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Cited by 18Biotin and Related StudiesPolysaccharides and Plant Cell WallsPlant Molecular Biology Research

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

Open access
Zheng, Shuai, Noack, Lise C., Khammy, Ouda, De Meyer, Andreas, Khan, Ghazanfar Abbas, De Winne, Nancy, Eeckhout, Dominique, Van Damme, Daniël, Persson, Staffan·2025·Nature Communications
DOI 10.1038/s41467-025-56192-3PMC PMC11747095

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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.

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Fig. 1 PUP-IT identifies protein interactors of RACK1 and TPLATE in plant cells. a Schematic of the PUP-IT proximity labeling approach [Created in BioRender. Persson, S. (2025) https://BioRender.com/z33f531]. FLAG-Pup(E) is attached to Prey protein mediated by PafA fused to a bait protein. b Vectors designed to express the FLAG-Pup(E) and PafA fused to GFP/AtRACK1a/NbRACK1, with cassettes for PafA and Pup(E) expressions present in one binary vector. c Venn diagram generated from enriched proteins of transiently expressed AtRACK1a and NbRACK1 as PafA-baits in N. benthamiana compared to PafA-GFP, respectively. d Molecular function enrichment of proteins in the overlap of (c). e A PPI network of putative RACK1 interactors that is also supported by interactions from the String database (https://string-db.org/). Nodes indicate proteins and edges indicate interactions, with line thickness indicating the strength of data support. f Vectors designed to express STREPII-FLAG-Pup(E) under a Dexamethasone (DEX)-inducible promoter, combined with constitutive expression of PafA fused to TPLATE. g iBAQ, a measure for the abundance of TPLATE subunits. iBAQ of TPLATE subunits were normalized to the bait, TPLATE, for the different experiments (as indicated below).
Fig. 1 PUP-IT identifies protein interactors of RACK1 and TPLATE in plant cells. a Schematic of the PUP-IT proximity labeling approach [Created in BioRender. Persson, S. (2025) https://BioRender.com/z33f531]. FLAG-Pup(E) is attached to Prey protein mediated by PafA fused to a bait protein. b Vectors designed to express the FLAG-Pup(E) and PafA fused to GFP/AtRACK1a/NbRACK1, with cassettes for PafA and Pup(E) expressions present in one binary vector. c Venn diagram generated from enriched proteins of transiently expressed AtRACK1a and NbRACK1 as PafA-baits in N. benthamiana compared to PafA-GFP, respectively. d Molecular function enrichment of proteins in the overlap of (c). e A PPI network of putative RACK1 interactors that is also supported by interactions from the String database (https://string-db.org/). Nodes indicate proteins and edges indicate interactions, with line thickness indicating the strength of data support. f Vectors designed to express STREPII-FLAG-Pup(E) under a Dexamethasone (DEX)-inducible promoter, combined with constitutive expression of PafA fused to TPLATE. g iBAQ, a measure for the abundance of TPLATE subunits. iBAQ of TPLATE subunits were normalized to the bait, TPLATE, for the different experiments (as indicated below).CC BY-NC-ND · PubMed Central
Fig. 2 Employing PUP-IT to identify proteins in cellulose synthesis. a, b Volcano plots show differently enriched proteins in the PafA-CC1 and PafA-LTI6B groups from experiments in N. benthamiana (a) and Arabidopsis seedlings (b), based on a two-sided t-test with permutation-based FDR (FDR = 0.05, S0 = 1). Orange dots represent proteins unique to the PafA-CC1 group, maroon dots represent high-abundance proteins, gray dots represent proteins with no significant differences, and blue dots represent proteins more abundant in the control. CSC and BEN1-related proteins are marked with larger dots. c Co-localization of BEN1-mCherry and EGFP-CC1 in stable transgenic Arabidopsis. Scale bar = 10 µm. d Fluorescence intensity along the transect in (c). Images were taken from cells in the root tip area of 5-day-old seedlings. e BEN1-mCherry was labeled with FLAG when co-expressed with FLAG-Pup(E) and PafA-CC1 in N. benthamiana. RFP trap was used for immunoprecipitation, and α-RFP (mCherry) or α-FLAG antibodies for western blot. p19-only samples served as control. f BEN1-mCherry was co-immunoprecipitated with EGFP-CC1 in N. benthamiana. GFP trap was used for immunoprecipitation, and α-RFP (mCherry) or α-GFP antibodies for western blot. EGFP samples served as the negative control, and p19-only samples as the empty control. Arrowheads point to proteins of interest: BEN1-mCherry (white), EGFP (yellow) and EGFP-CC1 (red). g CC1 is a transmembrane protein with its N terminal facing the cytosol and C terminal in the apoplast, C terminal truncation (∆C), N terminal truncation (∆N). h Membrane split-ubiquitin Y2H was used to detect interactions between BEN1 and CC1, with CC1 constructs designed as shown in (g). Colony growth percentages on selection media from three replicates. Values are mean + SD. Significance was determined by one-way ANOVA followed by a Tukey’s test (p < 0.05). i BiFC assay assessed the interaction between BEN1 and CC1, with CC1 constructs designed as shown in (g). Scale bar = 50 µm. j Relative YFP to RFP ratio was measured along the cell outlines in BiFC images. Values are mean ± SD. Significance was determined by one-way ANOVA followed by a Tukey’s test (p < 0.05, n = 15).
