GBPL3 localizes to the nuclear pore complex and functionally connects the nuclear basket with the nucleoskeleton in plants

Abstract

The nuclear basket (NB) is an essential structure of the nuclear pore complex (NPC) and serves as a dynamic and multifunctional platform that participates in various critical nuclear processes, including cargo transport, molecular docking, and gene expression regulation. However, the underlying molecular mechanisms are not completely understood, particularly in plants. Here, we identified a guanylate-binding protein (GBP)-like GTPase (GBPL3) as a novel NPC basket component in Arabidopsis. Using fluorescence and immunoelectron microscopy, we found that GBPL3 localizes to the nuclear rim and is enriched in the nuclear pore. Proximity labeling proteomics and protein-protein interaction assays revealed that GBPL3 is predominantly distributed at the NPC basket, where it physically associates with NB nucleoporins and recruits chromatin remodelers, transcription apparatus and regulators, and the RNA splicing and processing machinery, suggesting a conserved function of the NB in transcription regulation as reported in yeasts and animals. Moreover, we found that GBPL3 physically interacts with the nucleoskeleton via disordered coiled-coil regions. Simultaneous loss of GBPL3 and 1 of the 4 Arabidopsis nucleoskeleton genes CRWNs led to distinct development- and stress-related phenotypes, ranging from seedling lethality to lesion development, and aberrant transcription of stress-related genes. Our results indicate that GBPL3 is a bona fide component of the plant NPC and physically and functionally connects the NB with the nucleoskeleton, which is required for the coordination of gene expression during plant development and stress responses.

Proximity labelling allows to study novel factors in chloroplast developmenta

“…Despite successful application of TID-based proximity labelling in plant cells at different sub cellular localisations (Mair et al, 2019; Tang et al, 2022; Arora et al, 2020; Xu et al 2021), it has always been questioned if it would work in plastids of higher plant cells as well. Reports on the free biotin concentration suggested only marginal amounts inside chloroplasts (Badelt et al 1993).…”

TurboID reveals the proxiomes of CGE1, VIPP1, and VIPP2 inChlamydomonas reinhardtii

“…TurboID applications to identify protein-protein interaction networks in Arabidopsis and N. benthamiana used only 50-200 µM of exogenously added biotin but similar labeling times of 0.5-12 h, although strong protein biotinylation was observed already after as little as 15 min (Mair et al, 2019; Zhang et al, 2019; Xu et al, 2021; Tang et al, 2022; Wurzinger et al, 2022). While in these studies no effects on naturally biotinylated proteins was reported, we observed strong effects in Chlamydomonas with loss of natural protein biotinylation correlating with TurboID expression levels (Figures 2B, 2C, 3A, 4A; Supplemental Figure S7B).…”

“…In land plants, a desalting step was essential to remove excess biotin from protein extracts prior to incubation with streptavidin beads (Mair et al, 2019; Zhang et al, 2019; Arora et al, 2020; Xu et al, 2021; Tang et al, 2022; Wurzinger et al, 2022). This desalting step was required for Chlamydomonas only if biotin was added to crude membrane extracts.…”

“…In conclusion, TurboID-mediated PL has enabled the probing of known and new protein interaction networks in the nucleus, cytoplasm and at the plasma membrane of land plants with amazingly high sensitivity and specificity (Mair et al, 2019; Zhang et al, 2019; Arora et al, 2020; Xu et al, 2021; Tang et al, 2022). Our work and that of two parallel studies by Lau et al (2022) and Wurzinger et al (2022) add Chlamydomonas as another plant model and the chloroplast as another compartment amenable to the great power of TurboID-mediated PL.…”

“…Nevertheless, since its publication in 2018, TurboID has been used extensively to map proteomes and to identify protein-interaction networks in a variety of model organisms including mammalian cells (Cho et al, 2020), zebrafish (Xiong et al, 2021), or yeast (Larochelle et al, 2019). There are also first reports on the successful application of TurboID to land plants for the identification of interactors of plant immune receptor N (Zhang et al, 2019), stomatal transcription factor FAMA (Mair et al, 2019), nuclear transport receptor exportin 4 (Xu et al, 2021), nuclear pore complex protein GBPL3 (Tang et al, 2022), and the TPLATE complex (Arora et al, 2020) as well as for the mapping of the nuclear proteome (Mair et al, 2019).…”