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In vivo proximity-labeling with miniTurboID to screen for protein-protein interactions in the filamentous ascomycete Sordaria macrospora

ABSTRACT: The Biotin Identification (BioID) method is an in vivo labeling strategy for the identification of cellular microenvironments in animal cells, plants, yeast and filamentous fungi. With this method, the protein of interest (POI) is fused to a promiscuous biotin ligase. The ligase biotinylates proximal proteins in a cumulative manner, which are then captured from cell lysates by affinity-binding and identified by mass spectrometry. This allows the identification of POI co-localizing proteins, including weakly or transiently binding ones (Roux et al., 2012; Branon et al., 2018). In our recent research, we performed BioID experiments with the TurboID biotin ligase expressed in the filamentous fungus Sordaria macrospora (Hollstein et al., 2022). Here, we tested a smaller variant of TurboID biotin ligase called miniTurboID for its application S. macrospora. For a proof of principle, we used the well-characterized striatin interacting phosphatase and kinase (STRIPAK) complex as molecular microenvironment. In detail, the STRIPAK complex interactor 1 (SCI1) was fused to the codon-optimized TurboID or the miniTurboID biotin ligase. These fusion proteins complemented a sterile Δsci1 deletion strain to restore fertility. A BioID experiment with LC-MS analysis showed significant enrichment of the SmSTRIPAK components PRO11, SmMOB3 and PRO22 in all biological replicates of SCI1-TurboID and SCI1-miniTurboID, while being absent in the free TurboID control. This demonstrates that both, TurboID and the smaller variant miniTurboID, can be applied in the filamentous ascomycete S. macrospora. We provide a step by step protocol for the preparation of BioID samples for LC-MS analysis in the MethodsX article.

INSTRUMENT(S): Q Exactive HF

ORGANISM(S): Sordaria Macrospora

SUBMITTER: Oliver Valerius

LAB HEAD: Prof. Stefanie Pöggeler

PROVIDER: PXD061752 | Pride | 2025-05-12

REPOSITORIES: Pride

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Project description:Identifying specific protein interactors and spatially or temporally restricted local proteomes contributes significantly to our understanding of cellular processes in virtually all aspects of life. Obtaining such data is challenging, especially when the protein, cell type or event of interest is rare. In recent years, different proximity labeling techniques have been developed that have greatly improved our ability to tackle these questions. However, while effective in mammalian systems, use in plants has been extremely limited due to technical challenges. Recent technological improvements in the form of two highly active versions of the biotin ligase BirA* (TurboID and miniTurboID), prompted us to test this new system on two challenging but widely asked questions in plants: what are interaction partners of low-abundant proteins and what are organellar proteomes in rare and transient cell types. To address the second question, we used nuclei of developing stomatal guard cells, which express the transcription factor FAMA, as our test case. FAMA is a master regulator of guard cell development and promotes terminal differentiation of the guard cell precursor by both activating and repressing hundreds of genes. FAMA-expressing young guard cells are rare and restricted to the epidermis of developing aerial tissues, which makes them a good model system to test TurboID applicability under material-limiting conditions. For this experiment, young Arabidopsis seedlings expressing nuclear TurboID under the FAMA or UBIQUITIN10 (UBQ10) promoter were treated with biotin to label nuclear proteomes. Col-0 wild type was used as controls for unspecific binding of proteins to the beads. Biotinylated proteins were isolated by affinity purification with streptavidin-coupled beads and identified by LC-MS/MS. By targeting TurboID to the nucleus we were able to purify nuclear proteins with relatively high specificity. Enrichment of FAMA-stage specific proteins, when nuclear TurboID was driven by the FAMA compared to the UBQ10 promoter, supports general applicability of TurboID for identification of subcellular proteomes.

Project description:Identifying specific protein interactors and spatially or temporally restricted local proteomes contributes significantly to our understanding of cellular processes in virtually all aspects of life. Obtaining such data is challenging, especially when the protein, cell type or event of interest is rare. In recent years, different proximity labeling techniques have been developed that have greatly improved our ability to tackle these questions. However, while effective in mammalian systems, use in plants has been extremely limited due to technical challenges. Recent technological improvements in the form of two highly active versions of the biotin ligase BirA* (TurboID and miniTurboID), prompted us to test this new system on two challenging but widely asked questions in plants: what are interaction partners of low-abundant proteins and what are organellar proteomes in rare and transient cell types. To address the first question, we used the transcription factor FAMA as a test case. FAMA is a master regulator of guard cell development and promotes terminal differentiation of the guard cell precursor by both activating and repressing hundreds of genes. FAMA-expressing young guard cells are rare and restricted to the epidermis of developing aerial tissues, which makes them a good model system to test TurboID applicability under material-limiting conditions. For this experiment, young Arabidopsis seedlings expressing FAMA-TurboID were treated with biotin to label FAMA complexes. Col-0 wild type and seedlings expressing nuclear TurboID under the FAMA promoter were used as controls for unspecific binding of proteins to the beads and stochastic labeling of nuclear proteins, respectively. Biotinylated proteins were isolated by affinity purification with streptavidin-coupled beads and identified by LC-MS/MS. Analysis of proteins labeled by FAMA-TurboID fusions revealed known interactors of this late stomatal lineage specific transcription factor, as well as novel proteins that could explain its dual function as an activator and repressor. Comparison with proteins obtained from classical co-immunoprecipitation approaches with FAMA showed that proximity labeling is superior for identification of meaningful interaction partners of low-abundant proteins.

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In vivo proximity-labeling with miniTurboID to screen for protein-protein interactions in the filamentous ascomycete Sordaria macrospora

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