Uas dsred

CantonS, elav-GAL4, UAS-PKA-C1, UAS-DsRed, GMR52D11-lexA and P{UAS-CD4-spGFP1–10}3, P{lexAop-CD4-spGFP11}3 were obtained from the Bloomington Stock Center. Pka-C3^NIG.6117R was obtained from the National Institute of Genetics (NIG-Fly), Japan. natalisin-GAL4 was kindly provided by Y. Park and Y-J. Kim and is described in^{25 (link)}. PBacf07226 and PBacPka-C3^f00695 were obtained from the Exelixis Collection at the Harvard Medical School. UAS-PKA-C3 is described in^{21 (link)}. The constitutively inactive PKA-C3 construct was generated by replacing aspartate 397 (D³⁹⁷, see Supp. Figure 3) with alanine by site–directed mutagenesis using the site-directed mutagenesis kit from clontech. D³⁹⁷ is part of the highly conserved YRDLKPEN core sequence of the catalytic loop and mutations in the conserved aspartate have been shown to abolish or dramatically reduce the catalytic activity^{36 (link)–38 (link)}. The construct was tagged with HA. Flies were maintained on standard fly food under a 12:12 h light:dark cycle. Stocks were maintained at 18 °C while crosses and aging flies were maintained at 25 °C. The age of the flies in each experiment is indicated in the figures and figure legends.

Drosophila lines used in the experiments were hs-GAL4 (2077; the Bloomington Drosophila Stock Center), mef2-GAL4 (27390; the Bloomington Drosophila Stock Center), PINK1^B9 (34749; the Bloomington Drosophila Stock Center), park¹ (34747; the Bloomington Drosophila Stock Center), UAS-CISD RNAi (33925 and 104501; the Vienna Drosophila Resource Center), UAS-Itpr (30742; the Bloomington Drosophila Stock Center), UAS-Itpr RNAi (6484; the Vienna Drosophila Resource Center), UAS-ERGCaMP6-210 (83294; the Bloomington Drosophila Stock Center), UAS-GCaMP5G (42037; the Bloomington Drosophila Stock Center), UAS-Luciferase RNAi (31603; the Bloomington Drosophila Stock Center), and UAS-DsRed (6282; the Bloomington Drosophila Stock Center). The UAS-CISD WT-HA was generated by microinjecting pUAST-CISD-HA into w¹¹¹⁸ embryos. The RNAi lines for Parkin substrate genes used in Fig. 2a were described in Supplementary Table 1. All Drosophila stocks were maintained at 25 °C on the standard cornmeal-yeast-agar medium.