Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

Global profiling of plant nuclear membrane proteome in Arabidopsis

Abstract

The nuclear envelope (NE) is structurally and functionally vital for eukaryotic cells, yet its protein constituents and their functions are poorly understood in plants. Here, we combined subtractive proteomics and proximity-labelling technology coupled with quantitative mass spectrometry to understand the landscape of NE membrane proteins in Arabidopsis. We identified ~200 potential candidates for plant NE transmembrane (PNET) proteins, which unravelled the compositional diversity and uniqueness of the plant NE. One of the candidates, named PNET1, is a homologue of human TMEM209, a critical driver for lung cancer. A functional investigation revealed that PNET1 is a bona fide nucleoporin in plants. It displays both physical and genetic interactions with the nuclear pore complex (NPC) and is essential for embryo development and reproduction in different NPC contexts. Our study substantially enlarges the plant NE proteome and sheds new light on the membrane composition and function of the NPC.

Access options

Rent or Buy article

Get time limited or full article access on ReadCube.

from$8.99

All prices are NET prices.

Fig. 1: Identification of PNET candidates by subtractive proteomics.
Fig. 2: Identification of PNET candidates by proximity-labelling proteomics.
Fig. 3: Subcellular localization of new PNETs and comparative genomic analysis and coexpression analysis of PNETs.
Fig. 4: PNET1 is a bona fide plant nucleoporin.

Data availability

The data that support the results in this study are available from the corresponding author on reasonable request. All the MS proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE partner repository (identifier, PXD015919). All MS datasets are listed in Supplementary Table 10. Source data are provided with this paper.

References

  1. 1.

    Wilson, K. L. & Berk, J. M. The nuclear envelope at a glance. J. Cell Sci. 123, 1973–1978 (2010).

    CAS  PubMed  PubMed Central  Google Scholar 

  2. 2.

    Gomez-Cavazos, J. S. & Hetzer, M. W. Outfits for different occasions: tissue-specific roles of nuclear envelope proteins. Curr. Opin. Cell Biol. 24, 775–783 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

  3. 3.

    Smoyer, C. J. et al. Analysis of membrane proteins localizing to the inner nuclear envelope in living cells. J. Cell Biol. 215, 575–590 (2016).

    CAS  PubMed  PubMed Central  Google Scholar 

  4. 4.

    Zhou, X., Graumann, K., Wirthmueller, L., Jones, J. D. & Meier, I. Identification of unique SUN-interacting nuclear envelope proteins with diverse functions in plants. J. Cell Biol. 205, 677–692 (2014).

    CAS  PubMed  PubMed Central  Google Scholar 

  5. 5.

    Wang, H., Dittmer, T. A. & Richards, E. J. Arabidopsis CROWDED NUCLEI (CRWN) proteins are required for nuclear size control and heterochromatin organization. BMC Plant Biol. 13, 200 (2013).

    PubMed  PubMed Central  Google Scholar 

  6. 6.

    Zhao, Q., Brkljacic, J. & Meier, I. Two distinct interacting classes of nuclear envelope-associated coiled-coil proteins are required for the tissue-specific nuclear envelope targeting of Arabidopsis RanGAP. Plant Cell 20, 1639–1651 (2008).

    CAS  PubMed  PubMed Central  Google Scholar 

  7. 7.

    Zhou, X., Graumann, K., Evans, D. E. & Meier, I. Novel plant SUN–KASH bridges are involved in RanGAP anchoring and nuclear shape determination. J. Cell Biol. 196, 203–211 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

  8. 8.

    Schirmer, E. C., Florens, L., Guan, T., Yates, J. R. 3rd & Gerace, L. Nuclear membrane proteins with potential disease links found by subtractive proteomics. Science 301, 1380–1382 (2003).

    CAS  PubMed  Google Scholar 

  9. 9.

    Kim, D. I. et al. An improved smaller biotin ligase for BioID proximity labeling. Mol. Biol. Cell 27, 1188–1196 (2016).

    CAS  PubMed  PubMed Central  Google Scholar 

  10. 10.

    Tamura, K., Fukao, Y., Iwamoto, M., Haraguchi, T. & Hara-Nishimura, I. Identification and characterization of nuclear pore complex components in Arabidopsis thaliana. Plant Cell 22, 4084–4097 (2010).

    CAS  PubMed  PubMed Central  Google Scholar 

  11. 11.

    Goto, C., Tamura, K., Fukao, Y., Shimada, T. & Hara-Nishimura, I. The novel nuclear envelope protein KAKU4 modulates nuclear morphology in Arabidopsis. Plant Cell 26, 2143–2155 (2014).

    CAS  PubMed  PubMed Central  Google Scholar 

  12. 12.

