title: Two glycosylated vacuolar GFPs are new markers for ER-to-vacuole sorting.
abstract: Vacuolar Sorting Determinants (VSDs) have been extensively studied in plants but the mechanisms for the accumulation of storage proteins in somatic tissues are not yet fully understood. In this work we used two mutated versions of well-documented vacuolar fluorescent reporters, a GFP fusion in frame with the C-terminal VSD of tobacco chitinase (GFPChi) and an N-terminal fusion in frame with the sequence-specific VSD of the barley cysteine protease aleurain (AleuGFP). The GFP sequence was mutated to present an N-glycosylation site at the amino-acid position 133. The reporters were transiently expressed in Nicotiana tabacum protoplasts and agroinfiltrated in Nicotiana benthamiana leaves and their distribution was identical to that of the non-glycosylated versions. With the glycosylated GFPs we could highlight a differential ENDO-H sensitivity and therefore differential glycan modifications. This finding suggests two different and independent routes to the vacuole for the two reporters. BFA also had a differential effect on the two markers and further, inhibition of COPII trafficking by a specific dominant-negative mutant (NtSar1h74l) confirmed that GFPChi transport from the ER to the vacuole is not fully dependent on the Golgi apparatus.
authors.affiliation: Laboratory of Cell and Molecular Biology, University of Neuchâtel, Rue Emile-Argand 11, CH-2000 Neuchâtel, Switzerland; CNR-IGV, Institute of Plant Genetics, Thematic Center for the Preservation of Mediterranean Plant Biodiversity, via Nazionale 44, 75025 Policoro, MT, Italy.
authors.forename: Egidio
authors.initials: E
authors.lastname: Stigliano
authors.forename: Marianna
authors.initials: M
authors.lastname: Faraco
authors.forename: Jean-Marc
authors.initials: JM
authors.lastname: Neuhaus
authors.forename: Anna
authors.initials: A
authors.lastname: Montefusco
authors.forename: Giuseppe
authors.initials: G
authors.lastname: Dalessandro
authors.forename: Gabriella
authors.initials: G
authors.lastname: Piro
authors.forename: Gian-Pietro
authors.initials: GP
authors.lastname: Di Sansebastiano
mesh_terms: D022181:COP-Coated Vesicles; D002688:Chitinases / Q000378:metabolism*; D003546:Cysteine Endopeptidases / Q000378:metabolism; D057056:Cysteine Proteases / Q000378:metabolism; D004721:Endoplasmic Reticulum / Q000378:metabolism*; D006031:Glycosylation; D006056:Golgi Apparatus; D049452:Green Fluorescent Proteins / Q000378:metabolism*; D001467:Hordeum / Q000201:enzymology; D017038:Mannosyl-Glycoprotein Endo-beta-N-Acetylglucosaminidase / Q000378:metabolism; D009154:Mutation; D018515:Plant Leaves / Q000378:metabolism; D010940:Plant Proteins / Q000378:metabolism*; D011134:Polysaccharides / Q000378:metabolism; D021381:Protein Transport; D011523:Protoplasts / Q000378:metabolism; D014026:Nicotiana / Q000201:enzymology / Q000235:genetics / Q000378:metabolism*; D014617:Vacuoles / Q000378:metabolism*
chemical_list: D010940:Plant Proteins; D011134:Polysaccharides; D049452:Green Fluorescent Proteins; D002688:Chitinases; D017038:Mannosyl-Glycoprotein Endo-beta-N-Acetylglucosaminidase; D057056:Cysteine Proteases; D003546:Cysteine Endopeptidases; C046726:aleurain
keywords: GFP; Glycosylation; Golgi; Protoplast; Secretion; Vacuole; ctVSD