Biogenesis of mitochondrial β-barrel membrane proteins

Abstract

β-barrel membrane proteins in the mitochondrial outer membrane are crucial for mediating the metabolite exchange between the cytosol and the mitochondrial intermembrane space. In addition, the β-barrel membrane protein subunit Tom40 of the translocase of the outer membrane (TOM) is essential for the import of the vast majority of mitochondrial proteins encoded in the nucleus. The sorting and assembly machinery (SAM) in the outer membrane is required for the membrane insertion of mitochondrial β-barrel proteins. The core subunit Sam50, which has been conserved from bacteria to humans, is itself a β-barrel protein. The β-strands of β-barrel precursor proteins are assembled at the Sam50 lateral gate forming a Sam50-preprotein hybrid barrel. The assembled precursor β-barrel is finally released into the outer mitochondrial membrane by displacement of the nascent β-barrel, termed the β-barrel switching mechanism. SAM forms supercomplexes with TOM and forms a mitochondrial outer-to-inner membrane contact site with the mitochondrial contact site and cristae organizing system (MICOS) of the inner membrane. SAM shares subunits with the ER-mitochondria encounter structure (ERMES), which forms a membrane contact site between the mitochondrial outer membrane and the endoplasmic reticulum. Therefore, β-barrel membrane protein biogenesis is closely connected to general mitochondrial protein and lipid biogenesis and plays a central role in mitochondrial maintenance.

Keywords: Mco6; Mdm10; SAM; Sam35; Sam37; Sam50; mitochondria; outer membrane; sorting and assembly machinery; β‐barrel protein.

Fig. 1

β‐barrel membrane protein biogenesis in mitochondria and Gram‐negative bacteria. Mitochondrial β‐barrels are synthesized in the eukaryotic cytosol, translocated across the outer membrane by the TOM complex, and guided by the TIM transfer chaperones across the intermembrane space to the SAM complex, where they are inserted into the outer membrane. Bacterial β‐barrels are synthesized in the cytosol, translocated across the inner membrane by the SEC translocase, chaperoned through the periplasm by a system of chaperones, and inserted into the outer membrane by the BAM complex. Although mitochondrial and bacterial β‐barrels are synthesized in opposite compartments related to the outer membrane, they are conservatively inserted into the outer membrane from the intermembrane space/periplasmic side by the homologous Omp85 family proteins Sam50 and BamA respectively. BAM, β‐barrel assembly machinery; SAM, sorting and assembly machinery; TIM, translocase of the inner mitochondrial membrane; TOM, translocase of the outer membrane.

Fig. 2

Translocase of the inner mitochondrial membrane (TIM) transfer chaperone and Sam50 POTRA domain structures. The TIM transfer chaperones Tim9/Tim10 (and Tim8/13) form an alternating hexameric structure. Each subunit contains intramolecular disulfide bonds and conserved hydrophobic residues (in positions + and ‐ with respect to the residues involved in disulfide bond formation) in their N‐ and C‐terminal helices that form a precursor binding cleft. Sam50 contains one N‐terminal POTRA domain in the intermembrane space (IMS). POTRA β‐strand 2 is available for β‐strand augmentation by the substrate and thus contributes to β‐barrel membrane protein assembly. POTRA, polypeptide‐transport‐associated domain.

Fig. 3

Membrane insertion of β‐barrel precursors at the SAM complex by β‐barrel switching. The last C‐terminal β‐strand of the substrate, which contains a conserved β‐signal, binds to the SAM^dimer complex by pairing with the first β‐strand of Sam50a. While the β‐signal remains bound to Sam50a, additional β‐strands are sequentially inserted into the membrane from C‐ to N terminus, likely as β‐hairpins. This is termed the hybrid barrel stage. The growing substrate β‐barrel displaces Sam50b. Once the substrate barrel is fully folded, its terminal β‐strands pair, replacing the β‐signal pairing with Sam50a and closing the lateral gates of both β‐barrels. The newly assembled β‐barrel precursor is released into the outer membrane and switched by Sam50b to re‐form the SAM^dimer complex. Hy, hydrophobic residues; Po, polar residues; SAM, sorting and assembly machinery.

Fig. 4

Role of peripheral cytosolic subunits Sam35 and Sam37. Sam35 sits atop Sam50a, interacting with the barrel wall with its N terminus. Sam35 assists in substrate binding to Sam50a. Sam37 is similar to Sam35 in structure and its ability to bind mitochondrial β‐barrels from the cytosolic side. Sam37 contains an α‐protrusion which is able to bind into the lumen of the growing β‐barrel substrate, thereby stabilizing the β‐barrel during its formation.

Fig. 5

Tom40 assembly at SAM. Tom40 follows the general β‐barrel insertion pathway in terms of binding to Sam50a via a β‐signal and forming a hybrid barrel structure (upper panel). However, unlike other β‐barrels, Tom40 assembles with small α‐helical transmembrane proteins while bound to the SAM complex (lower panel). Tom5 and Tom6 assemble with the Tom40 precursor while still bound to SAM. Release of Tom40 requires another β‐barrel protein, Mdm10, which displaces the Tom40 substrate at SAM via interactions with Sam50a and Sam37. During the displacement process, Mdm10 delivers another small translocase of the outer membrane (TOM) protein, Tom7, to Tom40‐5‐6. Mdm10 is itself stabilized by an additional small membrane protein, Mco6. The final core TOM subunit, Tom22, is assembled with Tom40‐5‐6‐7, which allows for dimerization and formation of the mature TOM complex.

Fig. 6

Interactions of the sorting and assembly machinery (SAM) complex with other mitochondrial protein machineries. SAM forms a supercomplex with translocase of the outer membrane (TOM) through interactions of Sam37 and Tom22. This couples β‐barrel substrate translocation across and insertion into the outer membrane. SAM interaction with the α‐helical outer membrane insertase mitochondrial import machinery (MIM) facilitates small TOM subunit assembly with Tom40 at the SAM complex. Mdm10 is a subunit of both SAM and ERMES to regulate mitochondrial protein and lipid biogenesis. The Sam50 POTRA domain interacts with the MICOS complex at cristae junctions in the inner membrane and forms a mitochondrial contact site between the outer and the inner mitochondrial membranes. All of these interactions place SAM in a central position with regard to protein and lipid biogenesis in mitochondria. ERMES, endoplasmic reticulum‐mitochondria encounter structure; MICOS, mitochondrial contact site and cristae organizing system.