Drosophila ZDHHC8 palmitoylates scribble and Ras64B and controls growth and viability
Fig 5
dZDHHC8 affects Ras64B stability.
(A) Ras64B overexpression decreases viability of dZDHHC8 knockouts. Four days after L1 collection, the number of live Ras64B overexpressing larvae (hsG4; UAS-Ras64B), dZDHHC8 homozygous mutant larvae (KO1; hsG4) or larvae overexpressing Ras64B in dZDHHC8KO1 mutant background (KO1; hsG4; UAS-Ras64B) was determined. Ras64B overexpression in the dZDHHC8KO mutant background causes synergistic lethality. Error bars = stdev. n = 3x60 larvae/genotype. * ttest≤0.04. (B) ABE assay was performed on lysates from control (CT) and dZDHHC8 knockout (KO1) early L3 larvae expressing Ras64B under the control of heatshock-Gal4 24h after heatshock. KO1 larvae have less palmitoylated Ras64B-HA when compared to WT controls but also less total Ras64B-HA protein. (C) dZDHHC8 affects Ras64B stability. Protein levels of Ras64B-HA expressed with hsG4 in control (CT) and dZDHHC8 knockout (KO1) larvae were assayed by immunoblotting at different time points after heat-shock. ERK was used as a loading control. KO1 larvae have less Ras64B-HA protein than CT larvae at any time point. (D) Ras64B is palmitoylated on at least two cysteines. Larvae expressing HA-tagged wildtype Ras64B, or mutant Ras64B where each of the three cysteine residues present in Ras64B is mutated to alanine. All expression constructs are inserted in the same genomic locus using phiC31-directed integration, and their expression induced using hsG4 and collecting samples for ABE 24h after heat-shock. All Ras64B-HA C/A mutants are still palmitoylated, albeit less than WT Ras64B-HA. Note that Ras64B C46A protein levels are lower than wildtype Ras64B. (E-G) Ras64B is palmitoylated on all three cysteines C46, C120, C147. Assay performed as in (B). Each of the Ras64B-HA double cysteine mutants is still palmitoylated (E). Only mutating all three cysteines at the same time (F) or mutating the cysteine before and within the CAAX motif (G) abolishes palmitoylation. Note that protein levels of Ras64B versions carrying C46A are lower.