    Graumann, K., Runions, J. & Evans, D. E. Characterization of SUN-domain proteins at the higher plant nuclear envelope. Plant J. 61, 134–144 (2010).

    CAS  PubMed  Google Scholar 

  13. 13.

    Varas, J. et al. Absence of SUN1 and SUN2 proteins in Arabidopsis thaliana leads to a delay in meiotic progression and defects in synapsis and recombination. Plant J. 81, 329–346 (2015).

    CAS  PubMed  Google Scholar 

  14. 14.

    Zhou, X., Groves, N. R. & Meier, I. SUN anchors pollen WIP–WIT complexes at the vegetative nuclear envelope and is necessary for pollen tube targeting and fertility. J. Exp. Bot. 66, 7299–7307 (2015).

    CAS  PubMed  PubMed Central  Google Scholar 

  15. 15.

    Hung, V. et al. Proteomic mapping of cytosol-facing outer mitochondrial and ER membranes in living human cells by proximity biotinylation. eLife 6, e24463 (2017).

    PubMed  PubMed Central  Google Scholar 

  16. 16.

    Lambert, J. P., Tucholska, M., Go, C., Knight, J. D. & Gingras, A. C. Proximity biotinylation and affinity purification are complementary approaches for the interactome mapping of chromatin-associated protein complexes. J. Proteomics 118, 81–94 (2015).

    CAS  PubMed  Google Scholar 

  17. 17.

    Liu, X. et al. An AP-MS- and BioID-compatible MAC-tag enables comprehensive mapping of protein interactions and subcellular localizations. Nat. Commun. 9, 1188 (2018).

    PubMed  PubMed Central  Google Scholar 

  18. 18.

    Shin, J. J. H., Gillingham, A. K., Begum, F., Chadwick, J. & Munro, S. TBC1D23 is a bridging factor for endosomal vesicle capture by golgins at the trans-Golgi. Nat. Cell Biol. 19, 1424–1432 (2017).

    CAS  PubMed  PubMed Central  Google Scholar 

  19. 19.

    Conlan, B., Stoll, T., Gorman, J. J., Saur, I. & Rathjen, J. P. Development of a rapid in planta BioID system as a probe for plasma membrane-associated immunity proteins. Front. Plant Sci. 9, 1882 (2018).

    PubMed  PubMed Central  Google Scholar 

  20. 20.

    Khan, M., Youn, J. Y., Gingras, A. C., Subramaniam, R. & Desveaux, D. In planta proximity dependent biotin identification (BioID). Sci. Rep. 8, 9212 (2018).

    PubMed  PubMed Central  Google Scholar 

  21. 21.

    Lin, Q. et al. Screening of proximal and interacting proteins in rice protoplasts by proximity-dependent biotinylation. Front. Plant Sci. 8, 749 (2017).

    PubMed  PubMed Central  Google Scholar 

  22. 22.

    Mair, A., Xu, S. L., Branon, T. C., Ting, A. Y. & Bergmann, D. C. Proximity labeling of protein complexes and cell-type-specific organellar proteomes in Arabidopsis enabled by TurboID. eLife 8, e47864 (2019).

    PubMed  PubMed Central  Google Scholar 

  23. 23.

    Zhang, Y. et al. TurboID-based proximity labeling reveals that UBR7 is a regulator of N NLR immune receptor-mediated immunity. Nat. Commun. 10, 3252 (2019).

    CAS  PubMed  PubMed Central  Google Scholar 

  24. 24.

    Pawar, V. et al. A novel family of plant nuclear envelope-associated proteins. J. Exp. Bot. 67, 5699–5710 (2016).

    CAS  PubMed  Google Scholar 

  25. 25.

    Kosugi, S., Hasebe, M., Tomita, M. & Yanagawa, H. Systematic identification of cell cycle-dependent yeast nucleocytoplasmic shuttling proteins by prediction of composite motifs. Proc. Natl Acad. Sci. USA 106, 10171–10176 (2009).

    CAS  PubMed  Google Scholar 

  26. 26.

    Dou, X. Y. et al. AtTMEM18 plays important roles in pollen tube and vegetative growth in Arabidopsis. J. Integr. Plant Biol. 58, 679–692 (2016).

    CAS  PubMed  PubMed Central  Google Scholar 

  27. 27.

    Neelakandan, A. K. et al. Molecular characterization and functional analysis of Glycine max sterol methyl transferase 2 genes involved in plant membrane sterol biosynthesis. Plant Mol. Biol. 74, 503–518 (2010).

    CAS  PubMed  Google Scholar 

  28. 28.

    Yang, X. H., Xu, Z. H. & Xue, H. W. Arabidopsis membrane steroid binding protein 1 is involved in inhibition of cell elongation. Plant Cell 17, 116–131 (2005).

    CAS  PubMed  PubMed Central  Google Scholar 

  29. 29.

