title: An N-terminal dileucine motif directs two-pore channels to the tonoplast of plant cells.
abstract: Two-pore channels (TPCs) constitute a family of endolysosomal cation channels with functions in Ca²⁺ signaling. We used a mutational analysis to investigate the role of channel domains for the trafficking of the Arabidopsis TPC1 to the tonoplast, a process that is generally not well understood in plants. The results show that the soluble C-terminus was not essential for targeting but for channel function, while further C-terminal truncations of two or more transmembrane domains impaired protein trafficking. An N-terminal dileucine motif (EDPLI) proved to be critical for vacuolar targeting of TPC1, which was independent of the adaptor protein AP-3. Deletion or mutation of this sorting motif, which is conserved among TPCs caused redirection of the protein transport to the plasma membrane. An N-terminal region with a predicted α-helical structure was shown to support efficient vacuolar trafficking and was essential for TPC1 function. Similar to their localization in mammalian endosomes and lysosomes, MmTPC1 and MmTPC2 were targeted to small organelles and the membrane of the lytic vacuole, respectively, when expressed in plant cells. These results shed new light on the largely uncharacterized sorting signals of plant tonoplast proteins and reveal similarities between the targeting machinery of plants and mammals.
authors.affiliation: Department Biology, Friedrich-Alexander-Universität Erlangen-Nürnberg, Molecular Plant Physiology and Erlangen Center of Plant Science, Staudtstrasse 5, 91058 Erlangen, Germany.
authors.forename: Nina
authors.initials: N
authors.lastname: Larisch
authors.forename: Christina
authors.initials: C
authors.lastname: Schulze
authors.forename: Antony
authors.initials: A
authors.lastname: Galione
authors.forename: Petra
authors.initials: P
authors.lastname: Dietrich
mesh_terms: D033963:Adaptor Protein Complex 3 / Q000502:physiology; D017360:Arabidopsis / Q000378:metabolism; D029681:Arabidopsis Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D015220:Calcium Channels / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D020013:Calcium Signaling; D002460:Cell Line; D008856:Microscopy, Fluorescence; D018408:Patch-Clamp Techniques; D021382:Protein Sorting Signals; D021381:Protein Transport; D000097903:Two-Pore Channels
chemical_list: D033963:Adaptor Protein Complex 3; D029681:Arabidopsis Proteins; D015220:Calcium Channels; D021382:Protein Sorting Signals; C498240:TPC1 protein, Arabidopsis; D000097903:Two-Pore Channels