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Proxiome assembly of the plant nuclear pore reveals an essential hub for gene expression regulation

Nature Plants volume 10, pages 1005–1017 (2024)Cite this article

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Abstract

The nuclear pore complex (NPC) is vital for nucleocytoplasmic communication. Recent evidence emphasizes its extensive association with proteins of diverse functions, suggesting roles beyond cargo transport. Yet, our understanding of NPC’s composition and functionality at this extended level remains limited. Here, through proximity-labelling proteomics, we uncover both local and global NPC-associated proteome in Arabidopsis, comprising over 500 unique proteins, predominantly associated with NPC’s peripheral extension structures. Compositional analysis of these proteins revealed that the NPC concentrates chromatin remodellers, transcriptional regulators and mRNA processing machineries in the nucleoplasmic region while recruiting translation regulatory machinery on the cytoplasmic side, achieving a remarkable orchestration of the genetic information flow by coupling RNA transcription, maturation, transport and translation regulation. Further biochemical and structural modelling analyses reveal that extensive interactions with nucleoporins, along with phase separation mediated by substantial intrinsically disordered proteins, may drive the formation of the unexpectedly large nuclear pore proteome assembly.

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Fig. 1: Assembly of the nuclear basket proteome in Arabidopsis using PL proteomics.
Fig. 2: Recruitment of proteins involved in translation regulation to the cytoplasmic side of the nuclear pore.
Fig. 3: Assembly of the NPC-associated proteome in Arabidopsis.
Fig. 4: Extensive protein–protein interactions within the NPC-associated proteome.
Fig. 5: Phase separation contributes to the assembly of the nuclear pore-associated proteome.

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Data availability

Datasets that support the results in this study are available in the supplementary tables and source data. All the mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE partner repository (Identifier: PXD039253). The high-confidence predicted heterodimer complexes are available via Zenodo at https://doi.org/10.5281/zenodo.10023066 (ref. 45). Other available databases used in this study are listed in the above ‘Bioinformatics analysis’. Source data are provided with this paper.

Code availability

All scripts used in this study are available in GitHub at https://github.com/s-kyungyong/NPC_structure_prediction.

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Acknowledgements

This work was supported by the Young Taishan Scholars Program of Shandong Province (to Y.T.), the National Institutes of Health Director’s Award (1DP2AT011967-01, to K.K.), and funds from the US National Science Foundation (MCB 2049931, to Y.G.) and the USDA National Institute of Food and Agriculture (HATCH project CA-B-PLB-0243-H, to Y.G.).

Author information

Authors and Affiliations

  1. Department of Plant and Microbial Biology, University of California, Berkeley, CA, USA

    Yu Tang, Kyungyong Seong, Ksenia Krasileva & Yangnan Gu

  2. Institute of Advanced Agricultural Sciences, Peking University, Weifang, Shandong, China

    Yu Tang, Xiangyun Yang & Mao Ye

  3. Center for Plant Biology, School of Life Sciences, Tsinghua University, Beijing, China

    Aobo Huang, Mengting Li & Qiao Zhao

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Contributions

Y.T., Q.Z. and Y.G. designed the research. Y.T., A.H. and M.L. performed proximity-labelling proteomics experiments. K.S. and K.K. performed protein–protein interaction prediction using AlphaFold-Multimer and AF2Complex. Y.T., X.Y. and M.Y. performed protein localization and co-IP analysis. Y.T. performed all other experiments. Y.T. and Y.G. wrote the paper. All authors analysed the data, discussed the results and edited the manuscript.

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Correspondence to Yangnan Gu.

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Nature Plants thanks Sachihiro Matsunaga 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 Nuclear basket proxiome reveals robust association of NPC with the spliceosome.

a, Schematic diagrams illustrating the DNA constructs carried by transgenic Arabidopsis plants utilized for proximity labeling (PL) proteomics. The positions of FG repeats are marked with red stars on Nup50a and CG1. b, Bubble plot presenting the reanalysis of PL proteomic data obtained using different Nups as bait. The left side displays LFQ intensity values of Nup50a protein, while the right side features a heatmap showing normalized peptide spectrum match (PSM) values of Nup50a. Controls for Nup82 and Nup93a are biotin-treated NEAP1-BioID2 sample (Ctrl 1) and mock-treated YFP-BioID2 sample (Ctrl 2). For GBPL3, non-transformant plants (NT) served as control (Ctrl 1). Controls for KAKU4, Nup188, and PNET1 were NT plants (Ctrl 1) and mock-treated YFP-BioID2 samples (Ctrl 2). For CG1 and Nup54, NT plants (Ctrl 1) and mock-treated transgenic plants (Ctrl 2) were used as controls. c, Scatter plot showing candidates identified in the Nup82 proxiome. Known nucleoporins and nucleoskeleton proteins are labeled. Controls for ratiometric analysis included NEAP1-BioID2 and mock samples. Three biological replicates were utilized for each sample. Significantly enriched protein candidates, denoted by red dots, were selected using cutoffs p-value < 0.01, fold-change > 4, and PSM > 1. On the right, GO enrichment analysis and heatmaps displaying averaged PSM values of known nucleoporins and other candidates in the Nup82 proxiome are presented. d, GO enrichment analysis of the integrated nuclear basket proxiome consisting of 423 proteins. Representative GO terms are displayed. e, Gene expression patterns of Nup82, Nup50a, KAKU4, and GBPL3 in different tissues and developmental stages of Arabidopsis. Transcriptomic data were retrieved from the TraVA database (Transcriptome Variation Analysis, http://travadb.org/). For protein candidates identified by proximity labeling, statistical tests were two-sided t-tests without adjustment (b and c). Statistical tests used for GO analysis in (c and d) were one-sided Fisher’s Exact tests with multiple comparison adjustments.

