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Searching for the interaction partners in yeast cytosol of newly synthesized mitochondrial signal-anchored proteins

ABSTRACT: The aim of the mass spectrometry analysis was to identify interaction partners of newly synthesized precursors of mitochondrial outer membrane proteins. To that aim, HA-tagged proteins were synthesized in vitro in extract of yeast cells and pull-down with anti-HA beads was performed.

INSTRUMENT(S): Q Exactive

ORGANISM(S): Saccharomyces Cerevisiae (baker's Yeast)

TISSUE(S): Cell Culture

SUBMITTER: Mirita Franz-Wachtel

LAB HEAD: Boris Maček

PROVIDER: PXD031610 | Pride | 2022-08-09

REPOSITORIES: Pride

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Project description:The precise regulation of transcription requires the coordinated activity of numerous proteins and protein complexes. Although significant progress has been made in understanding the mechanisms that regulate transcription, many questions remain unresolved. Accurately defining the direct effects of transcriptional regulators is critical to addressing these questions. An effective approach for identifying the direct targets of transcriptional regulators is combining rapid protein depletion and quantification of newly synthesized RNA. The auxin-inducible degron (AID) system and thiol (SH)-linked alkylation for the metabolic sequencing of RNA (SLAM-seq) are powerful methods to rapidly degrade a target protein and directly quantify newly synthesized RNA, respectively. Both methods have been widely applied to study transcriptional regulation. To address unresolved questions in transcription, we assembled an end-to-end workflow to deplete proteins of interest using the AID system and measure newly synthesized RNA using SLAM-seq in the model eukaryote, Saccharomyces cerevisiae. We provide an open-source, step-by-step protocol to support rapid implementation of this workflow. We include methods for targeted protein degradation, 4-thiouracil (4tU) incorporation, rapid methanol fixation, RNA purification, RNA alkylation, mRNA-seq library construction, and data analysis. Additionally, we demonstrate that this workflow can help define the direct effects of transcriptional regulators using the bromodomain and extra-terminal domain (BET) proteins, Bdf1 and Bdf2, as an example. We provide evidence that data generated using this workflow is robust to normalization using whole-cell spike-in or total read counts, correlates well with 4tU-seq data, and identifies extensive differential expression due to the depletion of Bdf1 and Bdf2. Taken together, this workflow will help address outstanding questions underlying the molecular basis of transcriptional regulation and other processes in S. cerevisiae and other eukaryotes.

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Searching for the interaction partners in yeast cytosol of newly synthesized mitochondrial signal-anchored proteins

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