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Adaptor protein complex interaction map in Arabidopsis identifies P34 as a common stability regulator

Nature Plants volume 9, pages 355–371 (2023)Cite this article

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Abstract

Adaptor protein (AP) complexes are evolutionarily conserved vesicle transport regulators that recruit coat proteins, membrane cargoes and coated vesicle accessory proteins. As in plants endocytic and post-Golgi trafficking intersect at the trans-Golgi network, unique mechanisms for sorting cargoes of overlapping vesicular routes are anticipated. The plant AP complexes are part of the sorting machinery, but despite some functional information, their cargoes, accessory proteins and regulation remain largely unknown. Here, by means of various proteomics approaches, we generated the overall interactome of the five AP and the TPLATE complexes in Arabidopsis thaliana. The interactome converged on a number of hub proteins, including the thus far unknown adaptin binding-like protein, designated P34. P34 interacted with the clathrin-associated AP complexes, controlled their stability and, subsequently, influenced clathrin-mediated endocytosis and various post-Golgi trafficking routes. Altogether, the AP interactome network offers substantial resources for further discoveries of unknown endomembrane trafficking regulators in plant cells.

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Fig. 1: Integrative interaction network of six multimeric adaptor complexes and their accessory proteins.
Fig. 2: Selection from the AP interactome of putative regulators and their crosstalk in vesicle trafficking.
Fig. 3: GO term enrichment analysis of the AP-associated proteins.
Fig. 4: Validation of AP-5, AAK1, BAG4 and P34 protein interaction networks.
Fig. 5: P34 regulates the stability of AP-1, AP-2 and AP-4.
Fig. 6: P34 regulates vesicle trafficking depending on AP-1, AP-2 and AP-4.

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Data availability

The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE partner repository with the dataset identifier PXD035454 and 10.6019/PXD035454. Source data are provided with this paper.

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Acknowledgements

We thank I. Hwang, G. Jürgens and J. Pan for the kind gift of α-AP1G and α-AP2A, α-KNOLLE and α-AP2S antibodies, respectively, M. Sauer, S. Schneider, J. Friml, J. Lin and I. Hara-Nishimura for providing published materials, T. Jacobs for useful discussions and M. De Cock for help in preparing the manuscript. This work was supported by the Research Foundation-Flanders projects (G008416N, G0E5718N and 3G038020 to E.R.), the Belgian Science Policy Office for a postdoctoral fellowship (R.K.), the China Scholarship Council for predoctoral fellowships (P.W., X.Z. and R.W.) and the European Research Council T-Rex (project number 682436 to D.V.D.).

Author information

Author notes

  1. Rahul Kumar

    Present address: Department of Plant Sciences, University of Hyderabad, Hyderabad, India

  2. Anaxi Houbaert

    Present address: Department of Plant Molecular Biology, University of Lausanne, Lausanne, Switzerland

  3. These authors contributed equally: Peng Wang, Wei Siao.

  4. These authors jointly supervised this work: Wei Siao, Eugenia Russinova.

Authors and Affiliations

  1. Department of Plant Biotechnology and Bioinformatics, Ghent University, Ghent, Belgium

    Peng Wang, Wei Siao, Xiuyang Zhao, Deepanksha Arora, Ren Wang, Dominique Eeckhout, Jelle Van Leene, Rahul Kumar, Anaxi Houbaert, Nancy De Winne, Evelien Mylle, Michael Vandorpe, Steffen Vanneste, Geert De Jaeger, Daniël Van Damme & Eugenia Russinova

  2. Center for Plant Systems Biology, VIB, Ghent, Belgium

    Peng Wang, Wei Siao, Xiuyang Zhao, Deepanksha Arora, Ren Wang, Dominique Eeckhout, Jelle Van Leene, Rahul Kumar, Anaxi Houbaert, Nancy De Winne, Evelien Mylle, Michael Vandorpe, Steffen Vanneste, Geert De Jaeger, Daniël Van Damme & Eugenia Russinova

  3. Plant Physiology and Cell Biology, Swammerdam Institute for Life Sciences, University of Amsterdam, Amsterdam, the Netherlands