Extended Data Fig. 2 Comparative analysis of the CG1 proxiome and the GBPL3 proxiome.

The middle Venn diagram illustrates the overlaps and specific proteins probed by CG1 and GBPL3 using proximity labeling proteomics. The upper panel presents a combined GO analysis (red squares and blue edges) and protein-protein interaction analysis (gray edges, STRING) using the 53 overlapping preys probed by both CG1 and GBPL3. The lower panel displays GO analyses, and the GO terms of CG1 and GBPL3 specific preys are labeled with blue and yellow circles, respectively. The common GO term ‘RNA binding’ is positioned in the middle and labeled with a red square. Statistical tests used for GO analysis were one-sided Fisher’s Exact tests with multiple comparison adjustments.

Extended Data Fig. 3 Assembly of the NPC core scaffold proxiome.

a, Scatter plots displaying candidates identified in the Nup54 and Nup188 proxiomes. Mock-treated and non-transformant (NT) samples were used as controls for ratiometric analysis. Two biological replicates were used for the Nup54 proxiome, while three replicates were used for the Nup188 proxiome. Significantly enriched protein candidates were selected based on the following cutoffs: p-value < 0.1, fold-change > 2, and PSM > 5 for Nup54 proxiome; and p-value < 0.2, fold-change > 2, and PSM > 0.3 for Nup188 proxiome. Enriched candidates are represented by red dots and labeled in green text. Heatmaps illustrate the normalized average PSM values of the known nucleoporins. Statistical analyses were two-sided t-tests without adjustment. Underlying data can be found in Supplementary Table 5 and 6. b, Representative confocal images of the protein localization of Nup54-3xHA-miniTurbo and Nup188-BioID2-HA in transgenic plants by immunostaining using HA antibody. DAPI was used to stain the nucleus. The localization patterns have been repeated in three independent experiments with similar results. Bars = 10 μm. c, Assembly of the NPC core scaffold proxiome. Venn diagram representing the assembled NPC core scaffold proxiome probed by nucleoporins located at or near the NPC core. Known NPC and NE proteins are highlighted in red.

Extended Data Fig. 4 Predicted protein-protein interactions within the extended NPC proteome.

a, Pairwise interaction matrix of the 109 selected proteins within the NPC-associated proteome. The predicted interactions were generated using Alphafold-Multimer, and the interface scores derived from AF2Complex were plotted. The subgroup of proteins with strong interactions, located in the upper right corner of the matrix, is highlighted with dashed lines and green text label. b, Structure models depicting the predicted interactions between the following protein pairs: Nup93a and NDC1, Nup93b and NDC1, PNET1 and PNET6, GBPL3 and RAE1, and CRWN4 and MAD2. c, Nuclear membrane localization of selected candidates in NPC-associated proteome. GFP fusion constructs were transiently coexpressed with free mCherry in N. benthamiana. The localization patterns have been repeated in three independent experiments with similar results. Bars = 10 μm.

Extended Data Fig. 5 Nucleoporins and NPC-associated proteins are enriched in intrinsically disordered regions and FG-repeats.

a, Intrinsically disordered domains in selected Nups and NPC-associated proteins predicted by PONDR. b, Summary of NPC-associated FG repeats-containing proteins. Based on known Arabidopsis FG nucleoporins and previous investigations on FG repeats-containing proteins in animals, we used four criteria to determine FG repeats-containing proteins in Arabidopsis. Firstly, a functionally relevant FG repeat domain should contain a minimum of three FG repeats within 50 amino acids or at least four FG repeats within 200 amino acids. Secondly, Tyrosine-Glycine (YG) repeats are considered to function similar to FG repeats. Thirdly, we excluded candidates that possessed FG dipeptides within predicted transmembrane domains. Lastly, FG repeats were required to localize within predicted IDRs of a protein. By applying these criteria, we identified a total of 108 FG repeats-containing proteins in the NPC-associated proteome. D2P2 database and RNA-binding protein repertoire were used to determine whether they are also intrinsically disordered proteins (IDPs) and RNA-binding proteins (RBPs). RBPs were further divided into RBPs with high and low confidence, which were colored with light orange and dark orange, respectively. The bottom heatmap displays their average coexpression values with all known nucleoporins.

Supplementary information

Reporting Summary

Supplementary Information

Supplementary Table 1 Proteins identified by proximity-labelling proteomics using Nup50a as bait. Supplementary Table 2 The nuclear basket proteome assembled by proximity labelling. Supplementary Table 3 Comparison between nuclear basket proteome and MAC3b-associated proteins. Supplementary Table 4 Proteins identified by proximity-labelling proteomics using CG1 as bait. Supplementary Table 5 Proteins identified by proximity-labelling proteomics using Nup54 as bait. Supplementary Table 6 Proteins identified by proximity-labelling proteomics using Nup188 as bait. Supplementary Table 7 The extended nuclear pore complex-assocaited proteome assembled by proximity labelling. Supplementary Table 8 Functional categorization of nuclear pore complex-associated proteins. Supplementary Table 9 Co-expression analysis of nuclear pore complex-associated components with nucleoporin genes. Supplementary Table 10 Structural modelling-based prediction of protein–protein interactions between selected nucleoporins and NPC-associated proteins. Supplementary Table 11 List of intrinsically disordered proteins and RNA-binding proteins in the extended nuclear pore complex proteome. Supplementary Table 12 List of FG repeats-containing proteins in Arabidopsis. Supplementary Table 13 Primers used in this study.

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Unprocessed western blots.

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Tang, Y., Yang, X., Huang, A. et al. Proxiome assembly of the plant nuclear pore reveals an essential hub for gene expression regulation. Nat. Plants 10, 1005–1017 (2024). https://doi.org/10.1038/s41477-024-01698-9

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