TurboID-based proximity labelling reveals a connection between VPS34 and cellular homeostasis

Taotao Wang; Xinjing Li; Ningjing Liu; Yi Yang; Qingqiu Gong

doi:10.1016/j.jplph.2023.154100

TurboID-based proximity labelling reveals a connection between VPS34 and cellular homeostasis

J Plant Physiol. 2023 Oct:289:154100. doi: 10.1016/j.jplph.2023.154100. Epub 2023 Sep 20.

Authors

Taotao Wang¹, Xinjing Li¹, Ningjing Liu¹, Yi Yang¹, Qingqiu Gong²

Affiliations

¹ State Key Laboratory of Microbial Metabolism & Joint International Research Laboratory of Metabolic and Developmental Sciences, School of Life Sciences and Biotechnology, Shanghai Jiao Tong University, Shanghai, 200240, PR China.
² State Key Laboratory of Microbial Metabolism & Joint International Research Laboratory of Metabolic and Developmental Sciences, School of Life Sciences and Biotechnology, Shanghai Jiao Tong University, Shanghai, 200240, PR China. Electronic address: gongqingqiu@sjtu.edu.cn.

PMID: 37748420
DOI: 10.1016/j.jplph.2023.154100

Abstract

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.

Keywords: Glycolysis; Membrane trafficking; Phosphoinositide; Protein-protein interaction; TurboID; VPS34; Vacuole.