title: N-Glycomic and Microscopic Subcellular Localization Analyses of NPP1, 2 and 6 Strongly Indicate that trans-Golgi Compartments Participate in the Golgi to Plastid Traffic of Nucleotide Pyrophosphatase/Phosphodiesterases in Rice.
abstract: Nucleotide pyrophosphatase/phosphodiesterases (NPPs) are widely distributed N-glycosylated enzymes that catalyze the hydrolytic breakdown of numerous nucleotides and nucleotide sugars. In many plant species, NPPs are encoded by a small multigene family, which in rice are referred to NPP1-NPP6 Although recent investigations showed that N-glycosylated NPP1 is transported from the endoplasmic reticulum (ER)-Golgi system to the chloroplast through the secretory pathway in rice cells, information on N-glycan composition and subcellular localization of other NPPs is still lacking. Computer-assisted analyses of the amino acid sequences deduced from different Oryza sativa NPP-encoding cDNAs predicted all NPPs to be secretory glycoproteins. Confocal fluorescence microscopy observation of cells expressing NPP2 and NPP6 fused with green fluorescent protein (GFP) revealed that NPP2 and NPP6 are plastidial proteins. Plastid targeting of NPP2-GFP and NPP6-GFP was prevented by brefeldin A and by the expression of ARF1(Q71L), a dominant negative mutant of ADP-ribosylation factor 1 that arrests the ER to Golgi traffic, indicating that NPP2 and NPP6 are transported from the ER-Golgi to the plastidial compartment. Confocal laser scanning microscopy and high-pressure frozen/freeze-substituted electron microscopy analyses of transgenic rice cells ectopically expressing the trans-Golgi marker sialyltransferase fused with GFP showed the occurrence of contact of Golgi-derived membrane vesicles with cargo and subsequent absorption into plastids. Sensitive and high-throughput glycoblotting/mass spectrometric analyses showed that complex-type and paucimannosidic-type glycans with fucose and xylose residues occupy approximately 80% of total glycans of NPP1, NPP2 and NPP6. The overall data strongly indicate that the trans-Golgi compartments participate in the Golgi to plastid trafficking and targeting mechanism of NPPs.
authors.affiliation: Graduate School of Science and Technology, Niigata University, 2-8050 Ikarashi, Niigata, 950-2181 Japan.
authors.forename: Kentaro
authors.initials: K
authors.lastname: Kaneko
authors.affiliation: Graduate School of Science and Technology, Niigata University, 2-8050 Ikarashi, Niigata, 950-2181 Japan Department of Applied Biological Chemistry, Niigata University, 2-8050 Ikarashi, Niigata, 950-2181 Japan.
authors.forename: Takeshi
authors.initials: T
authors.lastname: Takamatsu
authors.affiliation: Graduate School of Science and Technology, Niigata University, 2-8050 Ikarashi, Niigata, 950-2181 Japan.
authors.forename: Takuya
authors.initials: T
authors.lastname: Inomata
authors.affiliation: Department of Applied Biological Chemistry, Niigata University, 2-8050 Ikarashi, Niigata, 950-2181 Japan.
authors.forename: Kazusato
authors.initials: K
authors.lastname: Oikawa
authors.affiliation: Graduate School of Science and Technology, Niigata University, 2-8050 Ikarashi, Niigata, 950-2181 Japan Department of Applied Biological Chemistry, Niigata University, 2-8050 Ikarashi, Niigata, 950-2181 Japan.
authors.forename: Kimiko
authors.initials: K
authors.lastname: Itoh
authors.affiliation: Graduate School of Advanced Life Science, Frontier Research Center for Post-genomic Science and Technology, Hokkaido University, Sapporo, 001-0021 Japan.
authors.forename: Kazuko
authors.initials: K
authors.lastname: Hirose
authors.affiliation: Graduate School of Advanced Life Science, Frontier Research Center for Post-genomic Science and Technology, Hokkaido University, Sapporo, 001-0021 Japan.
authors.forename: Maho
authors.initials: M
authors.lastname: Amano
authors.affiliation: Graduate School of Advanced Life Science, Frontier Research Center for Post-genomic Science and Technology, Hokkaido University, Sapporo, 001-0021 Japan.
authors.forename: Shin-Ichiro
authors.initials: S
authors.lastname: Nishimura
authors.affiliation: RIKEN Center for Sustainable Resource Science, Kanagawa, 230-0045 Japan.
authors.forename: Kiminori
authors.initials: K
authors.lastname: Toyooka
authors.affiliation: Laboratory of Plant Nutrition, Faculty of Agriculture, Kyushu University, Fukuoka, 812-8581 Japan.
authors.forename: Ken
authors.initials: K
authors.lastname: Matsuoka
authors.affiliation: Instituto de Agrobiotecnología (CSIC, UPNA, Gobierno de Navarra), Mutiloako etorbidea zenbaki gabe, 31192 Mutiloabeti, Nafarroa, Spain.
authors.forename: Javier
authors.initials: J
authors.lastname: Pozueta-Romero
authors.affiliation: Graduate School of Science and Technology, Niigata University, 2-8050 Ikarashi, Niigata, 950-2181 Japan Department of Applied Biological Chemistry, Niigata University, 2-8050 Ikarashi, Niigata, 950-2181 Japan t.mitsui@agr.niigata-u.ac.jp.
authors.forename: Toshiaki
authors.initials: T
authors.lastname: Mitsui
mesh_terms: D020823:ADP-Ribosylation Factor 1 / Q000235:genetics / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D020126:Brefeldin A / Q000494:pharmacology; D002736:Chloroplasts / Q000378:metabolism / Q000648:ultrastructure; D004721:Endoplasmic Reticulum / Q000378:metabolism / Q000648:ultrastructure; D017930:Genes, Reporter; D054794:Glycomics; D006031:Glycosylation; D006056:Golgi Apparatus / Q000378:metabolism / Q000648:ultrastructure; D005810:Multigene Family; D012275:Oryza / Q000201:enzymology* / Q000235:genetics / Q000648:ultrastructure; D010727:Phosphoric Diester Hydrolases / Q000235:genetics / Q000378:metabolism*; D010940:Plant Proteins / Q000235:genetics / Q000378:metabolism; D018087:Plastids / Q000378:metabolism / Q000648:ultrastructure; D021381:Protein Transport / Q000187:drug effects; D011755:Pyrophosphatases / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins; D016415:Sequence Alignment
chemical_list: D010940:Plant Proteins; D011993:Recombinant Fusion Proteins; D020126:Brefeldin A; D010727:Phosphoric Diester Hydrolases; D011755:Pyrophosphatases; C021899:nucleotide pyrophosphatase; D020823:ADP-Ribosylation Factor 1
keywords: Glycoprotein; Golgi; Membrane traffic; N-glycome; Oryza sativa; Plastid