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シロイヌナズナζクラスホスホリパーゼDの細胞生物学的研究

島村, 亮太 京都大学 DOI:10.14989/doctor.k23599

2022.01.24

概要

本研究では、シロイヌナズナのζクラスホスホリパーゼD(PLD)であるPLDζ1およびPLDζ2に対して形質転換植物体を用いた細胞生物学的解析が行われ、それらの細胞内局在性およびその局在性に必要なタンパク質領域が解明された。

PLDは、生体膜を構成するグリセロリン脂質を加水分解しホスファチジン酸を生成することにより、膜脂質の代謝、膜交通、細胞骨格の再構成、細胞内シグナル伝達などの細胞内動的過程において重要な役割を果たす。高等植物では、PLD は生長・分化や環境応答を含む様々な生命現象に関与することが知られている。しかし、高等植物は多数のPLDをコードしており、それらの多くについて細胞内局在性が不明であるため、PLDがどのような細胞内動的過程を介してそれぞれの生命現象に関わるのかについては殆ど明らかにされてこなかった。

そこで、シロイヌナズナのPX-PH-PLDであるPLDζ1およびPLDζ2の細胞内局在性についての解析が行われた。それらPLDの蛍光融合タンパク質と様々な細胞内コンパートメントマーカーとの共局在性を調べたところ、自身の遺伝子のプロモーターにより発現したPLDζ1蛍光融合タンパク質はトランスゴルジネットワーク(TGN)全体に特異的に局在することが分かった。同様に自身の遺伝子のプロモーターにより発現したPLDζ2蛍光融合タンパク質は液胞膜、TGNの一部および多胞体を含む膜構造に局在することが分かった。

次に、PLDζ1およびPLDζ2の細胞内局在性に関与するタンパク質領域の解析が行われた。上記の局在パターンは、PLDζ1およびPLDζ2のPX-PHドメインを含むN末部分タンパク質の蛍光融合タンパク質でも再現されたことから、PX-PHドメインを含むN末領域がこれらの細胞内局在性を決定しているものと結論された。人工的発現誘導プロモーターを用いた実験において、PLDζ2のN末部分蛍光融合タンパク質は、まずTGNに局在し、次第に液胞膜への蓄積が増加する傾向が見られた。さらに、PLDζ2のN末端の39アミノ酸領域が液胞膜への積極的な蓄積に必要であること、PLDζ1のN末部分タンパク質のN末端領域をその39アミノ酸領域で置き換えると同様の液胞膜への蓄積が起こることなどが明らかになった。これらの結果から、PLDζ1およびPLDζ2はともにTNG膜上にリクルートされ、その後PLDζ2のみがそのN末端領域に依存して液胞膜上へ移動することが示唆された。

以上、PLDζ1とPLDζ2はTGNから液胞膜へのポストゴルジ膜交通経路に沿って部分的に重複して局在し、機能することによって様々な生理学的現象に関わるものと結論された。

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