial OM. Collectively, our findings suggest that the membrane assembly of various β-barrel proteins depends to a different extent on POTRA domains and periplasmic chaperones.

Effects of Periplasmic Chaperones and Membrane Thickness on BamA-Catalyzed Outer-Membrane Protein Folding

“…This is consistent with recent kinetic simulations showing that when Skp foldase rates are set to be equivalent to those mediated by SurA, the experimentally observed severity of the ∆ surA phenotype is not reproduced [20] . Interestingly, a recent study in yeast demonstrated that expression of E. coli Skp in the mitochondrial intermembrane space assists the assembly of some E. coli OMPs (OmpX, PhoE) into the mitochondrial OM, but not others (OmpA) [54] , consistent with the view that chaperones and BAM components perform different roles in OMP assembly, depending on substrate [55] .…”

Membrane protein insertion through a mitochondrial β-barrel gate

“…The large diversity of bacterial β-barrel proteins and the involvement of multiple POTRA domains and accessory Bam proteins (5, 15, 51, 60) raise the possibility that additional precursor-specific folding pathways may complement the central mechanism of β-signal exchange and sorting via the lateral gate elucidated here. For example assembly of oligomeric β-barrels in bacteria might be stalled at the BAM complex until all subunits are assembled (65), similar to the arrest of shortened precursor constructs of monomeric β-barrels (Fig.…”

“…The hidden-Markov model based homology search identified templates in the PDB with good p- and E-values. This included structures from FhaC [PDB code: 4QL0 (51)] and TamA [PDB code: 4C00 (18)] as well as several structures from BamA [PDB codes: 4K3B and 4K3C (16); 4C4V (21); 4N75 (22) and 5EKQ (23)], which exhibit considerable variations in the interaction between the β1 and β16-strands. A Sam50 model was calculated from each template using Modeller (72).…”

“…In addition to the β-barrel channel, Sam50 possesses two major characteristic elements, an N-terminal polypeptide transport associated (POTRA) domain exposed to the intermembrane space and a highly conserved loop 6 that extends from the cytosolic side of the β-barrel. (i) Whereas bacterial BamA proteins contain several POTRA domains that interact with β-barrel precursors and are crucial for precursor transfer from the periplasm into the outer membrane (17, 46–49), Sam50 contains a single POTRA domain that is not essential for cell viability (13, 50, 51). Disulfide formation between the Por1 precursor and Sam50 β-strands 1 and 16 was not blocked in mitochondria lacking the entire POTRA domain (fig.…”

Landscape of Eukaryotic Transmembrane Beta Barrel Proteins

“…TOM40 and SAM50 together have representatives in 97.93 % of the studied proteomes (1042 out of 1064 proteomes) and this reflects their crucial biological role in the translocation of OMPs into the MOM in all eukaryotes. This process begins with the translation of mitochondrial OMPs in cytosolic ribosomes and then follows their import into the target organelles 61,62,97 . Mitochondrial proteins reach the mitochondrial surface and then are translocated into the intermembrane space across the outer membrane via the translocase of the MOM (TOM) complex 45,98 .…”