Pupylation-based proximity labeling reveals regulatory factors in cellulose biosynthesis in Arabidopsis
Shuai Zheng, Lise C. Noack, Ouda Khammy, Andreas De Meyer, Ghazanfar Abbas Khan, Nancy De Winne, Dominique Eeckhout, Daniël Van Damme, Staffan Persson
Abstract
Knowledge about how and where proteins interact provides a pillar for cell biology. Protein proximity-labeling has emerged as an important tool to detect protein interactions. Biotin-related proximity labeling approaches are by far the most commonly used but may have labeling-related drawbacks. Here, we use pupylation-based proximity labeling (PUP-IT) as a tool for protein interaction detection in plants. We show that PUP-IT readily confirmed protein interactions for several known protein complexes across different types of plant hosts and that the approach increased detection of specific interactions as compared to biotin-based proximity labeling systems. To further demonstrate the power of PUP-IT, we used the system to identify protein interactions of the protein complex that underpin cellulose synthesis in plants. Apart from known complex components, we identified the ARF-GEF BEN1 (BFA-VISUALIZED ENDOCYTIC TRAFFICKING DEFECTIVE1). We show that BEN1 contributes to cellulose synthesis by regulating both clathrin-dependent and -independent endocytosis of the cellulose synthesis protein complex from the plasma membrane. Our results highlight PUP-IT as a powerful proximity labeling system to identify protein interactions in plant cells.