Figure 1.
In vivo neuron-specific PB/MS in Drosophila for detection of proteins interacting with active Rab2 and the DCV membrane protein VMAT. (A) Schematic illustrating the effect of Rab2 loss on axonal transport of DCVs in flies (Lund et al., 2021). In WT motor axons, DCVs (blue spheres) are transported bidirectionally with similar anterograde and retrograde flux. In Rab2 null mutants, retrograde flux is strongly reduced, while anterograde flux is partially reduced. (B) Volcano plot showing the fold change and Student’s t test statistics of biotinylated protein label free quantification (LFQ) intensities from flies with pan-neuronal expression of TurboID-Rab2Q65L (elav > 2xHA-TurboID-Rab2Q65L) relative to TurboID-Rab2S20N (elav > 2xHA-TurboID-Rab2S20N). Proteins significantly upregulated in the active Rab2 condition (fold change > 2, FDR-adjusted P value < 0.05) are highlighted in red, and known Rab2 effectors in light blue (see also Fig. S1). (C) Distribution in subcellular neuronal compartments of proteins specifically enriched in flies expressing TurboID-Rab2Q65L compared with TurboID-Rab2S20N. EE, early endosomes; RE, recycling endosomes; LE, late endosomes; Lyso, lysosomes; Autoph, autophagosomes; Presyn, presynapse. (D) Representative STED image showing the distribution of ILP2-GFP and HA-tagged TurboID-VMAT fusion protein in motor axons in peripheral nerve A7 of third instar larva. Scale bars: left, 500 nm; right, 500 nm, 100 nm (inset). (E) Student’s t test of biotinylated protein LFQ intensities from flies with pan-neuronal expression of TurboID-VMAT (elav > ILP2-GFP, TurboID-HA-VMAT) relative to free cytosolic TurboID (elav > ILP2-GFP, TurboID). Proteins significantly enriched in the TurboID-HA-VMAT condition (fold change > 2, FDR-adjusted P value < 0.05) are highlighted in red. (F) Venn diagram of the overlap between proteins enriched both in flies expressing TurboID-Rab2Q65L and TurboID-VMAT. (G) Relationship between proteins significantly enriched in TurboID-Rab2Q65L and TurboID-VMAT flies, when ranked by enrichment level. Spearman’s rank correlation is 0.422, P < 10−6.

In vivo neuron-specific PB/MS in Drosophila for detection of proteins interacting with active Rab2 and the DCV membrane protein VMAT. (A) Schematic illustrating the effect of Rab2 loss on axonal transport of DCVs in flies (Lund et al., 2021). In WT motor axons, DCVs (blue spheres) are transported bidirectionally with similar anterograde and retrograde flux. In Rab2 null mutants, retrograde flux is strongly reduced, while anterograde flux is partially reduced. (B) Volcano plot showing the fold change and Student’s t test statistics of biotinylated protein label free quantification (LFQ) intensities from flies with pan-neuronal expression of TurboID-Rab2Q65L (elav > 2xHA-TurboID-Rab2Q65L) relative to TurboID-Rab2S20N (elav > 2xHA-TurboID-Rab2S20N). Proteins significantly upregulated in the active Rab2 condition (fold change > 2, FDR-adjusted P value < 0.05) are highlighted in red, and known Rab2 effectors in light blue (see also Fig. S1). (C) Distribution in subcellular neuronal compartments of proteins specifically enriched in flies expressing TurboID-Rab2Q65L compared with TurboID-Rab2S20N. EE, early endosomes; RE, recycling endosomes; LE, late endosomes; Lyso, lysosomes; Autoph, autophagosomes; Presyn, presynapse. (D) Representative STED image showing the distribution of ILP2-GFP and HA-tagged TurboID-VMAT fusion protein in motor axons in peripheral nerve A7 of third instar larva. Scale bars: left, 500 nm; right, 500 nm, 100 nm (inset). (E) Student’s t test of biotinylated protein LFQ intensities from flies with pan-neuronal expression of TurboID-VMAT (elav > ILP2-GFP, TurboID-HA-VMAT) relative to free cytosolic TurboID (elav > ILP2-GFP, TurboID). Proteins significantly enriched in the TurboID-HA-VMAT condition (fold change > 2, FDR-adjusted P value < 0.05) are highlighted in red. (F) Venn diagram of the overlap between proteins enriched both in flies expressing TurboID-Rab2Q65L and TurboID-VMAT. (G) Relationship between proteins significantly enriched in TurboID-Rab2Q65L and TurboID-VMAT flies, when ranked by enrichment level. Spearman’s rank correlation is 0.422, P < 10−6.

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