title: Assembly and sorting of the tonoplast potassium channel AtTPK1 and its turnover by internalization into the vacuole.
abstract: The assembly, sorting signals, and turnover of the tonoplast potassium channel AtTPK1 of Arabidopsis (Arabidopsis thaliana) were studied. We used transgenic Arabidopsis expressing a TPK1-green fluorescent protein (GFP) fusion or protoplasts transiently transformed with chimeric constructs based on domain exchange between TPK1 and TPK4, the only TPK family member not located at the tonoplast. The results show that TPK1-GFP is a dimer and that the newly synthesized polypeptides transiently interact with a thus-far unidentified 20-kD polypeptide. A subset of the TPK1-TPK4 chimeras were unable to assemble correctly and these remained located in the endoplasmic reticulum where they interacted with the binding protein chaperone. Therefore, TPK1 must assemble correctly to pass endoplasmic reticulum quality control. Substitution of the cytosolic C terminus of TPK4 with the corresponding domain of TPK1 was sufficient to allow tonoplast delivery, indicating that this domain contains tonoplast sorting information. Pulse-chase labeling indicated that TPK1-GFP has a half-life of at least 24 h. Turnover of the fusion protein involves internalization into the vacuole where the GFP domain is released. This indicates a possible mechanism for the turnover of tonoplast proteins.
authors.affiliation: Istituto di Biologia e Biotecnologia Agraria, Consiglio Nazionale delle Ricerche, 20133 Milan, Italy.
authors.forename: Marie
authors.initials: M
authors.lastname: Maîtrejean
authors.forename: Michael M
authors.initials: MM
authors.lastname: Wudick
authors.forename: Camilla
authors.initials: C
authors.lastname: Voelker
authors.forename: Bhakti
authors.initials: B
authors.lastname: Prinsi
authors.forename: Bernd
authors.initials: B
authors.lastname: Mueller-Roeber
authors.forename: Katrin
authors.initials: K
authors.lastname: Czempinski
authors.forename: Emanuela
authors.initials: E
authors.lastname: Pedrazzini
authors.forename: Alessandro
authors.initials: A
authors.lastname: Vitale
mesh_terms: D017360:Arabidopsis / Q000166:cytology* / Q000235:genetics / Q000378:metabolism*; D029681:Arabidopsis Proteins / Q000737:chemistry / Q000378:metabolism*; D003600:Cytosol / Q000378:metabolism; D004705:Endocytosis; D004721:Endoplasmic Reticulum / Q000378:metabolism; D049452:Green Fluorescent Proteins / Q000378:metabolism; D008970:Molecular Weight; D010455:Peptides / Q000378:metabolism; D030821:Plants, Genetically Modified; D015221:Potassium Channels / Q000737:chemistry / Q000378:metabolism*; D024683:Potassium Channels, Tandem Pore Domain / Q000737:chemistry / Q000378:metabolism*; D011485:Protein Binding; D055503:Protein Multimerization; D017434:Protein Structure, Tertiary; D021381:Protein Transport; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D014617:Vacuoles / Q000378:metabolism*
chemical_list: D029681:Arabidopsis Proteins; C503455:KCO1 protein, Arabidopsis; D010455:Peptides; D015221:Potassium Channels; D024683:Potassium Channels, Tandem Pore Domain; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins