Human mitochondrial Lon protease initiates unidirectional degradation from either substrate terminus

The ATP-dependent Lon protease (LonP1) unfolds and progressively degrades damaged or redundant proteins in the mitochondrial matrix. However, how LonP1 recognizes substrates remains unclear. Here, we investigated LonP1 degradation of the mitochondrial transcription factor TFAM, a physiological substrate. Using engineered TFAM variants carrying bulky moieties at either terminus, we show that degradation can initiate from either the N- or the C-terminus, with no evidence for internal initiation. Once engaged, substrate processing proceeds unidirectionally. Nanogold labeling experiments trapped TFAM termini within the N-domain cavity of LonP1, providing direct evidence that substrates enter through the N-domain assembly. A high-resolution cryo-EM structure of LonP1 in a transition-state-like complex with Mg ²⁺ , ADP, and AlF ₃ revealed substrate density within the axial channel with resolved main-chain carbonyl and Cα moieties, yet the peptide can be modelled equally well in either direction. Mass spectrometry of degradation products further supports initiation from either substrate terminus while confirming unidirectional processive translocation. These observations explain how LonP1 combines broad substrate specificity with selective recognition of flexible termini, avoiding initiation at internal flexible loops while enabling efficient ATP-dependent unfolding and degradation.