CoMR: an integrative scoring pipeline for Comprehensive Mitochondrial proteome Reconstruction across eukaryotes

Mitochondrial proteome reconstruction from eukaryotic sequence data typically relies on prediction of mitochondrial targeting signals (MTSs). However, MTS predictors are primarily trained on model organisms and may perform poorly in phylogenetically divergent lineages or in organisms with atypical or reduced targeting sequences. Accurate reconstruction therefore requires integration of complementary sources of evidence beyond targeting prediction alone. We developed CoMR (Comprehensive Mitochondrial Reconstructor), an integrative workflow that combines targeting prediction, curated homology searches, large-scale similarity searches, and automated phylogenetic analysis within a unified scoring framework. Benchmarking on the model yeast Saccharomyces cerevisiae yielded strong discriminatory performance (ROC-AUC = 0.92), exceeding standalone TargetP2 prediction (ROC-AUC = 0.72). In the divergent anaerobic protist Paratrimastix pyriformis, CoMR maintained robust performance (ROC-AUC = 0.86) validated with an experimental proteome despite extreme class imbalance, achieving a precision-recall AUC of 0.183 ([~]78-fold enrichment over random expectation and [~]10-fold improvement over TargetP2). Ablation analyses demonstrate that predictive performance is robust to individual evidence-layer removal and that the relative contribution of homology sources depends on phylogenetic context. Together, these results show that integrative evidence scoring improves mitochondrial proteome reconstruction across both model and non-model eukaryotes.