    Romanauska, A. & Kohler, A. The inner nuclear membrane is a metabolically active territory that generates nuclear lipid droplets. Cell 174, 700–715 (2018).

    CAS  PubMed  PubMed Central  Google Scholar 

  30. 30.

    Poulet, A., Probst, A. V., Graumann, K., Tatout, C. & Evans, D. Exploring the evolution of the proteins of the plant nuclear envelope. Nucleus 8, 46–59 (2017).

    CAS  PubMed  Google Scholar 

  31. 31.

    Gu, Y. et al. Nuclear pore permeabilization is a convergent signaling event in effector-triggered immunity. Cell 166, 1526–1538 (2016).

    CAS  PubMed  PubMed Central  Google Scholar 

  32. 32.

    Obayashi, T., Aoki, Y., Tadaka, S., Kagaya, Y. & Kinoshita, K. ATTED-II in 2018: a plant coexpression database based on investigation of the statistical property of the mutual rank index. Plant Cell Physiol. 59, e3 (2018).

    PubMed  Google Scholar 

  33. 33.

    Fujitomo, T., Daigo, Y., Matsuda, K., Ueda, K. & Nakamura, Y. Critical function for nuclear envelope protein TMEM209 in human pulmonary carcinogenesis. Cancer Res. 72, 4110–4118 (2012).

    CAS  PubMed  Google Scholar 

  34. 34.

    Goto, C., Hashizume, S., Fukao, Y., Hara-Nishimura, I. & Tamura, K. Comprehensive nuclear proteome of Arabidopsis obtained by sequential extraction. Nucleus 10, 81–92 (2019).

    CAS  PubMed  PubMed Central  Google Scholar 

  35. 35.

    Wang, S. et al. A noncanonical role for the CKI-RB-E2F cell-cycle signaling pathway in plant effector-triggered immunity. Cell Host Microbe 16, 787–794 (2014).

    CAS  PubMed  PubMed Central  Google Scholar 

  36. 36.

    Gu, Y. The nuclear pore complex: a strategic platform for regulating cell signaling. New Phytol. 219, 25–30 (2018).

    PubMed  Google Scholar 

  37. 37.

    Mosalaganti, S. et al. In situ architecture of the algal nuclear pore complex. Nat. Commun. 9, 2361 (2018).

    PubMed  PubMed Central  Google Scholar 

  38. 38.

    Gu, Y. & Innes, R. W. The KEEP ON GOING protein of Arabidopsis recruits the ENHANCED DISEASE RESISTANCE1 protein to trans-Golgi network/early endosome vesicles. Plant Physiol. 155, 1827–1838 (2011).

    CAS  PubMed  PubMed Central  Google Scholar 

  39. 39.

    Calikowski, T. T. & Meier, I. Isolation of nuclear proteins. Methods Mol. Biol. 323, 393–402 (2006).

    CAS  PubMed  Google Scholar 

  40. 40.

    Zargar, S. M. et al. Quantitative proteomics of Arabidopsis shoot microsomal proteins reveals a cross-talk between excess zinc and iron deficiency. Proteomics 15, 1196–1201 (2015).

    CAS  PubMed  Google Scholar 

  41. 41.

    Kumar, S., Stecher, G. & Tamura, K. MEGA7: molecular evolutionary genetics analysis version 7.0 for bigger datasets. Mol. Biol. Evol. 33, 1870–1874 (2016).

    CAS  PubMed  PubMed Central  Google Scholar 

  42. 42.

    Wisecaver, J. H. et al. A global coexpression network approach for connecting genes to specialized metabolic pathways in plants. Plant Cell 29, 944–959 (2017).

    CAS  PubMed  PubMed Central  Google Scholar 

  43. 43.

    Gu, Y. & Innes, R. W. The KEEP ON GOING protein of Arabidopsis regulates intracellular protein trafficking and is degraded during fungal infection. Plant Cell 24, 4717–4730 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

Download references

Acknowledgements

We thank S. McCormick for a critical reading of the manuscript. This work was supported by funding from the Innovative Genomics Institute (IGI) at UC Berkeley and the Tsinghua-Peking Joint Center for Life Sciences.

Author information

Affiliations

Authors

Contributions

Y.T., A.H. and Y.G. designed the research. Y.T. and A.H. performed the experiments. T.Y. and Y.G. wrote the paper. All authors analysed the data, discussed the results and edited the manuscript.

Corresponding author

Correspondence to Yangnan Gu.

Ethics declarations

Competing interests

The authors declare no competing interests.

