Dataset Information

Phosphoproteomic Effects of Acute Depletion of PP2A Regulatory Subunit Cdc55

ABSTRACT: Protein phosphatase regulatory subunits are increasingly recognized as promising drug targets. In the absence of an existing drug, inducible degradation provides a means of predicting candidate targets. We here employed auxin-inducible degradation of S. cerevisiae PP2A regulatory subunit Cdc55 in combination with quantitative phosphoproteomics. A prevalence of hyperphosphorylated phosphopeptides and enrichment of proteins containing the PP2A consensus sequence indicates that the approach successfully identified direct PP2A-Cdc55 targets. PRM follow up of DDA-results confirmed that vacuolar amino acid transporters are among the proteins most strongly affected by Cdc55 depletion.

ORGANISM(S): Saccharomyces Cerevisiae

SUBMITTER: Michael Plank

PROVIDER: PXD019534 | panorama | Wed Jan 27 00:00:00 GMT 2021

REPOSITORIES: PanoramaPublic

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Phosphoproteomic Effects of Acute Depletion of PP2A Regulatory Subunit Cdc55.

Plank Michael M Berti Marina M Loewith Robbie R

Proteomics 20201013 1

Protein phosphatase regulatory subunits are increasingly recognized as promising drug targets. In the absence of an existing drug, inducible degradation provides a means of predicting candidate targets. Here auxin-inducible degradation of Saccharomyces cerevisiae PP2A regulatory subunit Cdc55 in combination with quantitative phosphoproteomics is employed. A prevalence of hyperphosphorylated phosphopeptides indicates that the approach successfully identified direct PP2A<sup>Cdc55</sup> targets. P ...[more]

PMID: 32970932

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Project description:Purpose: The exosome plays major roles in RNA processing and surveillance but the in vivo target range and substrate acquisition mechanisms remain unclear. We applied an in vivo cross-linking technique coupled with deep sequencing (CRAC) that captures transcriptome-wide interactions between individual yeast exosome subunits and their targets in a living cell. Methods: We apply CRAC to HTP-tagged proteins (HTP: His6 - TEV cleavage site - two copies of the z-domain of Protein A): Two nucleases (Rrp44, Rrp6) and two structural subunits (Rrp41, Csl4) of the yeast exosome. At least two independent experiments were performed in each case and analyzed separately. We performed CRAC on wild-type (WT) Rrp44 and two catalytic mutants, rrp44-endo (D91N, E120Q, D171N, D198N) and rrp44-exo (D551N). We further developed CRAC using cleavable proteins (split-CRAC) to compare endonuclease and exonuclease targets of Rrp44. Plasmids designed for split-CRAC contain a PreScission protease cleavage site (PP) inserted between aa 241 and 242 in the RRP44 ORF to allow in vitro cleavage of purified protein, and a His6 tag to select the respective cleaved fragment. Results: Analysis of wild-type Rrp44 and catalytic mutants showed that both the CUT and SUT classes of noncoding RNA, snoRNAs and, most prominently, pre-tRNAs and other Pol III transcripts are targeted for oligoadenylation and exosome degradation. Unspliced pre-mRNAs were also identified as targets for Rrp44 and Rrp6. CRAC performed using cleavable proteins (split-CRAC) revealed that Rrp44 endonuclease and exonuclease activities cooperate on most substrates. Mapping oligoadenylated reads suggests that the endonuclease activity may release stalled exosome substrates. Rrp6 was preferentially associated with structured targets, which frequently did not associate with the core exosome. This indicates that substrates can follow multiple pathways to the nucleases. Conclusion: Our study represents the first transcriptome-wide map of substrates for the yeast exosome nuclease complex. Identification of targets for individual exosome subunits in wild-type and mutant yeast cells.

Dataset Information

Phosphoproteomic Effects of Acute Depletion of PP2A Regulatory Subunit Cdc55

Publications

Phosphoproteomic Effects of Acute Depletion of PP2A Regulatory Subunit Cdc55.

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