title: Vacuolar transport in tobacco leaf epidermis cells involves a single route for soluble cargo and multiple routes for membrane cargo.
abstract: We tested if different classes of vacuolar cargo reach the vacuole via distinct mechanisms by interference at multiple steps along the transport route. We show that nucleotide-free mutants of low molecular weight GTPases, including Rab11, the Rab5 members Rha1 and Ara6, and the tonoplast-resident Rab7, caused induced secretion of both lytic and storage vacuolar cargo. In situ analysis in leaf epidermis cells indicates a sequential action of Rab11, Rab5, and Rab7 GTPases. Compared with Rab5 members, mutant Rab11 mediates an early transport defect interfering with the arrival of cargo at prevacuoles, while mutant Rab7 inhibits the final delivery to the vacuole and increases cargo levels in prevacuoles. In contrast with soluble cargo, membrane cargo may follow different routes. Tonoplast targeting of an α-TIP chimera was impaired by nucleotide-free Rha1, Ara6, and Rab7 similar to soluble cargo. By contrast, the tail-anchored tonoplast SNARE Vam3 shares only the Rab7-mediated vacuolar deposition step. The most marked difference was observed for the calcineurin binding protein CBL6, which was insensitive to all Rab mutants tested. Unlike soluble cargo, α-TIP and Vam3, CBL6 transport to the vacuole was COPII independent. The results indicate that soluble vacuolar proteins follow a single route to vacuoles, while membrane spanning proteins may use at least three different transport mechanisms.
authors.affiliation: Centre for Plant Sciences, Faculty of Biological Sciences, University of Leeds, Leeds LS2 9JT, United Kingdom.
authors.forename: Francesca
authors.initials: F
authors.lastname: Bottanelli
authors.forename: Ombretta
authors.initials: O
authors.lastname: Foresti
authors.forename: Sally
authors.initials: S
authors.lastname: Hanton
authors.forename: Jürgen
authors.initials: J
authors.lastname: Denecke
mesh_terms: D060054:Agrobacterium / Q000235:genetics / Q000378:metabolism; D029681:Arabidopsis Proteins / Q000235:genetics / Q000378:metabolism; D001692:Biological Transport / Q000502:physiology; D022181:COP-Coated Vesicles / Q000235:genetics / Q000378:metabolism; D002135:Calcium-Binding Proteins; D002352:Carrier Proteins / Q000235:genetics / Q000378:metabolism; D020558:GTP Phosphohydrolases / Q000235:genetics / Q000378:metabolism*; D006056:Golgi Apparatus / Q000378:metabolism; D008565:Membrane Proteins / Q000378:metabolism; D019441:Plant Epidermis / Q000201:enzymology / Q000235:genetics / Q000378:metabolism; D018515:Plant Leaves / Q000201:enzymology / Q000235:genetics / Q000378:metabolism / Q000382:microbiology; D030821:Plants, Genetically Modified; D011523:Protoplasts / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism; D014026:Nicotiana / Q000201:enzymology / Q000378:metabolism* / Q000382:microbiology; D014617:Vacuoles / Q000201:enzymology / Q000378:metabolism*
chemical_list: D029681:Arabidopsis Proteins; C576221:CBL6 protein, Arabidopsis; D002135:Calcium-Binding Proteins; D002352:Carrier Proteins; D008565:Membrane Proteins; D011993:Recombinant Fusion Proteins; D020558:GTP Phosphohydrolases