Mitochondrial outer membrane proteins (β-OMPs) serve as the first line of communication with the cellular milieu. A crucial β-OMP, the sorting and assembly machinery Sam50, is a 16-stranded transmembrane β-barrel highly conserved in all eukaryotes. Sam50 dysregulation is lethal; yet, molecular elements that regulate Sam50 remain poorly understood. Here, we identify and characterize residues that regulate Sam50 structure and function. Using single-molecule electrophysiology, in vivo function, and stability measurements, we demonstrate that the POlypeptide-TRansport Associated domain is dispensable for Sam50. Complete characterization of the folding mechanism using 165 Xaa→Ala substitutions reveals that Sam50 folds through parallel pathways, with at least two transition states. The folding nucleus is towards the N-terminus, whereas frustrated folding at the C-terminal region kinetically traps the structure. We correlate this unexpected folding of Sam50 with its SAM-assisted assembly. Additionally, our per-residue stability measurements show that destabilizing hotspots in Sam50 are linked to its gating function. Our findings suggest how the dynamic structure of Sam50 offers a functional advantage, with specific residues that regulate folding, stability, and function, also determine the protein’s sensitivity to mutations. Here, authors map how stepwise assembly of the mitochondrial Sam50 involves parallel folding pathways. They identify residue-level contributors to β-barrel stability, and link energetics with in vivo function of this essential membrane chaperone.
Ravi et al. (Thu,) studied this question.