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植物液胞タンパク質の細胞内輸送に関わる細胞質因子の解析

植物液泡蛋白细胞内运输涉及的细胞质因子分析

基本信息

批准号：
12740446
负责人：
嶋田知生
金额：
$ 1.41万
依托单位：
Kyoto University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Encouragement of Young Scientists (A)
财政年份：
2000
资助国家：
日本
起止时间：
2000 至 2001
项目状态：
已结题

项目摘要

輸送レセプターPV72はタイプ1の膜タンパク質で,ルーメンドメインで液胞タンパク質と結合しその細胞内輸送を行っている.PV72のC末端37アミノ酸は細胞質側に出ており,他の因子と相互作用することにより,PV72の細胞内局在性や機能を制御していると考えられる.本研究では,PV72の細胞質ドメインに注目し,輸送を担う輸送装置の面から,液胞タンパク質の細胞内輸送の分子機構を解析した.輸送レセプターPV72のルーメンドメインをGFPに置換したコンストラクトをシロイヌナズナ培養細胞に一過的に発現させた.その結果,細胞質中にドット状の蛍光パターンが見られた.培養細胞の内在性のレセプターを蛍光抗体法で検出すると,GFP蛍光と一致したことから,本コンストラクトは正しい細胞内局在性を反映していると考えられた.一過的発現でドット状の蛍光パターンになった後に,細胞を薬剤ブレフェルジンAで処理すると,GFP蛍光はネットワーク状になった.さらに,GFP蛍光はRGP1タンパク質の蛍光抗体法によるパターンと一致した.これらの結果から,本レセプターはゴルジ体に局在していることが判明した.PV72の細胞質ドメインと相互作用する因子を探索するために,上記GFPコンストラクトのデリーションシリーズを作製し解析した.その結果,細胞質ドメインを削るとゴルジ体には留まれず,GFPが細胞膜まで運ばれることが判明した.C末端35アミノ酸まで削っても同様の結果であった.このことから,細胞質ドメインのN末付近の配列が何らかの因子と相互作用しゴルジ体局在化に必要でると考えられた.細胞質ドメインのチロシンモチーフのチロシンをアラニンに置換すると,液胞に到達しやすくなることが判明した.このことから,チロシンモチーフが細胞質因子と相互作用し,PV72のゴルジ体へのリサイクルに効いている可能性が考えられた.

In this paper, we study the effects of different concentrations of PV72 on the membrane, the temperature of the liquid cell, the temperature of the cell, the temperature, the temperature, the temperature, In this study, PV72 cells pay close attention to the equipment, the liquid cell is responsible for the analysis of the molecular mechanism of intracellular transport. Please send your PV72 information to you. You may need to know that the GFP settings are correct. You need to learn how to improve the quality of your cell. The results show that there is no light in the cell. In the culture of the intrinsic nature of the cell, the light antibody method is used to detect the internal sex of the cell. The GFP antibody method is consistent with the results of the photoantibody method. As soon as you see the light, the light. GFP, RGP1, RGP1, photoantibody, photoantibody. According to the results of the results, the Department of physical Education has determined that the interaction between PV72 and PV72 cells has been detected. The factors have been explored, and the data have been analyzed in the GFP environment. The results showed that there was no significant difference between the two groups. The results showed that there was no significant difference between the two groups. The results showed that there was no significant difference between the two groups. The results showed that there was no significant difference between the two groups. The results showed that there was no significant difference between the two groups. The results showed that there was no significant difference between the two groups. The results showed that there was no significant difference between the two groups. The results showed that the results were the same as those at the end of the C terminal. We need to know how to arrange the interaction between different factors. I don't know what to do. I don't know. I don't know what to do. The possibility of cell interaction is different from that of PV72.