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

Abstract

Regulation of organ shape and size is an important area of research in developmental biology in both plants and animals. The shape of a leaf is determined primarily by endogenous factors such as gene function. Only a small number of such genes have been identified till now, the mutation of which perturbs leaf shape. Our laboratory previously carried out a forward genetic screen and identified additional leaf shape genes in Arabidopsis. Important among these mutants is the tarani (tni) mutant with cup-shaped leaves as opposed to the flat leaves in wildtype. Apart from changes in leaf shape, the tni mutant also exhibited additional phenotypic changes including tri-cotyledonous seedlings, fewer lateral roots and reduced gravitropic response. Map-based cloning identified TNI as the ubiquitin-specific protease 14 (UBP14) protein, which cleaves polyubiquitin chains into mono-ubiquitin. TNI/UBP14 has an N terminal ZnF-UBP domain followed by a ubiquitin hydrolase domain and two consensus C terminal UBA domains. A similar ZnF domain is also present in many other transcription factors. The ZnF-UBP and UBA domains serve as protein-protein interaction (PPI) interfaces in various components of the ubiquitin-proteasomal machinery. TNI/ UBP14 is localised to the nucleus, suggesting that it modulates gene function by interaction with proteins in the nuclear compartment, including transcription factors. The pleiotropic phenotype observed in the tni mutant resembles that of other mutants with a defect in auxin response and proteasomal function, suggesting that TNI interacts with and acts upon proteins involved in diverse biological pathways. We have undertaken biochemical approaches to identify the interacting partners of TNI. First, a yeast two-hybrid screen was performed using TNI as bait and a subset of 197 Arabidopsis transcription factors as prey. Second, a pulldown experiment was carried out from the total protein lysate of a transgenic plant expressing Myc-tagged TNI protein using a commercially available anti-Myc antibody, followed by the identification of interacting proteins through mass spectrometry. Third, a transgenic line that expresses biotin ligase fused to TNI protein was generated and total protein from this line was used to pulldown TNI and identify interacting proteins. Interaction of TNI and a selected few interacting protein thus identified was validated by binary methods and genetic interaction studies of their mutant individuals. Through this study, we highlight the significance of performing different approaches in protein-protein interaction studies.