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Global profiling of plant nuclear membrane proteome in Arabidopsis

Nature Plants volume 6, pages 838–847 (2020)Cite this article

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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.

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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.

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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.

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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

Authors and Affiliations

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

    Yu Tang & Yangnan Gu

  2. Tsinghua-Peking Joint Center for Life Sciences, Center for Plant Biology, School of Life Sciences, Tsinghua University, Beijing, China

    Aobo Huang

  3. Innovative Genomics Institute, University of California, Berkeley, CA, USA

    Yangnan Gu

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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.

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Correspondence to Yangnan Gu.

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The authors declare no competing interests.

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Peer review information Nature Plants thanks Tokuko Haraguchi 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 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.

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Unprocessed western blots.

Source Data Fig. 2

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Source Data Fig. 3

Statistical source data.

Source Data Fig. 4

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Statistical source data.

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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

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