Towards Mechanistic Understanding of Mitochondrial β-Barrel Biogenesis: Structural Studies of the Sorting and Assembly Machinery

Abstract

Mitochondria are essential eukaryotic organelles and play a vital role in many cellular processes, including ATP production, lipid synthesis, and apoptosis. The majority of the mitochondrial proteome is translated in the cytosol and imported into the mitochondria as unfolded precursors. Outer membrane β-barrel proteins are imported by the translocase of the outer membrane (TOM complex) then folded and inserted into the membrane by the sorting and assembly machinery (SAM complex). The SAM complex is composed of three subunits: a β-barrel core (Sam50) that spans the mitochondrial outer membrane, and two accessory subunits (Sam35 and Sam37) that associate on the cytosolic side of the membrane. We recently solved cryoEM structures of the SAM complex in detergent and lipid nanodiscs to resolutions of 3.0Å and 3.4Å, respectively. These structures confirm that Sam50 is a sixteen strand β-barrel and that Sam35 and Sam37 are located on the cytosolic side of the membrane. The N-terminus of Sam35 interacts with the cytosolic loops of Sam50, blocking cytosolic access to the Sam50 β-barrel. The cytosolic domain of Sam37 forms extensive interactions with Sam35, stabilizing Sam35 association with Sam50. In the intermembrane space, Sam37 contributes a β-strand to the Sam50 N-terminal polypeptide transport-associated (POTRA) domain. Interestingly, both Sam37 and the Sam50 POTRA domain are implicated in precursor release. While our structure agrees with current biochemical knowledge of the SAM complex, it remains unclear how the β-signal is recognized by the SAM complex. Current work aims to characterize the interaction of the β-signal with the SAM complex subunits.