Additional information

Peer review information Nature Plants thanks Tokuko Haraguchi and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Extended data

Extended Data Fig. 1 Subcellular localization of PNET candidates identified by subtractive proteomics.

a, Subcellular localization patterns of PNET1 through PNET5 in leaf epidermal cells during transient expression in N. benthamiana. b, Subcellular localization patterns of other PNET candidates (Fig. 1e, lower panel) in leaf epidermal cells during transient expression in N. benthamiana. YFP fusion directions are as indicated. Free mCherry was coexpressed with YFP constructs. The localization patterns have been repeated in two independent experiments with similar results. Bars = 10 μm.

Extended Data Fig. 2 Nup93a preys identified by conventional IP-MS.

a, Nuclear localization of Nup50c-YFP in leaf epidermal cells during transient expression in N. benthamiana. Free mCherry was coexpressed. The nucleus (N) is marked with arrowhead. The localization pattern has been repeated in two independent experiments with similar results. Bar = 10 μm. b, Nup93a-BioID2-HA preys identified by conventional immunoprecipitation (IP) using HA antibody followed by LFQMS. YFP-BioID2-HA and HA-BioID2-NEAP1 plants were used as controls for ratiometric analysis. The integrated peptide intensity data of all samples were normalized and subjected to ratiometric analysis and plotting. Nup93 preys are defined by cutoffs fold-change > 2 and p-value < 0.05 compared to both controls (linear model F-test, n = 2 biologically independent samples). c, Statistics for Nup93a preys identified by the conventional IP-MS approach in b.

Extended Data Fig. 3 Profiling of PNET proteins by proximity-labeling proteomics.

a, Proximity labeling scheme using four known PNET proteins tagged with BioID2. b, Nuclear fractionation was performed using transgenic seedlings expressing 35S promoter-driven and HA-BioID2-tagged SUN1, NEAP1, WIP1, and SINE1, respectively. The total protein extract, fractionation from the nuclear pellet (N), and fractionation from nuclei-depleted supernatant (ΔN) were immunoblotted with antibodies against actin, HA, and histone H3. Similar results have been obtained twice. c, d, Heatmaps of normalized average PSM values of known NE proteins (c) and new PNET candidates (d) identified by proximity-labeling proteomics using different baits. Colored dots indicate significant enrichment of proteins (red for p-value < 0.01 and orange for p-value < 0.05, linear model F-test, n = 3 biologically independent samples) compared to controls. e, Fluorescence images of YFP-tagged PNET6 through PNET13 in leaf epidermal cells upon transient expression in N. benthamiana. The localization patterns have been repeated in two independent experiments with similar results. Bars = 10 μm. Source data

Extended Data Fig. 4 Phylogenetic analysis and multiple sequence alignment of PNET1 and its homologs.

a, Phylogenetic analysis of PNET1 and its homologs in 11 eukaryotic species, including 9 plant species (with green background) and 2 animal species (with orange background), using protein sequences and MEGA7.0 software. b, Multiple sequence alignment of PNET1 and its homologs using ClustalW. Predicted transmembrane regions were outlined by red boxes.

Extended Data Fig. 5 Physical and genetic interactions of PNET1/6 with nucleoporins.

a, Fluorescence images of BiFC between PNET1 and Nup35, Nup88, Nup93a, Nup58, and Nup155. The indicated BiFC constructs and free mCherry were coexpressed in N. benthamiana. The localization patterns have been repeated in two independent experiments with similar results. Bars = 10 μm. b, Interactions of PNET6 with nucleoporins using BiFC assay performed in N. benthamiana. The relative BiFC intensity was obtained by normalizing BiFC fluorescence using averaged expression levels of the corresponding Nup-YFP measured in separate experiments. Results are presented as boxplots with first quartile, median, and third quartile (n = 12). Ac, accessory nucleoporin; IRC, inner ring complex; FG, nucleoporins that contain Phe-Gly repeats; ORC, outer ring complex; Linker, linker nucleoporins. Membrane, membrane nucleoporins. c, Quantification of silique length of six-week-old plants. Data are presented as mean ± standard deviation (n = 15). Similar results have been obtained by an independent batch of samples. Source data

Supplementary information

Reporting Summary

Supplementary Tables

Supplementary Tables 1–10.

Source data

Source Data Fig. 1

Statistical source data.

Source Data Fig. 2

Unprocessed western blots.

Source Data Fig. 2

Statistical source data.

Source Data Fig. 3

Statistical source data.

Source Data Fig. 4

Statistical source data.

Source Data Extended Data Fig. 3

Unprocessed western blots.

Source Data Extended Data Fig. 3

Statistical source data.

Source Data Extended Data Fig. 5

Statistical source data.

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Tang, Y., Huang, A. & Gu, Y. Global profiling of plant nuclear membrane proteome in Arabidopsis. Nat. Plants 6, 838–847 (2020). https://doi.org/10.1038/s41477-020-0700-9

Download citation

Further reading

Search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing