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.
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.
复制标题
DOI：
10.1093/pcp/pcw089
复制
发表时间：
2016-08
影响因子：
4.9
通讯作者：
Mitsui T
中科院分区：
文献类型：
--
作者：
Kaneko K;Takamatsu T;Inomata T;Oikawa K;Itoh K;Hirose K;Amano M;Nishimura S;Toyooka K;Matsuoka K;Pozueta-Romero J;Mitsui T
核苷酸焦磷酸酶/磷酸二酯酶（NPPs）是广泛分布的n -糖基化酶，可催化多种核苷酸和核苷酸糖的水解分解。在许多植物物种中，npp由一个小的多基因家族编码，在水稻中被称为NPP1-NPP6。虽然最近的研究表明，水稻细胞中n -糖基化的NPP1通过分泌途径从内质网(ER) -高尔基体系统转运到叶绿体，但关于其他NPP1的n -聚糖组成和亚细胞定位的信息仍然缺乏。计算机辅助分析了从不同的水稻npp编码cdna中推断出的氨基酸序列，预测所有npp都是分泌糖蛋白。用共聚焦荧光显微镜观察表达NPP2和NPP6与绿色荧光蛋白（GFP）融合的细胞，发现NPP2和NPP6是质体蛋白。brefeldin A和ARF1（Q71L）的表达阻止了NPP2 - gfp和NPP6 - gfp的质体靶向，ARF1是adp -核糖基化因子1的显性负突变体，可阻止内质网向高尔基体的转运，表明NPP2和NPP6从内质网-高尔基体转运到质体室。对异位表达反式高尔基标记物唾液基转移酶与GFP融合的转基因水稻细胞的共聚焦激光扫描显微镜和高压冷冻/冷冻替代电镜分析显示，高尔基衍生的膜囊泡与货物接触，随后被吸收到质体中。高通量糖印迹/质谱分析表明，具有聚焦残基和木糖残基的复合物型和少毛甘露苷型聚糖约占NPP1、NPP2和NPP6总聚糖的80%。总体数据有力地表明，跨高尔基区室参与了NPPs的高尔基体转运和靶向机制。
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.