Elucidation and application of a novel mechanism regulating vacuolar localization of plant proteins

调节植物蛋白液泡定位的新机制的阐明和应用

基本信息

批准号：
17380198
负责人：
NAKAYAMA Toru
金额：
$ 9.54万
依托单位：
Tohoku University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (B)
财政年份：
2005
资助国家：
日本
起止时间：
2005 至 2006
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-17380198/
关键词：
vacuole transport cell plant secondary metabolism flavonoids organelle enzyme protein

项目摘要

Aureusidin synthase, a polyphenol oxidase (PPO), specifically catalyzes the oxidative formation of aurones from chalcones, which are plant flavonoids, and is responsible for the yellow coloration of snapdragon flowers. All known PPOs have been found to be localized in plastids, whereas flavonoid biosynthesis is thought to take place in cytoplasm (or on the cytoplasmic surface of ER). However, primary structural characteristics and some molecular properties of aureusidin synthase contradict the enzyme's localization in plastids and cytoplasm. In this study, the subcellular localization of this enzyme in petal cells of the yellow snapdragon was investigated. Sucrose-density gradient and differential centrifugation analyses suggested that the enzyme (the 39-kDa mature form) is not located in plastids or on the ER. Transient assays using a green fluorescent protein (GFP) chimera fused with the putative propeptide of the PPO precursor suggested that the enzyme was localized within vacuole lumen. We also found that the necessary information for the vacuolar targeting of the PPO was encoded within the 53-residue N-terminal sequence (NTPP), but not in C-terminal sequence of the precursor. The NTPP-mediated ER-to-Golgi trafficking to vacuoles was confirmed by means of the co-expression of an NTPP-GFP chimera fused with a dominant negative mutant of Sari GTPase of Arabidopsis or that with mRFP-fused Glogi marker (H^+-translocating inorganic pyrophosphatase of Arabidopsis). We identified a sequence-specific vacuolar sorting determinant in the NTPP of the precursor. This provides the first example of the biosynthesis of a flavonoid skeleton in vacuoles. This metabolic compartmentation should serve as a strategy for overcoming the biochemical instability of the precursor chalcones in the cytoplasm, thus leading to the efficient accumulation of aurones in the flower.

金黄色素蛋白合酶，一种多酚氧化酶（PPO），特异性地催化了来自植物类黄酮的Chalcone的过敏的氧化形成，并负责Snapdragon花的黄色。已经发现所有已知的PPO都位于质体中，而类黄酮的生物合成被认为发生在细胞质（或ER的细胞质表面）中。然而，金黄色葡萄球菌素合酶的主要结构特征和某些分子特性与酶在质体和细胞质中的定位相矛盾。在这项研究中，研究了该酶在黄色Snapdragon的花瓣细胞中的亚细胞定位。蔗糖密度梯度和差异离心分析表明，酶（39 kDa成熟形式）不在质体或ER中。使用绿色荧光蛋白（GFP）嵌合体与PPO前体的推定前肽融合的瞬时测定表明该酶位于液泡腔内。我们还发现，PPO的液泡靶向的必要信息是在53个残留的N末端序列（NTPP）中编码的，但不在前体的C末端序列中进行编码。 NTPP介导的ER到Golgi运输到液泡的运输是通过与拟南芥莎丽GTPase的主要负突变体融合的NTPP-GFP Chimera的共表达，或与MRFP-FUSE的Glogi Marker（H^+translication-tripplistic tramperate clogy plogi Marker）（H^+ - 替代拟南天异酶的无线电磷酸酶）。我们确定了前体NTPP中的序列特异性液泡分选簇。这提供了液泡中类黄酮骨骼的生物合成的第一个例子。这种代谢区分室应作为克服细胞质中前体chalcone的生化不稳定的策略，从而导致极光在花中的有效积累。

项目成果

期刊论文数量（0）

专著数量（0）

科研奖励数量（0）

会议论文数量（0）

专利数量（0）

Localization of a flavonoid biosynthetic polyphenol pxidase in vacuoles

类黄酮生物合成多酚氧化酶在液泡中的定位

DOI：
发表时间：
2006
期刊：
Plant J. 45
影响因子：
0
作者：
Kazuishi Makino;Yasuhiro Hiroki;Yasumasa Hamada;Haobo Guo et al.;Eiichiro Ono et al.
通讯作者：
Eiichiro Ono et al.

Localization of a flavonoid biosynthetic polyphenol oxidase in vacuoles

DOI：
10.1111/j.1365-313x.2005.02625.x
发表时间：
2006-01-01
期刊：
PLANT JOURNAL
影响因子：
7.2
作者：
Ono, E;Hatayama, M;Nakayama, T
通讯作者：
Nakayama, T

Menaquinone-specific prenyl reductase from the pyperthermophilic archaeon Archaeoglobus fulgidus

来自高温古菌 Archaeoglobus fulgidus 的甲基萘醌特异性异戊二烯还原酶

DOI：
发表时间：
2005
期刊：
J. Bacteriol. 187
影响因子：
0
作者：
Haobo Guo;et al.;Hisashi Hemmi et al.
通讯作者：
Hisashi Hemmi et al.

UDP-Glucuronic Acid : Anthocyanin Glucuronosyltranferase from Red Daisy (Bellis perennis) Flowers ENZYMOLOGY AND PHYLOGENETICS OF A NOVEL GLUCURONOSYLTRANSFERASE INVOLVED IN FLOWER PIGMENT BIOSYNTHESIS

UDP-葡萄糖醛酸：来自红雏菊（Bellis perennis）花的花青素葡萄糖醛酸基转移酶参与花色素生物合成的新型葡萄糖醛酸基转移酶的酶学和系统发育学

DOI：
发表时间：
2005
期刊：
J.Biol.Chem. 280
影响因子：
0
作者：
Haobo Guo;et al.;Hisashi Hemmi et al.;Jian Liu et al.;Eiichiro Ono et al.;Hisashi Hemmi et al.;Jian Liu et al.;Eiichiro Ono et al.;Shin'ya Sawada et al.
通讯作者：
Shin'ya Sawada et al.

Processing, catalytic activity and crystal structures of kumamolisin-As with an engineered active site