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

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.

Fig 1. Drosophila S2R+ cell palmitoylome evaluation.

(A) Acyl-RAC dataset subjected to different filtering to identify proteins that are unlikely to be palmitoylated ("non-palmitoylated", FDR > = 0.1 or FC < 2), palmitoylated proteins with "normal" confidence ("NC", FDR < 0.1 and FC < 20 and FC > = 2), palmitoylated proteins with "high" confidence ("HC", FDR <0.1 and FC > = 20). (B) Overlap of our acyl-RAC dataset, consisting of the S2R+ palmitoylome proteins (normal and high confidence) and proteins not considered to be palmitoylated, with the larval palmitoylome from Strassburger et al 2019 [20]. (C) Barplots indicate the fraction (%) of proteins that have a mammalian ortholog (left), that have a mammalian ortholog that is palmitoylated (center) and that have a mammalian ortholog that is palmitoylated with “high” confidence (right) in each of the protein sets defined above. The palmitoylation status of the mammalian orthologs was obtained from the SwissPalm database. (D) Barplot that shows the fraction (%) of proteins predicted to be palmitoylated by CSS-Palm in each of the protein sets.

Fig 2. Most significantly enriched GO terms in the Drosophila S2R+ cell palmitoylome proteins.

Each bar represents the enrichment score (-log10(FDR p-value) of a GO term in the dataset and they are separated in three main categories: cellular component (black), biological process (dark grey) and molecular function (light grey). These terms were manually selected to be the most representative and avoiding redundancy. Full list can be found in S4 Table.

Fig 3. Validation of DHHC-BioID system using a known interaction.

(A) Alignment of several orthologues and paralogues of SNAP25 in the region flanking dHip recognition motif [51]. Red: conserved Proline essential for the interaction with Hip14 and mutated to Alanine in dSNAP25* (P125A, 25*). (B) Western Blot representative of co-overexpression of CG6017 (dHip14, used as positive control) or CG5620 (App) or CG6618 (Patsas) as BioID fusion proteins with either FLAG-dSNAp25 wt (25) or FLAG-dSNAP25 P125A (25*). Note that biotinylation signal of the band relative to FLAG-SNAP25 is not present in the mutant (25*), indicative of specific interaction with the enzyme fused with BioID, as quantified in (C). (D, E) Western Blots representative of co-overexpression of FLAG-dSNAP24 (24) or dSNAP25 (25) with a subset of 10 different DHHC-BioID. Relative biotinylation of the substrates was quantified in (F). (B, D, E) Representative blots are shown. FLAG-dSNAP24/25/25* were detected using an anti-FLAG primary antibody (upper panel) and biotin was detected using a Streptavidin probe (lower panel). Biotinylation and FLAG-tagged proteins were detected on the same blot membrane using two different fluorophores on a Li-Cor Odyssey. (C, F) Quantification of dSNAP relative biotinylation signal, normalized by the signal of the respective dSNAP in wt S2R+ cells, was estimated according to material and methods. Results from three independent experiments are shown as means +/- standard deviations. Asterisks (*) or section signs (§) indicate statistically significant differences in relative biotinylation of dSNAP25 or dSNAP24 respectively in the negative control (S2R+) using a one-way ANOVA with Dunnett’s multiple comparison test (* and § P<0.05; ** and §§ P<0.01).

Fig 4. DHHC-PAT interactomes.

(A) Venn diagram showing the overlap between the NC and HC fractions of the Drosophila palmitoylome of S2R+ cells and the DHHC-PAT-BioID interactome, which is also divided in low and high confidence interactors, depending on whether they were determined by one experiment alone or by both; (B) number of proteins (interactors) enriched by DHHC-PAT-BioID over the control. (C-G) percentage of DHHC-PAT-BioID interactors that (C) are palmitoylated, (D) are transmembrane proteins, (E) are palmitoylated transmembrane proteins, (F) contain the Hip14 interaction motif and (G) contain the Hip14 interaction motif and are palmitoylated. (B-G) ‘all interactors’ refers to proteins that are significantly enriched in the respective DHHC-PAT-BioID cell line over control cells; ‘<7 DHHC-PATs refers’ to interactors that are significantly enriched in the respective DHHC-PAT-BioID cell line over control cells and are not enriched in seven or more other DHHC-PAT-BioID cell lines.