title: Rice plastidial N-glycosylated nucleotide pyrophosphatase/phosphodiesterase is transported from the ER-golgi to the chloroplast through the secretory pathway.
abstract: A nucleotide pyrophosphatase/phosphodiesterase (NPP) activity that catalyzes the hydrolytic breakdown of ADP-glucose (ADPG) has been shown to occur in the plastidial compartment of both mono- and dicotyledonous plants. To learn more about this enzyme, we purified two NPPs from rice (Oryza sativa) and barley (Hordeum vulgare) seedlings. Both enzymes are glycosylated, since they bind to concanavalin A, stain with periodic acid-Schiff reagent, and are digested by Endo-H. A complete rice NPP cDNA, designated as NPP1, was isolated, characterized, and overexpressed in transgenic plants displaying high ADPG hydrolytic activity. Databank searches revealed that NPP1 belongs to a functionally divergent group of plant nucleotide hydrolases. NPP1 contains numerous N-glycosylation sites and a cleavable hydrophobic signal sequence that does not match with the N-terminal part of the mature protein. Both immunocytochemical analyses and confocal fluorescence microscopy of rice cells expressing NPP1 fused with green fluorescent protein (GFP) revealed that NPP1-GFP occurs in the plastidial compartment. Brefeldin A treatment of NPP1-GFP-expressing cells prevented NPP1-GFP accumulation in the chloroplasts. Endo-H digestibility studies revealed that both NPP1 and NPP1-GFP in the chloroplast are glycosylated. Collectively, these data demonstrate the trafficking of glycosylated proteins from the endoplasmic reticulum-Golgi system to the chloroplast in higher plants.
authors.affiliation: Laboratory of Plant and Microbial Genome Control, Department of Applied Biological Chemistry, Niigata University, Niigata 950-2181, Japan.
authors.forename: Yohei
authors.initials: Y
authors.lastname: Nanjo
authors.forename: Hiromasa
authors.initials: H
authors.lastname: Oka
authors.forename: Noriko
authors.initials: N
authors.lastname: Ikarashi
authors.forename: Kentaro
authors.initials: K
authors.lastname: Kaneko
authors.forename: Aya
authors.initials: A
authors.lastname: Kitajima
authors.forename: Toshiaki
authors.initials: T
authors.lastname: Mitsui
authors.forename: Francisco José
authors.initials: FJ
authors.lastname: Muñoz
authors.forename: Milagros
authors.initials: M
authors.lastname: Rodríguez-López
authors.forename: Edurne
authors.initials: E
authors.lastname: Baroja-Fernández
authors.forename: Javier
authors.initials: J
authors.lastname: Pozueta-Romero
mesh_terms: D000595:Amino Acid Sequence; D001483:Base Sequence; D002736:Chloroplasts / Q000201:enzymology*; D018076:DNA, Complementary; D004591:Electrophoresis, Polyacrylamide Gel; D004721:Endoplasmic Reticulum / Q000201:enzymology*; D006031:Glycosylation; D006056:Golgi Apparatus / Q000201:enzymology*; D006868:Hydrolysis; D008969:Molecular Sequence Data; D012275:Oryza / Q000201:enzymology*; D010727:Phosphoric Diester Hydrolases / Q000302:isolation & purification / Q000378:metabolism*; D018087:Plastids / Q000201:enzymology*; D021381:Protein Transport; D011755:Pyrophosphatases / Q000302:isolation & purification / Q000378:metabolism*
chemical_list: D018076:DNA, Complementary; D010727:Phosphoric Diester Hydrolases; D011755:Pyrophosphatases