Dataset Information

Establishment of TurboID proximity proteomics in the filamentous fungus Sordaria macrospora

ABSTRACT: Biotin identification (BioID) is a proximity-dependent labeling technique to study protein-protein co-localization in vivo. Although BioID has been applied in animal cells, plants and yeast, the method remained to be established in filamentous fungi. In this study, we established BioID for the filamentous fungus Sordaria macrospora using the well-characterized striatin interacting phosphatase and kinase (STRIPAK) complex as a proof of principle. In detail, the STRIPAK complex interactor 1 (SCI1) was fused to a codon-optimized TurboID biotin ligase. This SCI1-TurboID fusion protein complemented the Δsci1 deletion strain phenotype. The identification of the already known SmSTRIPAK components PRO11, SmMOB3, SmPP2Ac1 and PRO22 in a BioID experiment with SCI1-TurboID demonstrated its successful application in S. macrospora. The technique will provide a powerful proteomics tool for fungal biologists.

INSTRUMENT(S): Q Exactive HF

ORGANISM(S): Sordaria Macrospora

SUBMITTER: Oliver Valerius

LAB HEAD: Stefanie Pöggeler

PROVIDER: PXD034217 | Pride | 2022-10-25

REPOSITORIES: Pride

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Publications

Establishment of in vivo proximity labeling with biotin using TurboID in the filamentous fungus Sordaria macrospora.

Hollstein Lucas S LS Schmitt Kerstin K Valerius Oliver O Stahlhut Gertrud G Pöggeler Stefanie S

Scientific reports 20221022 1

Proximity-dependent biotin identification (BioID) has emerged as a powerful methodology to identify proteins co-localizing with a given bait protein in vivo. The approach has been established in animal cells, plants and yeast but not yet in filamentous fungi. BioID relies on promiscuous biotin ligases fused to bait proteins to covalently label neighboring proteins with biotin. Biotinylated proteins are specifically enriched through biotin affinity capture from denatured cell lysates and subseque ...[more]

PMID: 36272986

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Project description:The in vivo labeling technique Split-Biotin identification (Split-BioID, De Munter et al., 2017, Schopp et al., 2017, Cho et al., 2020) was used to capture the common microenvironment of Bre5 and the ribosomal protein Rps2 (uS5). Biotinylated proteins were enriched from cell lysates using affinity purification. After SDS-PAGE, proteins were digested in-gel with trypsin and the resulting peptides were analyzed by liquid chromatography-mass spectrometry (LC-MS) for identification and relative quantification against controls. An aliquot of the cell lysate that was used for the affinity purification was taken as an input control and analyzed as well by LC-MS to account for possible differences in the input abundance of enriched proteins. Stable isotope labeling with amino acids in cell culture (SILAC) was used for relative quantification of proteins according to the 2nSILAC approach (Dannenmaier et al., 2018). To evaluate the labeling efficiency, an aliquot of each culture was harvested separately prior to the pooling of the cells for the Split-BioID experiment. Cell lysates from these samples were used for SDS-PAGE, and a part of each gel lane was used for an in-gel digest of proteins. These peptides were also analyzed with LC-MS and the percentages of SILAC-labeled peptides (based on MSMS counts) were calculated. The captured proteins belong to a common cellular microenvironment of Bre5 and Rps2. References: Cho, K.F., Branon, T.C., Rajeev, S., Svinkina, T., Udeshi, N.D., Thoudam, T., Kwak, C., Rhee, H.W., Lee, I.K., Carr, S.A., Ting, A.Y. (2020). Split-TurboID enables contact-dependent proximity labeling in cells. Proc Natl Acad Sci U S A. 117, 12143-12154. Dannenmaier, S., Stiller, S.B., Morgenstern, M., Lübbert, P., Oeljeklaus, S., Wiedemann, N., Warscheid, B. (2018). Complete Native Stable Isotope Labeling by Amino Acids of Saccharomyces cerevisiae for Global Proteomic Analysis. Anal Chem 90, 10501-10509. De Munter, S., Görnemann, J., Derua, R., Lesage, B., Qian, J., Heroes, E., Waelkens, E., Van Eynde, A., Beullens, M., Bollen, M. (2017). Split-BioID: a proximity biotinylation assay for dimerization-dependent protein interactions. FEBS Lett 591, 415-424. Schopp, I.M., Amaya Ramirez, C.C., Debeljak, J., Kreibich, E., Skribbe, M., Wild, K., Béthune, J. (2017). Split-BioID a conditional proteomics approach to monitor the composition of spatiotemporally defined protein complexes. Nat Commun 8,15690.

Project description:As biopharmaceuticals, recombinant proteins have become indispensable tools in medicine. An increasing demand, not only in quantity but also in diversity, drives the constant development and improvement of production platforms. The N-glycosylation pattern on biopharmaceuticals plays important roles in activity, serum half-life and immunogenicity. Therefore, production platforms with tailored protein N-glycosylation are of great interest. Plant-based systems have already demonstrated their potential to produce pharmaceutically relevant recombinant proteins, although their N-glycan patterns differ from those in humans. Plants have shown great plasticity towards the manipulation of their glycosylation machinery, and some have already been glyco-engineered in order to avoid the attachment of plant-typical, putatively immunogenic sugar residues. This resulted in complex-type N-glycans with a core structure identical to the human one. Compared to humans, plants lack the ability to elongate these N-glycans with β1,4-linked galactoses and terminal sialic acids. However, these modifications, which require the activity of several mammalian enzymes, have already been achieved for Nicotiana benthamiana and the moss Physcomitrella. Here, we present the first step towards sialylation of recombinant glycoproteins in Physcomitrella, human β1,4-linked terminal N-glycan galactosylation, which was achieved by the introduction of a chimeric β1,4-galactosyltransferase (FTGT). This chimeric enzyme consists of the moss α1,4-fucosyltransferase transmembrane domain, fused to the catalytic domain of the human β1,4-galactosyltransferase. Stable FTGT expression led to the desired β1,4-galactosylation. However, additional pentoses of unknown identity were also observed. The nature of these pentoses was subsequently determined by Western blot and enzymatic digestion followed by mass spectrometric analysis and resulted in their identification as α-linked arabinoses. Since a pentosylation of β1,4-galactosylated N-glycans was reported earlier, e.g. on recombinant human erythropoietin produced in glyco-engineered Nicotiana tabacum, this phenomenon is of a more general importance for plant-based production platforms. Arabinoses, which are absent in humans, may prevent the full humanization of plant-derived products. Therefore, the identification of these pentoses as arabinoses is important as it creates the basis for their abolishment to ensure the production of safe biopharmaceuticals in plant-based systems.

Dataset Information

Establishment of TurboID proximity proteomics in the filamentous fungus Sordaria macrospora

Publications

Establishment of in vivo proximity labeling with biotin using TurboID in the filamentous fungus Sordaria macrospora.

Similar Datasets

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