Identification and Characterization of Interacting Proteins of TARANI/ Ubiquitin Specific Protease-14 in Arabidopsis thaliana

Ubiquitin proteases play a crucial role in protein degradation and turnover by regulating the cleavage of polyubiquitin chains. TARANI/UBIQUITIN SPECIFIC PROTEASE-14 (TNI/UBP14) specifically cleaves Lys-48-linked and linear polyubiquitin chains into mono-ubiquitins. The tni mutant exhibits pleiotropic phenotypes, including cup-shaped leaves, tri-cotyledons, reduced lateral roots, and increased petal number, though the underlying mechanisms driving these phenotypes remain unclear. In this study, we generated TNI transgenic lines and employed immunoprecipitation mass spectrometry, proximity labelling, and yeast two-hybrid screening to identify TNIs interacting proteins. These analyses revealed 92 interactors involved in diverse biological processes, including protein and carbohydrate metabolism, light signalling, and intracellular transport. Subcellular localization analysis showed that many of the interacting proteins are located in the nucleus and cytoplasm, suggesting that TNIs nuclear localization may regulate gene function. We further validated the in planta biological significance of ULTRAPETALA 2 and HASPIN KINASE as key interacting partners of TNI. These findings uncover previously uncharacterized functions of TNI/UBP14, shedding light on its central role in cellular processes and providing insights into its regulatory mechanisms--an area that has remained largely unexplored until now. Summary statementThe proteins that interact with the TARANI/ Ubiquitin protease 14 in vivo have been identified using immunoprecipitation mass-spectrometry methods. Identification of non-overlapping targets highlight the importance of using diverse protein identification methods.