Molecular mechanisms of tonoplast K+ channels degradation in Arabidopsis thaliana: a model for tonoplast proteostasis

The aim of this project was to acquire new information on the mechanisms and signals responsible for the vacuolar degradation of the tonoplast potassium channel AtTPK1 as a model for the proteostasis of tonoplast proteins. We examined two aspects related to the degradation of this protein: whether ubiquitination is a necessary step to trigger AtTPK1 degradation and whether the degradation into the vacuole occurs via a multivesicular bodies-mediated pathway or by direct internalization. We have excluded a role of ubiquitination of the three Lys residues that, based in silico predictions, were considered the most likely sites for ubiquitination. We also excluded a role of a Cys residue that could in theory be palmitoylated. Treatments on AtTPK1-GFP expressing plants with the inhibitor Wortmannin, which alters the MVB to vacuole traffic, seem to favor a direct internalization of the channel into the vacuole, but the slow turnover rate of TPK1 can have hampered an accurate measurement of the effect of the inhibitor. These results are therefore preliminary, but we have set up an in vivo assay in intact plants that can be further refined by performing longer treatments. Our results also showed an effect of cycloheximide treatment that may be useful to define the routes of degradation.Finally we have prepared plants that coexpress two different tonoplast proteins; these will allow an accurate comparison of turnover rates of different proteins located at the same membrane.