PROTEOSTASIS OF THE TONOPLAST:SYNTHESIS, SORTING AND TURNOVER OF THE POTASSIUM CHANNEL ATTPK1

The vacuole of plant cells plays a major role in fundamental plant functions such as turgor maintenance, storage of metabolites, sequestration of deleterious compounds and accumulation of seed storage proteins. These functions depend directly or indirectly on proteins residing in the tonoplast, the vacuolar membrane. Indeed, the tonoplast contains many proteins responsible for the transport of salts and small solutes, and proteins involved in membrane fusion and remodelling that allow delivery of macromolecules by vesicle traffic. To fulfil these roles, tonoplast proteins must be correctly synthesised, sorted to the tonoplast and eventually turned over. The present Ph.D. project aims to study these mechanisms using the potassium channel TPK1 from Arabidopsis thaliana as a model. Transgenic Arabidopsis plants expressing a fusion between TPK1 and green fluorescent protein (TPK1-GFP) were produced. The correct tonoplast localisation of TPK1-GFP was confirmed by fluorescent microscopy and subcellular membrane fractionation. The intracellular traffic of TPK1-GFP is Golgi-dependent: disruption of Golgi-mediated traffic inhibited its tonoplast delivery. TPK1-GFP rapidly formed homodimers after its synthesis. This assembly is not triggered by interactions between the cytosolic domains, neither does it depends on the formation of a disulfide bond or the presence of potassium ions in the selective filter, as instead shown for a number of mammalian and viral K+ channels. By pulse-chase labelling with radioactive amino acids, it was also observed that newly synthesized TPK1-GFP transiently interacts with a small, unidentified 20-22 kDa polypeptide before forming dimers. The sorting signal responsible for TPK1 tonoplast delivery was investigated using GFP-tagged chimeric constructs based on AtTPK1 and AtTPK4, the homolog potassium channel located at the plasma membrane. When the cytosolic C-terminal domain of AtTPK4 was substituted with the corresponding one of AtTPK1, the chimera was delivered to the tonoplast, indicating that the TPK1 domain contains information for tonoplast sorting. A number of TPK1-TPK4 chimeras that we constructed were instead retained in the endoplasmic reticulum (ER). It was demonstrated that these constructs are assembly-defective and extensively interact with the chaperone BiP, a major component of the ER quality control machinery. These results indicate that TPK1 must assemble correctly to be delivered to its destination. Pulse-chase labelling showed that TPK1-GFP has a half-life of at least 24 hours. The GFP moiety, which is attached to the cytosolic C-terminal domain of TPK1, eventually accumulated as a soluble fragment in the vacuolar lumen. This observation indicates that degradation mechanism of this, and possibly other, tonoplast proteins involves an internalisation process and proteolytic degradation within the vacuolar lumen.