Molecular assembly pathway of mitochondrial SAM50 in native membranes

Abstract

The major outer mitochondrial membrane chaperone is the Sorting and Assembly Machinery (SAM). SAM is a trimeric complex, with the transmembrane β-barrel Sam50 forming the core component. It functions in the sorting and trafficking of β-barrel precursors into the mitochondrial outer membrane, and thus is indispensable for cell survival. Details of the energetics governing the structure and function of the SAM complex, and the explicit molecular role(s) of Sam50 still need to be unraveled. Using a consolidated approach involving thermodynamic and kinetic measurements, we now deduce unprecedented molecular insight on the assembly of Sam50 in native membranes. We measure the per-residue contribution of the Sam50 primary sequence to its folding and thermodynamic stability, using an exhaustive alanine scanning mutagenesis strategy in phosphatidylethanolamine-doped phosphatidylcholine-rich membranes. Our findings using φ-value analysis reveal two on-pathway early kinetic folding intermediates that populate prior to the complete assembly of Sam50 in the lipid bilayer. The early intermediates possess structured N- and C-terminal strands, whereas the central strands remain unfolded or form non-native contacts. We observe a β-augmentation mechanism involving the pre-folded terminal strands in the late intermediate, which is followed by a slow rearrangement process to yield a folded and functional Sam50 β-barrel. Measurements of thermodynamic stability of the end-state β-barrel reveal localized destabilization of the N-terminal strands and a scaffold anchoring role for the C-terminal region, which bear direct implications in the chaperoning mechanism of Sam50. By linking our findings from per-residue energetics with the mechanism of Sam50 action, we provide a thermodynamic perspective for Sam50 function in mitochondria.