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.

Routes to the Tonoplast: The Sorting of Tonoplast Transporters in Arabidopsis Mesophyll Protoplasts

“…Examples are the tonoplast two pore K + channels (TPKs), which were shown to contain targeting information in their cytosolic C termini both in rice (Oryza sativa; Isayenkov et al, 2011) and Arabidopsis (Maîtrejean et al, 2011). For the tonoplast-localized syntaxin VESICLE-ASSOCIATED MEMBRANE PROTEIN711, an N-terminal longin domain was demonstrated to be necessary for its correct sorting (Uemura et al, 2005).…”

Multiple N-Glycans Cooperate in the Subcellular Targeting and Functioning of Arabidopsis KORRIGAN1

“…The SYP61-CFP transgene was introduced into GFP-KOR1 lines by genetic crosses. Markers for transient expression were RFP-SYP61 for the TGN (Lam et al, 2009), OFP-ER for the ER , and TPK1-RFP for tonoplasts (Batistic, 2012), originally described as GFP fusion (Maîtrejean et al, 2011).…”

How are tonoplast proteins degraded?

“…We concluded that TPK1-GFP degradation requires the protein to reach the tonoplast and involves an internalization step (as the TPK1-GFP. 6 These structures are usually brighter than the tonoplast, indicating that they could be limited by a double membrane or contain a specific accumulation of our fusion protein (Fig. 2).…”

“…We recently investigated the mechanism involved in the degradation of a model tonoplast protein in fully developed leaf tissue. 6 When GFP was fused to the cytosolic C-terminus of the tonoplast Tandem-Pore Potassium channel AtTPK1 the resulting chimeric protein (TPK1-GFP) was correctly delivered to the tonoplast in transgenic Arabidopsis thaliana plants and still functional. By pulse-chase analysis, subcellular fractionation and protease protection experiments we demonstrated that intact TPK1-GFP progressively disappeared with a half-life time of 24 h and that a soluble degradation fragment, recognized by the GFP antiserum, accumulates in parallel in the vacuolar lumen.…”