Pupylation-based proximity labelling reveals regulatory factors in cellulose biosynthesis in Arabidopsis

Abstract Knowledge about how and where proteins interact provides a pillar for cell biology. Protein proximity-labelling has emerged as an important tool to detect protein interactions. Here, biotin-related proximity labelling approaches are by far the most commonly used, but may have labelling-related drawbacks. Here, we use pupylation-based proximity labelling (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 labelling 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 labelling system to identify protein interactions in plant cells.