Fig. 8: UBE2D/eff knockdown upregulates PEX protein levels in Drosophila skeletal muscle.

TMT mass spectrometry identifies protein changes induced in Drosophila skeletal muscle by knockdown of eff (the sole Drosophila UBE2D1/2/3 homolog), by overexpression of human UBE2D2 (hUBE2D2), and by eff/UBE2D knockdown rescued by the concomitant expression of hUBE2D2, compared to controls (GFP RNAi and mcherry overexpression). a Overexpression of human UBE2D2 in Drosophila muscles has limited impact on the peroxisomal proteome, apart from a significant decline in Pex11 and Pex13 protein levels. b UBE2D/eff knockdown increases the abundance of peroxisomal proteins, including Pex11 and Pex13. c Rescue experiments with hUBE2D2 overexpression concomitant to UBE2D/eff knockdown, compared to mock rescue with cherry overexpression: hUBE2D2 overexpression prevents the upregulation of Pex11, Pex13, and other peroxisomal proteins by UBE2D/eff RNAi. However, some of the peroxisomal proteins that are upregulated by UBE2D/eff knockdown (e.g., Pex1) are not affected, presumably because human hUBE2D2 only partially compensates for Drosophila UBE2D/eff loss. In (a-c), the x-axis displays the Log₂FC whereas the y-axis reports the significance, -Log₁₀(P value). d Alongside a reduction in Drosophila eff/UBE2D abundance, eff/UBE2D RNAi increases Pex11 and Pex13 protein levels (yellow) compared to controls (gray) but these are rescued by concomitant hUBE2D2 expression (light orange). Overexpression of hUBE2D2 by itself (dark orange) reduces Pex11 protein levels. N = 3 biological replicates/group with mean ±SEM, *P < 0.05, **P < 0.01, ***P < 0.001; ns= not significant (one-way ANOVA). In these comparisons, the number of UAS transgenes was kept equal to avoid Gal4 titration effects. e, f Immunostaining of Drosophila skeletal muscle with DAPI (to detect nuclei; blue), phalloidin (to detect F-actin; red), and eYFP with a PTS1 peroxisome targeting signal (eYFP-PTS1; green) to identify peroxisomes with functional protein import. g Knockdown of UBA1 and of eff/UBE2D increases the number of eYFP-PTS1+ puncta, indicative of functional peroxisomes, compared to control RNAi. h No changes in the size of peroxisomes are found. In (g, h), the n (biological replicates/group) is indicated in the figure, together with the mean ± SD, *P < 0.05 and ***P < 0.001 (one-way ANOVA). Altogether, these results indicate that the knockdown of UBE2D/eff and UBA1 increases the number of peroxisomes with correctly-imported eYFP-PTS1 in Drosophila skeletal muscle (e-h). Source data are provided in the Source data file.