Molecular mechanisms of tonoplast protein degradation

Protein turnover is fundamental both for development and cellular homeostasis. We are studying the possible mechanisms involved in the degradation of a tonoplast protein, the Arabidopsis potassium channel TPK1, previously shown as degraded upon internalization into the vacuole. In order to investigate if TPK1 degradation occurs via a multivesicular bodies -mediated pathway, or via a direct pathway, where the internalization event would occur directly at the vacuolar membrane, we are using two different approaches, based on site-directed mutagenesis of TPK1 lysine codons or on specific traffic inhibitors. First results of pulse-chase experiments on tobacco protoplasts expressing wild-type or three lysine deprived TPK1 showed no differences in the TPK1 turnover, suggesting that the ubiquitination of TPK1 does not occur, or at least does not involve the lysine residues we supposed. Site-directed mutagenesis of other TPK1 lysine codons is in progress, as well as in vivo treatments with tyrphostin A23 and wortmannin of Arabidopsis plants overexpressing wild-type TPK1. Supported by Italian Ministry of Education, Universities and Research (PRIN2010CSJX4F).