    Ruud A. Korver & Christa Testerink

  4. Laboratory of Plant Physiology, Wageningen University & Research, Wageningen, the Netherlands

    Christa Testerink

  5. Department of Biomolecular Medicine, Ghent University, Ghent, Belgium

    Kris Gevaert

  6. Center for Medical Biotechnology, VIB, Ghent, Belgium

    Kris Gevaert

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  1. Peng Wang
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Contributions

W.S., P.W., X.Z. and E.R. initiated the project and designed experiments. W.S., X.Z., R.K., A.H. and N.D.W. did cloning for TAP–MS and AP–MS. D.E., J.V.L., K.G. and G.D.J. performed the MS work and analysed data. W.S. and P.W. did the interactome validation. P.W. performed all the P34 work. E.M. did microscopy. R.A.K. and C.T. contributed materials. D.A., M.V. and D.V.D. did the PL. R.W. and S.V. performed the PIN2 immunolabelling. W.S., P.W. and E.R. wrote the manuscript. All authors revised the manuscript.

Corresponding authors

Correspondence to Wei Siao or Eugenia Russinova.

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The authors declare no competing interests.

Peer review

Peer review information

Nature Plants thanks Takashi Ueda, Michael Sauer and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

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Extended data

Extended Data Fig. 1 Dot plot matrix of selected proteins from the AP/TPC interactome.

Quantitative dot plot matrix covering core AP/TPC subunits and a selection of proteins linked to endocytosis/vesicle trafficking. The colour hue of the nodes corresponds with the abundance of each prey in a given experiment, calculated by subtracting the average normalized spectral abundance factor (NSAF) in the control dataset from the average NSAF (bait) of each prey [NSAF (bait - ctrl.)]. The size of the dots reflects the relative abundance of each prey over the different experiments. The identification of each bait protein is shown by an asterisk.

Extended Data Fig. 2 Protein sequence alignment of P34 in eukaryotes.

Amino acid sequence alignment of the P34/AAGAB family in At, Arabidopsis thaliana; Hs, Homo sapiens; Mm, Mus musculus; Ce, Caenorhabditis elegans; Sc, Saccharomyces cerevisiae; Dm, Drosophila melanogaster. The sequences were aligned with the CLC Main Workbench (Qiagen). The colour intensity reflects how conserved a particular position is in the alignment. Dark orange and dark purple represents 100% and 0% identity, respectively.

Extended Data Fig. 3 Protein sequence alignment of AAK1 in eukaryotes.

Amino acid sequence alignment of the NAK family in At, Arabidopsis thaliana; Hs, Homo sapiens; Mm, Mus musculus; Dm, Drosophila melanogaster; Ce, Caenorhabditis elegans, and Sc, Saccharomyces cerevisiae. The sequences were aligned with CLC Main Workbench (Qiagen). The colour intensity reflects how conserved the particular position is in the alignment. Dark orange and dark purple represent 100% and 0% identity, respectively.

Extended Data Fig. 4 rBiFC analysis of AP-5, AAK1 and BAG4.

rBiFC assay of AP-5 subunits (a), AAK1 (b) and BAG4 (c) with different AP subunits quantified in Fig. 4b,e,h, respectively. Scale bars, 20 µm.

Extended Data Fig. 5 rBiFC and co-immunoprecipitation (co-IP) analyses of P34.

rBiFC assays of P34 with different AP subunits (quantified in Fig. 4k) (a,b), with AAK1 and BAG4 (d). c,e, Quantification of rBiFC (YFP/RFP) in (b) and (d), respectively. n = 15, n, number of cells analysed. The significant differences were determined by one way Brown-Forsythe and Welch ANOVA tests combined with Dunnett T3 multiple comparisons test. **P ≤ 0.01, ***P ≤ 0.001; ns, not significant. The bounds of the boxes represent the 25th to 75th percentiles, the center line of the box and the whiskers indicate the median, the minimum and the maximum values, respectively. All individual values were plotted. rBiFC experiments were repeated twice and one representative experiment is shown. Scale bars, 50 µm. f, Validation of the interactions between P34, AP3B, and AP4E by co-IP in tobacco leaves transiently expressing the p35S:P34-mCherry, p35S:AP3B-GFP and p35S:AP4E-GFP constructs. g, co-IP analysis in ap4m-2 protoplasts transiently expressing p35S:P34-GFP. P34-GFP was pulled down with a GFP-trap and AP1G and AP2A were detected with the α-AP1G and α-AP2A antibodies. The p35S:GFP construct was used as a negative control (f,g), The two western blots were repeated two times. One representative experiment is shown.

