DynamiT · Dynamics of transporters dependent on ubiquitin in plants : mechanisms, roles in plant nutrition and beyond

Dynamics of transporters dependent on ubiquitin in plants : mechanisms, roles in plant nutrition and beyond

The mechanisms controlling the localization and the levels of cell-surface proteins including nutrient transporters are largely unknown in plants. To identify and characterize the mechanisms at stakes, we are using a plant iron transporter named IRT1 as a model protein. We recently uncovered that the modification of IRT1 with a small protein called ubiquitin, a process named ubiquitination, controls its abundance at the cell surface and its overall localization in the cell. The goal of the Career Integration Grant (CIG) DynamiT was i) to characterize the mechanisms and identify factors driving IRT1 ubiquitination, ii) to highlight its functional importance in the intracellular dynamics of IRT1 and iii) to shed light on its contribution to growth and development. Within the framework of the DynamiT project, we have demonstrated that IRT1 ubiquitination is regulated by the availability in its secondary metal substrates that are aspecifically transported by IRT1. Upon high concentrations of zinc, manganese, cobalt and cadmium in soils, IRT1 is decorated with ubiquitin moieties, allowing its removal from the cell-surface and targeting to intracellular stores or degradation factories to avoid detrimental accumulation of highly-reactive or toxic metals in the cell. We demonstrated that the IDF1 protein was responsible for the ubiquitination of IRT1, and for plant responses to metal toxicity. We further showed that IRT1 needs to be modified by another modification prior to its ubiquitination by IDF1. Indeed, we uncovered that IRT1 is phosphorylated by the CIPK23 kinase in response to metal excess in soils. The phosphorylation of IRT1 triggers the recruitment of IDF1 and the degradation of IRT1. In addition, we unraveled the intricate mechanisms by which plant perceive metal excess to regulate IRT1. We shed light on the ability of IRT1 itself to sense metals. Using metal-binding motif in its primary sequence, IRT1 directly perceives the concentration of metals in a cell. When metals are accumulating in tissues, IRT1 is loaded with metals and this allows the recruitment of CIPK23 and the degradation of IRT1. To identify more regulators of IRT1 dynamics, we performed a genetic screen looking for mutants unable to degrade IRT1 in conditions of metal excess. We have already identified several promising mutants that are currently being characterized. Our findings shed light on the ability of IRT1 to act as a hybrid between a transporter and a receptor, also called transceptor, directly sensing its highly-reactive non-iron metal substrates in the cytosol. The molecular framework uncovered allowing IRT1 to perceive and trigger its own degradation provides a unique insight into the regulation of metal transporters. The knowledge gained from DynamiT will certainly serve as a basis for transferring knowledge about the dynamics of other plant plasma membrane proteins, but also to establish crop plants with interesting agricultural traits to limit for example the accumulation of noxious metals in plants that are at the basis of the food chain. In addition, homologs of IRT1 are found from bacteria to mammals, indicating that our findings will likely allow great progress in metal transport in non-plant communities. Besides the detailed mechanisms allowing the regulation of IRT1 by metals, we also developed a number of ground-breaking tools within DynamiT. First, we implemented high-resolution imaging of cell surface proteins in plants using a technique called TIRF, previously used in yeast and mammals. This allows us to precisely monitor the dynamics of a protein in the cell and will be instrumental to track the influence of metals and ubiquitination of IRT1, but also of any cell surface protein. In parallel, we developed large-scale screening approaches to identify proteins able to mediate the ubiquitination of plasma membrane proteins using yeast as a model. We have screened over 12,000 proteins and are currently confirming the hits identified for a subset of model plasma membrane proteins. Finally, we have also performed a genetic screen looking for mutants unable to properly target an artificially ubiquitinated protein to the vacuole. This last approach is complementary to the genetic screen looking for mutants impaired in IRT1 trafficking and should identify the general endocytic factors required for ubiquitinated protein sorting in the cell. These approaches will provide unique insight into the dynamics of membrane proteins, from our ability to image these events to the fine mechanisms at stake.

Data: CORDIS, © European Union