Fig. 2 Employing PUP-IT to identify proteins in cellulose synthesis. a, b Volcano plots show differently enriched proteins in the PafA-CC1 and PafA-LTI6B groups from experiments in N. benthamiana (a) and Arabidopsis seedlings (b), based on a two-sided t-test with permutation-based FDR (FDR = 0.05, S0 = 1). Orange dots represent proteins unique to the PafA-CC1 group, maroon dots represent high-abundance proteins, gray dots represent proteins with no significant differences, and blue dots represent proteins more abundant in the control. CSC and BEN1-related proteins are marked with larger dots. c Co-localization of BEN1-mCherry and EGFP-CC1 in stable transgenic Arabidopsis. Scale bar = 10 µm. d Fluorescence intensity along the transect in (c). Images were taken from cells in the root tip area of 5-day-old seedlings. e BEN1-mCherry was labeled with FLAG when co-expressed with FLAG-Pup(E) and PafA-CC1 in N. benthamiana. RFP trap was used for immunoprecipitation, and α-RFP (mCherry) or α-FLAG antibodies for western blot. p19-only samples served as control. f BEN1-mCherry was co-immunoprecipitated with EGFP-CC1 in N. benthamiana. GFP trap was used for immunoprecipitation, and α-RFP (mCherry) or α-GFP antibodies for western blot. EGFP samples served as the negative control, and p19-only samples as the empty control. Arrowheads point to proteins of interest: BEN1-mCherry (white), EGFP (yellow) and EGFP-CC1 (red). g CC1 is a transmembrane protein with its N terminal facing the cytosol and C terminal in the apoplast, C terminal truncation (∆C), N terminal truncation (∆N). h Membrane split-ubiquitin Y2H was used to detect interactions between BEN1 and CC1, with CC1 constructs designed as shown in (g). Colony growth percentages on selection media from three replicates. Values are mean + SD. Significance was determined by one-way ANOVA followed by a Tukey’s test (p < 0.05). i BiFC assay assessed the interaction between BEN1 and CC1, with CC1 constructs designed as shown in (g). Scale bar = 50 µm. j Relative YFP to RFP ratio was measured along the cell outlines in BiFC images. Values are mean ± SD. Significance was determined by one-way ANOVA followed by a Tukey’s test (p < 0.05, n = 15).CC BY-NC-ND · PubMed Central
Fig. 3 BEN1 impacts cellulose synthesis. a Five-day-old seedlings grown on 1/2 MS media were transferred to soil and grown for 18 days in the greenhouse. Scale bar = 2 cm. b Rosette leaves were harvested from plants as those in (a) and cellulose levels were measured. Values are mean ± SD. Significance was determined by one-way ANOVA followed by a Tukey’s test (p < 0.05, n = 3). c Three-day-old seedlings grown on 1/2 MS media were transferred to media with 2 nM isoxaben for an additional 4 days. Scale bar = 0.5 cm (root elongation), scale bar = 0.5 mm (root tip area). d Root elongation after transfer to plates as indicated. Values are mean +/− SD. Significance was determined by one-way ANOVA followed by a Tukey’s test (p < 0.05, n ≥ 15). e Venus-CC1 and YFP-CESA6 in ben1 or wild-type were imaged following BFA (50 μM for 30 min) or DMSO (control) treatments. Scale bar = 20 µm. f BFA body distribution across cells in the root area, percentage area (% area; upper panel) and count per mm2 (lower panel) were used for statistical analyses. Values are mean±SD. Significance was determined by a two-sided t-test (* p < 0.05, ** p < 0.01, images from independent seedlings n > 5). g Venus-CC1 in ben1 or wild-type backgrounds were imaged following CHX + ES9-17/DMSO + BFA treatment (CHX 50 µM for 60 min, followed by ES9-17 30 µM /DMSO for 30 min, then BFA 50 µM for 30 min.). Scale bar = 20 µm. h BFA body distribution was reflected by the number of BFA bodies per cell; ≥100 cells were counted from images of independent seedlings n > 5 for each experiment repeated four times. Data from each replicate is shown in a different color. Significance was determined by the Kruskal-Wallis test, followed by pairwise comparison using Wilcoxon test, *** p < 0.001.
Fig. 3 BEN1 impacts cellulose synthesis. a Five-day-old seedlings grown on 1/2 MS media were transferred to soil and grown for 18 days in the greenhouse. Scale bar = 2 cm. b Rosette leaves were harvested from plants as those in (a) and cellulose levels were measured. Values are mean ± SD. Significance was determined by one-way ANOVA followed by a Tukey’s test (p < 0.05, n = 3). c Three-day-old seedlings grown on 1/2 MS media were transferred to media with 2 nM isoxaben for an additional 4 days. Scale bar = 0.5 cm (root elongation), scale bar = 0.5 mm (root tip area). d Root elongation after transfer to plates as indicated. Values are mean +/− SD. Significance was determined by one-way ANOVA followed by a Tukey’s test (p < 0.05, n ≥ 15). e Venus-CC1 and YFP-CESA6 in ben1 or wild-type were imaged following BFA (50 μM for 30 min) or DMSO (control) treatments. Scale bar = 20 µm. f BFA body distribution across cells in the root area, percentage area (% area; upper panel) and count per mm2 (lower panel) were used for statistical analyses. Values are mean±SD. Significance was determined by a two-sided t-test (* p < 0.05, ** p < 0.01, images from independent seedlings n > 5). g Venus-CC1 in ben1 or wild-type backgrounds were imaged following CHX + ES9-17/DMSO + BFA treatment (CHX 50 µM for 60 min, followed by ES9-17 30 µM /DMSO for 30 min, then BFA 50 µM for 30 min.). Scale bar = 20 µm. h BFA body distribution was reflected by the number of BFA bodies per cell; ≥100 cells were counted from images of independent seedlings n > 5 for each experiment repeated four times. Data from each replicate is shown in a different color. Significance was determined by the Kruskal-Wallis test, followed by pairwise comparison using Wilcoxon test, *** p < 0.001.CC BY-NC-ND · PubMed Central

