Neisserial Omp85 Protein Is Selectively Recognized and Assembled into Functional Complexes in the Outer Membrane of Human Mitochondria

Abstract

As a consequence of their bacterial origin, mitochondria contain ␤-barrel proteins in their outer membrane (OMM). These proteins require the translocase of the outer membrane (TOM) complex and the conserved sorting and assembly machinery (SAM) complex for transport and integration into the OMM. The SAM complex and the ␤-barrel assembly machinery (BAM) required for biogenesis of ␤-barrel proteins in bacteria are evolutionarily related. Despite this homology, we show that bacterial ␤-barrel proteins are not universally recognized and integrated into the OMM of human mitochondria. Selectivity exists both at the level of the TOM and the SAM complex. Of all of the proteins we tested, human mitochondria imported only ␤-barrel proteins originating from Neisseria sp., and only Omp85, the central component of the neisserial BAM complex, integrated into the OMM. PorB proteins from different Neisseria, although imported by the TOM, were not recognized by the SAM complex and formed membrane complexes only when functional Omp85 was present at the same time in mitochondria. Omp85 alone was capable of integrating other bacterial ␤-barrel proteins in human mitochondria, but could not substitute for the function of its mitochondrial homolog Sam50. Thus, signals and machineries for transport and assembly of ␤-barrel proteins in bacteria and human mitochondria differ enough to allow only a certain type of ␤-barrel proteins to be targeted and integrated in mitochondrial membranes in human cells.Mitochondria are organelles of bacterial origin, surrounded by an outer (OMM) 4 and an inner membrane (IMM). The OMM contains ␤-barrel proteins, a class of pore-forming proteins additionally found only in chloroplasts and Gram-negative bacteria (1, 2). Similar to the majority of other mitochondrial proteins, ␤-barrel proteins are synthesized in the cytosol and have to be imported into mitochondria with the help of the translocase of the outer mitochondrial membrane (TOM) complex. For the membrane integration and assembly into complexes, ␤-barrel proteins require additional proteinaceous machinery in the OMM. This is so called sorting and assembly machinery (SAM), also known as topogenesis of outer membrane ␤-barrel proteins (TOB complex) (3, 4).The central component of the SAM complex is Sam50/ Tob55, a protein with a function that has been conserved from bacteria to human (4 -6). Other components of the SAM complex include the Sam35/Tob38/Tom38 (7-9) and Sam37/ Mas37/Tom37 (3, 10) proteins in yeast, and Metaxin 1 and Metaxin 2 (11) in mammalian mitochondria.The signals in ␤-barrel proteins that are recognized by the TOM complex belong to internal targeting signals and are not yet fully understood. However, a specific signal has been identified in the C-terminal part of mitochondrial ␤-barrel proteins that directs them to the SAM complex to be properly sorted and integrated into the OMM (12). Unlike in yeast, this signal in mammalian ␤-barrel proteins is always present at the extreme C terminus of the protein, and the addition of even a shor...

Granzyme B enters the mitochondria in a Sam50-, Tim22- and mtHsp70-dependent manner to induce apoptosis

“…Freshly purified yeast mitochondria were incubated with or without GB before gentle treatment with proteinase K in the absence or presence of a swelling step to disrupt the mitochondrial outer membrane to analyze its submitochondrial localization. 30,31 Similarly to the mitochondrial matrix marker Tim44, some of the GB is protected from proteolysis even after disruption of the outer mitochondrial membrane, indicating that it is in the matrix (Figure 1d), in agreement with GB ability to cleave the matrix subunits of complex I and the mito-FRET reporter (Figure 1c).…”

Evolutionary Conservation in Biogenesis of β-Barrel Proteins Allows Mitochondria to Assemble a Functional Bacterial Trimeric Autotransporter Protein

“…Thus, they followed a route shared with mitochondrial ␤-barrel proteins (18). Similarly, the pathogenic bacterial PorB can target mitochondria in mammalian cells (19,20). Moreover, we could demonstrate by reciprocal approach that expression of mitochondrial porin in E. coli cells resulted in a BamA-dependent assembly of the protein in the bacterial OM (21).…”

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

“…Nevertheless, functional expression of bacterial β-barrel proteins in eukaryotic cells suggests that during this adaptation process the ability of mitochondria to facilitate the assembly of prokaryotic β-barrel proteins was maintained15161718. A closer look at the biogenesis pathways of β-barrel proteins reveals that many characteristics are shared among Gram-negative bacteria and mitochondria.…”