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

Kentaro Kaneko, Takeshi Takamatsu, Takuya Inomata, Kazusato Oikawa, Kimiko Itoh, Kazuko Hirose, Maho Amano, Shin Ichiro Nishimura, Kiminori Toyooka, Ken Matsuoka, Javier Pozueta-Romero, Toshiaki Mitsui

Research output: Contribution to journalArticle

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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.

Original languageEnglish
Pages (from-to)1610-1628
Number of pages19
JournalPlant and Cell Physiology
Volume57
Issue number8
DOIs
Publication statusPublished - Aug 1 2016

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nucleotide pyrophosphatase
Glycomics
pyrophosphatases
Plastids
Phosphoric Diester Hydrolases
traffic
plastids
nucleotides
Green Fluorescent Proteins
rice
green fluorescent protein
Endoplasmic Reticulum
Polysaccharides
endoplasmic reticulum
Confocal Microscopy
polysaccharides
ADP-Ribosylation Factor 1
Nucleotides
Sialyltransferases
Brefeldin A

All Science Journal Classification (ASJC) codes

  • Physiology
  • Plant Science
  • Cell Biology

Cite this

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. / Kaneko, Kentaro; Takamatsu, Takeshi; Inomata, Takuya; Oikawa, Kazusato; Itoh, Kimiko; Hirose, Kazuko; Amano, Maho; Nishimura, Shin Ichiro; Toyooka, Kiminori; Matsuoka, Ken; Pozueta-Romero, Javier; Mitsui, Toshiaki.

In: Plant and Cell Physiology, Vol. 57, No. 8, 01.08.2016, p. 1610-1628.

Research output: Contribution to journalArticle

Kaneko, Kentaro ; Takamatsu, Takeshi ; Inomata, Takuya ; Oikawa, Kazusato ; Itoh, Kimiko ; Hirose, Kazuko ; Amano, Maho ; Nishimura, Shin Ichiro ; Toyooka, Kiminori ; Matsuoka, Ken ; Pozueta-Romero, Javier ; Mitsui, Toshiaki. / 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. In: Plant and Cell Physiology. 2016 ; Vol. 57, No. 8. pp. 1610-1628.
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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.",
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AU - Kaneko, Kentaro

AU - Takamatsu, Takeshi

AU - Inomata, Takuya

AU - Oikawa, Kazusato

AU - Itoh, Kimiko

AU - Hirose, Kazuko

AU - Amano, Maho

AU - Nishimura, Shin Ichiro

AU - Toyooka, Kiminori

AU - Matsuoka, Ken

AU - Pozueta-Romero, Javier

AU - Mitsui, Toshiaki

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