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Extended Data Fig. 6 BiFC analysis of NECAP-1 and the putative AP2M cargos discovered by PL-MS.

a, rBiFC assay of NECAP-1 with different AP-1 and AP-2 subunits. Scale bars, 50 µm. b, Cytoscape model summarizing the interactions between various subunits and NECAP-1. Edge colours indicate the analysis method. Node colours correspond to the different complexes and protein families, red and blue, for AP-1 and AP-2, respectively. c, Quantification of rBiFC (YFP/RFP) in (a). rBiFC experiments were repeated twice and one representative experiment is shown. n = 15. d, rBIFC assay of AP2M interaction with KNOLLE and FORMIN-LIKE PROTEIN 6 (FH6), observed by PL-MS with AP2M as bait. The SHAGGY-like kinase BIN2 was used as negative control. Whereas AP2M interacts with KNOLLE evenly along the plasma membrane, the interaction between AP2M and FH6 is less intense and concentrated in plasma membrane-associated punctate. AP2M and BIN2 do not visually interact and only background and chlorophyll fluorescence are observed. e, Quantification of rBiFC (YFP/RFP PM intensity ratio) in (d) clearly shows significant differences between AP2M-KNOLLE and AP2M-BIN2. The punctate signal in the AP2M-FH6 combination is not sufficiently strong to yield a statistical difference compared to the control. n, number of cells analysed (c,e). The significant differences (c,e) were determined by one way Brown-Forsythe and Welch ANOVA tests with Dunnett T3 multiple comparisons test. ***P < 0.001; ns, not significant. The bounds of the boxes represent the 25th to 75th percentiles, the center line of the box indicates and the whiskers indicate the median, the minimum and the maximum values, respectively. All individual values were plotted. Scale bar, 50 µm.

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Extended Data Fig. 7 Genotype of the P34-CRISPR lines.

a, Schematic representation of the CRISPR editing on the P34 gene in the p34 CRISPR mutants. Blue, red, and green indicate insertion, nucleotide exchange, and mutated amino acids, respectively. The arrows mark the sites of the guide RNA (gRNA) sequences. b, Protein sequence alignment of P34 (wild type, Col-0) and P34(Δ) by means of the CLC Main Workbench (Qiagen). Hyphen and green letters indicate deletion and mutated amino acids, respectively. Asterisks mark the stop codons.

Extended Data Fig. 8 Phenotype of the P34-CRISPR lines.

a, Rosettes of wild type (Col-0), p34-1(+/-), p34-2(+/-), p34-3(Δ/-), p34-3(Δ/Δ) mutants, and p34-1(-/-) (lines #9 and #12) and p34-3(Δ/-) (lines #4 and #5) mutants complemented with the pP34:gP34-GFP construct grown in the soil for 4 weeks. Scale bar, 20 mm. b, Quantification the rosette leaf area of each genotype show in (a). Three independent experiments were combined, each with 8-9 plants per genotype. ***P ≤ 0.001 (one-way ANOVA test); ns, not significant. The bounds of the boxes represent the 25th to 75th percentiles, the center line of the box and the whiskers indicate the median, the minimum and the maximum values, respectively. All individual values were plotted. c, Aborted seeds in the siliques of genotypes in (b). Arrows indicate the aborted seeds. d, Quantification of the aborted seeds in (c). χ2 values were calculated with the χ2 test. n number of seeds analysed. e, Embryogenesis of the wild type and the p34-2(+/-) mutant. Differential interference contrast (DIC) microscopy of cleared whole-mount seeds at 6, 7 and 14 DAP (days after pollination). The ratio indicates the ratio of observed embryo stage/total embryos. Scale bars, 20 μm. The quantification data are combined from two experiments. Representative images from one experiment are shown. f, Phenotype of 7-day-old seedlings grown on ½MS. Scale bar, 10 mm. g, Primary root length of seedlings shown in (f). All the individual values were plotted and the center line represents the median. Twenty roots per genotype were measured. ***P ≤ 0.001 (one-way ANOVA test).

