与动物不同,植物和酵母仅具有III类磷脂酰肌醇3-激酶(PI3KC3)。它的脂质产品,磷脂酰肌醇3-磷酸酯(PtdIns-3-P,PI3P),组织细胞内运输途径,如自噬体形成,多囊体(MVB)形成,从跨高尔基网络(TGN)到晚期高尔基的逆向运输,以及自噬体与MVB和液泡之间的融合事件。植物PI3KC3的催化亚基由必需基因液泡蛋白分选34(VPS34)编码。尽管VPS34在细胞稳态和植物发育中的重要性,缺乏VPS34相互作用组。在这里我们使用了TurboID,一种酶催化的邻近标记(PL)方法,描述拟南芥VPS34的近端相互作用组。TurboID催化空间受限的生物素化,并通过亲和纯化与质谱联用,实现了273种蛋白质的VPS34特异性富集。相互作用组证实了VPS34在内溶酶体运输中的已知功能。有趣的是,碳水化合物代谢是最丰富的基因本体论(GO)术语,包括丙糖部分的糖酵解酶和在叶绿体三糖出口和蔗糖生物合成中起作用的酶。VPS34与糖酵解酶甘油醛-3-磷酸脱氢酶(GAPDH,GAPC1/2)在植物中得到了验证。还验证了VPS34与质膜H-ATPaseAHA2之间的相互作用,这是膜电位的主要决定因素。我们的研究将PI3KC3与碳水化合物代谢和膜电位联系起来,维持细胞稳态的两个关键过程。
Unlike animals, plants and yeasts only have a class III phosphatidylinositol 3-kinase (PI3KC3). Its lipid product, phosphatidylinositol 3-phosphate (PtdIns-3-P, PI3P), organizes intracellular trafficking routes such as autophagosome formation, multivesicular body (MVB) formation, retro-transport from trans-Golgi network (TGN) to late Golgi, and the fusion events between autophagosomes and MVBs and the vacuole. The catalytic subunit of plant PI3KC3 is encoded by the essential gene Vacuolar Protein Sorting 34 (
VPS34). Despite the importance of
VPS34 in cellular homeostasis and plant development, a
VPS34 interactome is lacking. Here we employed TurboID, an enzyme-catalyzed proximity labelling (PL) method, to describe a proximal interactome of Arabidopsis
VPS34. TurboID catalyzed spatially restricted biotinylation and enabled VPS34-specific enrichment of 273 proteins from affinity purification coupled with mass spectrometry. The interactome confirmed known functions of
VPS34 in endo-lysosomal trafficking. Intriguingly, carbohydrate metabolism was the most enriched Gene Ontology (GO) term, including glycolytic enzymes in the triose portion and enzymes functioning in chloroplast triose export and sucrose biosynthesis. The interaction between
VPS34 and the glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH, GAPC1/2) was validated in planta. Also verified was the interaction between VPS34 and the plasma membrane H+-ATPase AHA2, a primary determinant of membrane potential. Our study links PI3KC3 to carbohydrate metabolism and membrane potential, two key processes that maintain cellular homeostasis.