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Authors
Zheng, S., Noack, L. C., Khammy, O., De Meyer, A., Khan, G. A., De Winne, N., Eeckhout, D., Van Damme, D., & Persson, S.
Affiliations
  • University of Copenhagen (DK)
  • Deakin University (AU)
  • Ghent University (BE)
Corresponding author
Persson, Staffan
ORCID
Venue
Nature Communications
Publisher
Springer Science and Business Media LLC
Published
2025-01-20
Volume
16
Issue
1
Pages
872-872
ISSN
2041-1723 (ISSN-L)
DOI
10.1038/s41467-025-56192-3
PMC
PMC11747095
OpenAlex
W4406609724
Cited by
18
References
55
Citations by year
2026: 12 · 2025: 6
FWCI
8.99
Type
journal article
Language
en
Open access
Licence
Locations
  • Nature Communications (publishedVersion, cc-by-nc-nd)
  • Research Square (acceptedVersion, cc-by)
  • PubMed Central (publishedVersion, cc-by-nc-nd)
  • PubMed (publishedVersion)
  • Research at the University of Copenhagen (University of Copenhagen) (publishedVersion, cc-by-nc-nd)
  • Ghent University Academic Bibliography (Ghent University) (submittedVersion, cc-by-nc-nd)
  • RePEc: Research Papers in Economics (submittedVersion)
  • DOAJ (DOAJ: Directory of Open Access Journals) (submittedVersion, cc-by-sa)
  • Figshare (submittedVersion, cc-by-nc-nd)
Funders
  • Novo Nordisk Fonden (19OC0056076, 20OC0060564, 23OC0086341)
  • Villum Fonden (25915)
Topics
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Cell Biology › Biotin and Related Studies
  • Life Sciences › Agricultural and Biological Sciences › Plant Science › Polysaccharides and Plant Cell Walls
  • Life Sciences › Agricultural and Biological Sciences › Plant Science › Plant Molecular Biology Research
Keywords
protein-protein interaction, cellulose biosynthesis, Arabidopsis thaliana, ARF-GEF, clathrin-mediated endocytosis, clathrin-independent endocytosis, plasma membrane, plant cell wall, protein complexes
MeSH terms
Biotin, Cell Membrane, Cellulose, Clathrin, Endocytosis, Glucosyltransferases, Staining and Labeling, Arabidopsis, Protein Interaction Mapping, Arabidopsis Proteins

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