UNLABELLED: Post-translational modifications (PTMs) modulate protein functions, with ubiquitylation a pre-eminent example, and playing major roles in protein turnover. Ubiquitylation utilises a ligase enzyme cascade for conjugation of ubiquitin to client proteins, of which there are a large number in humans and lesser numbers in unicellular eukaryotes. The Cullin-RING ligases are amongst the most complex ligase subfamily and are present across the eukaryote lineage. We have reconstructed the evolution of cullin-RING E3 ubiquitin ligases across eukaryotes and experimentally determined the composition of six of seven cullin complexes in trypanosomatids. We find considerable diversity within cullins and reconstruct at least four ancestral pan-eukaryotic subfamilies. Furthermore, we identify expansions of cullin client adaptor protein families, novel client adaptors and demonstrate client specificity in trypanosomatids. We also find evidence for increasing complexity within client adaptors, suggesting ongoing expansion of adapter architecture. Finally, we show that turnover of ornithine decarboxylase (TbODC), an important target of the trypanocide eflornithine, is mediated by TbCul-A/CUL-1. These studies highlight lineage-specific aspects of cullin E3 ligases and their contributions towards eukaryotic complexity. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1038/s41598-025-32077-9.

Department of Parasitology Charles University Prague Faculty of Science Vestec 252 42 Czech Republic

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

School of Life Sciences University of Dundee Dundee UK

Sygnature Discovery The Discovery Building BioCity Pennyfoot Street Nottingham NG1 1GR UK

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