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Extended Data Fig. 9 CRISPR efficiency and different phenotypes of the p34 mutants.

a, Gene editing efficiency analysis of three random 9-day-old plants of p34iCRISPR-1 and p34iCRISPR-2 after induction with 10 μM β-oestradiol (Est). The sequencing results were analysed with the online software Inference of CRISPR Edits (ICE) (https://ice.synthego.com/#/). b, P34-GFP localization in roots of 5-day-old p35S:gP34-GFP/p34iCRISPR-1 plants grown on DMSO and 10 μM Est. Arrows indicate the remaining P34-GFP signal. Scale bar, 10 µm. c, Protein levels of AP1G, AP2A and AP2S in p34-3 mutants analysed by immunoblotting with α-AP1G, α-AP2S, α-AP2A, α-CHC, α-TPLATE and α-Tubulin, The experiments were repeated three times. One representative experiment is shown. d, Quantification of protein levels shown in (c). The protein level was normalized to tubulin. e, Confocal images of the 5-day-old pP34:gP34-GFP/p34-1(-/-) (line #12) root cells stained with FM4-64 (2 μM, 40 min). Scale bar, 10 µm. The imaging was repeated three times. One representative experiment is shown. f, FM4-64 uptake in the p34-3 mutants. g, Fluorescence intensity ratio of the relative intracellular-to-plasma membrane (PM) FM4-64 signal. All the individual value were plotted and the center line represents the median. ***P ≤ 0.001 (one-way ANOVA test); ns, not significant. The P values versus the Col-0 control for p34(Δ/-) = 0.0004, p34(Δ/Δ) = 0.6980 and ap2m-2 < 0.0001. n = 30, n number of cells analysed, Scale bar, 10 µm.

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Extended Data Fig. 10 Vesicular trafficking pathways affected in the p34 mutants.

a, secRFP localization in roots of 5-day-old Col-0 and p34iCRISPR-1 plants grown on DMSO and 10 μM β-oestradiol (Est). b, Immunofluorescence staining of KNOLLE in the root meristem of Col-0, p34iCRISPR-1 and p34iCRISPR-2 plants treated with 10 μM Est. c, PIN2-GFP localization in the roots of 5-day-old Col-0 and p34iCRISPR-1 plants grown on DMSO and 10 μM Est in the dark. d, Percentage of epidermal cells with GFP signals in the vacuoles as shown in (c), Error bars represent SD. e, Morphology of the lytic vacuoles in root cells of 7-day-old Col-0, p34iCRISPR-1 and p34iCRISPR-2 plants grown on DMSO and 10 μM Est. Images were taken after 2 h of staining with 4 μM FM4-64. Scale bars, 10 µm (a-c,e), The imaging of all genotypes was repeated three times. One representative experiment is shown.

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Supplementary information

Reporting Summary

Supplementary Tables 1–5

Supplementary Table 1. Proteomic analysis of adaptor complex interactome in Arabidopsis cell suspension cultures. Supplementary Table 2. GO analysis of adaptor complexes interactome in Arabidopsis. Supplementary Table 3. Summary of protein–protein interactions tested by rBiFC assay. Supplementary Table 4. Oligonucleotides used in this study. Supplementary Table 5. Accession numbers used for sequence alignment.

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Wang, P., Siao, W., Zhao, X. et al. Adaptor protein complex interaction map in Arabidopsis identifies P34 as a common stability regulator. Nat. Plants 9, 355–371 (2023). https://doi.org/10.1038/s41477-022-01328-2

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