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

• 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

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.

Aureusidin合成酶是一种多酚氧化酶(PPO)，它专门催化查尔酮氧化形成极光，查尔酮是一种植物类黄酮，是导致金鱼草花黄色的原因。所有已知的PPO都定位于叶绿体中，而类黄酮的生物合成被认为发生在细胞质中(或在ER的细胞质表面)。然而，金黄色葡萄糖素合成酶的一级结构特征和一些分子性质与该酶在质体和细胞质中的定位相矛盾。本研究对该酶在黄鱼花瓣细胞中的亚细胞定位进行了研究。蔗糖密度梯度分析和差速离心法分析表明，该酶(39 kDa成熟型)不存在于叶绿体或内质网中。利用绿色荧光蛋白(GFP)嵌合体与PPO前体的前肽融合的瞬时分析表明，该酶定位于液泡腔内。我们还发现，PPO液泡靶向所需的信息编码在53个残基的N-末端序列(NTPP)中，而不是在前体的C-末端序列中。通过NTPP-GFP嵌合体与拟南芥SarI GTP酶的显性负突变体或与MRFP融合的GLOGI标记(拟南芥的H~+-转位无机焦磷酸酶)的共表达，证实了NTPP介导的内质网到高尔基体向液泡的转运。我们在前体的NTPP中确定了一个序列特异性的空泡分选决定因素。这提供了第一个在液泡中生物合成类黄酮骨架的例子。这种代谢区划应作为一种策略，以克服细胞质中前体查尔酮的生化不稳定性，从而导致极光在花中有效积累。

项目成果

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

• 发表时间: 2006
• 期刊: Plant J. 45
• 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

Yellow flowers generated by expression of the aurone biosynthetic pathway

• DOI: 10.1073/pnas.0604246103
• 发表时间: 2006-07-18
• 期刊: PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
• 影响因子: 11.1
• 作者: Ono, Eiichiro;Fukuchi-Mizutani, Masako;Tanaka, Yoshikazu
• 通讯作者: Tanaka, Yoshikazu

Menaquinone-specific prenyl reductase from the hyperthermophilic archaeon Archaeoglobus fulgidus

• DOI: 10.1128/jb.187.6.1937-1944.2005
• 发表时间: 2005-03-01
• 期刊: JOURNAL OF BACTERIOLOGY
• 影响因子: 3.2
• 作者: Hemmi, H;Takahashi, Y;Nishino, T
• 通讯作者: Nishino, T

Amperometric detection of the bacterial metabolic regulation with a microbial array chip.

• DOI: 10.1016/j.bios.2004.08.039
• 发表时间: 2005-07
• 期刊: Biosensors & bioelectronics
• 影响因子: 12.6
• 作者: K. Nagamine;Nobuto Matsui;T. Kaya;T. Yasukawa;H. Shiku;T. Nakayama;T. Nishino;T. Matsue