Figure 4.

Hsp90 interaction affects PINK1 subcellular distribution in Hela cells. A) Subcellular distribution of WT and L347P PINK1 in Hela cells is not noticeably different. C = cytosolic fraction; M = mitochondrial fraction. B) Subcellular distribution of mito-Δ151 PINK1 shows the loss of cytosolic PINK1 when cells are treated with 1 μM 17-AAG, a Hsp90 inhibitor, for 4 hours. C) mito-L347P PINK1 localizes to cytosol and mitochondria. D) Mito-L347P PINK1 accumulates more in the mitochondria than mito-Δ151 PINK1, with little change to the cytosol distribution. E) Quantification from three independent experiments showing more PINK1 in mitochondrial fraction with L347P mutation. P = 0.025. Increase in PINK1 in the cytosolic fraction with L347P is not statistically significant. Mean ± SEM, n = 3. Statistical significance was calculated with ANOVA and Fisher's PLSD post-hoc test. F) L347P mutation reduces PINK1 interaction with Hsp90 by co-immunoprecipitation. GFP does not co-immunoprecipitate with Hsp90. G) Summary diagram depicting PINK1 protein structures and the role of each component in PINK1 topology and subcellular distribution. See Discussion for more details.