The S-palmitoylome and DHHC-PAT interactome of Drosophila melanogaster S2R+ cells indicate a high degree of conservation to mammalian palmitoylomes

PLoS One. 2022 Aug 12;17(8):e0261543. doi: 10.1371/journal.pone.0261543. eCollection 2022.

Abstract

Protein S-palmitoylation, the addition of a long-chain fatty acid to target proteins, is among the most frequent reversible protein modifications in Metazoa, affecting subcellular protein localization, trafficking and protein-protein interactions. S-palmitoylated proteins are abundant in the neuronal system and are associated with neuronal diseases and cancer. Despite the importance of this post-translational modification, it has not been thoroughly studied in the model organism Drosophila melanogaster. Here we present the palmitoylome of Drosophila S2R+ cells, comprising 198 proteins, an estimated 3.5% of expressed genes in these cells. Comparison of orthologs between mammals and Drosophila suggests that S-palmitoylated proteins are more conserved between these distant phyla than non-S-palmitoylated proteins. To identify putative client proteins and interaction partners of the DHHC family of protein acyl-transferases (PATs) we established DHHC-BioID, a proximity biotinylation-based method. In S2R+ cells, ectopic expression of the DHHC-PAT dHip14-BioID in combination with Snap24 or an interaction-deficient Snap24-mutant as a negative control, resulted in biotinylation of Snap24 but not the Snap24-mutant. DHHC-BioID in S2R+ cells using 10 different DHHC-PATs as bait identified 520 putative DHHC-PAT interaction partners of which 48 were S-palmitoylated and are therefore putative DHHC-PAT client proteins. Comparison of putative client protein/DHHC-PAT combinations indicates that CG8314, CG5196, CG5880 and Patsas have a preference for transmembrane proteins, while S-palmitoylated proteins with the Hip14-interaction motif are most enriched by DHHC-BioID variants of approximated and dHip14. Finally, we show that BioID is active in larval and adult Drosophila and that dHip14-BioID rescues dHip14 mutant flies, indicating that DHHC-BioID is non-toxic. In summary we provide the first systematic analysis of a Drosophila palmitoylome. We show that DHHC-BioID is sensitive and specific enough to identify DHHC-PAT client proteins and provide DHHC-PAT assignment for ca. 25% of the S2R+ cell palmitoylome, providing a valuable resource. In addition, we establish DHHC-BioID as a useful concept for the identification of tissue-specific DHHC-PAT interactomes in Drosophila.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Acyltransferases* / genetics
  • Animals
  • Drosophila / metabolism
  • Drosophila melanogaster* / genetics
  • Drosophila melanogaster* / metabolism
  • Lipoylation / physiology
  • Mammals / metabolism
  • Protein Processing, Post-Translational

Substances

  • Acyltransferases

Grant support

Felix T. Wieland got the following grant from DFG (Deutsche Forschungsgemeinschaft / German Research Foundation): grant: Wi654/11-1 funder: German Research Foundation (Deutsche Forschungsgemeinschaft, DFG) URL: https://www.dfg.de Robert B. Russel got the following grants: grant: 031A537C funder: de.NBI URL: www.denbi.de grant: 2018-05882 funder: Swedish Research Council (VR) URL: https://www.vr.se Juan Carlos González-Sánchez salary was partly funded (he was NOT employed by the Swedish Research Council, the grant contained money for salaries through which J C G S was partially paid) through the following grant to Robert B. Russel grant: 2018-05882 funder: Swedish Research Council (VR) URL: https://www.vr.se The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.