BACKGROUND: Diplonemids constitute an abundant and geographically widespread but little-studied group of marine protists. A hallmark of this lineage, the kinetoplastid sister group within Euglenozoa, is a mitochondrial genome comprising numerous small circular DNA molecules that carry fragments of mitochondrial genes. Complex RNA processing of the corresponding transcripts involves numerous ligation and RNA editing steps in the production of mature RNA species. To assess the diplonemid mitochondrial proteome and, in particular, to search for proteins that might mediate RNA processing, we undertook a comprehensive in silico analysis to predict candidate mitochondrial proteins in the type species Diplonema papillatum. RESULTS: Using sequence similarity searches in conjunction with a mitochondrial targeting pipeline, we identified at least 1878 candidate nucleus-encoded mitochondrial proteins in addition to 16 mitochondrion-encoded proteins described previously. Despite the highly unconventional nature of the mitochondrial genome in D. papillatum, its mitochondrial proteome (mitoproteome) contains virtually all the functionally most important proteins that are ubiquitous among aerobic mitochondria, and several novel proteins that have been recruited in the euglenozoan last common ancestor to augment complexes involved in coupled electron transport oxidative phosphorylation and mitochondrial ribosome formation. Notably, we identified several individual proteins and multi-protein families that are candidates for RNA ligation and editing enzymes. CONCLUSIONS: This first comprehensive mitoproteome data for a diplonemid, together with published mitoproteome data for other members of Discoba, allows us to make inferences about marked changes in mitochondrial structure and function that have occurred since the divergence of diplonemids and other euglenozoans from the last common discobid ancestor. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1186/s12864-025-12233-1.

Département de Biochimie and Robert Cedergren Center for Bioinformatics and Genomics Université de Montréal Montréal QC Canada

Department of Biochemistry and Molecular Biology and Institute for Comparative Genomics Dalhousie University Halifax NS Canada

Faculty of Sciences University of South Bohemia České Budějovice Czech Republic

Institute of Parasitology Biology Centre Czech Academy of Sciences České Budějovice Czech Republic

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