Even with a loss of Hsp90 binding, we reasoned that the intact transmembrane domain was enough to prevent PINK1 L347P from completely entering the mitochon dria. Therefore, we constructed more and expressed mito 151 PINK1 where we exchanged the PINK1 MLS with that of cytochrome b2 to isolate the effect of TM out of the equation and to focus on Hsp90 interac tion. We compared the subcellular distribu tion of mito 151 PINK1 in the absence and presence of Hsp90 inhibitor, 17 AAG. We observed that in the presence of 17 AAG, mito 151 PINK1 loses its cytosolic distribution with slight reduction in mito chondrial PINK1. We also noticed that the PINK1 pro tein sizes are slightly different between cytosol and mitochondria, although we are unsure of the explana tion behind this size shift.

It has been reported that matrix localized PINK1 appears as a doublet either through post translational modification or this size dif ference may arise from PINK1 having entered the mito chondria to have its MLS cleaved off by mitochondrial matrix protease. In addition to the Hsp90 inhibitor experiment, we constructed mito L347P PINK1 and compared its subcellular distribution to mito 151 PINK1. When we compared the cytosol mitochondria distribution between mito 151 PINK1 and mito L347P PINK1, there was significantly more mito L347P PINK1 than mito 151 PINK1 in the mitochondria. Lastly, we confirmed the Hsp90 interaction by co immunoprecipitation and found a reduction in Hsp90 binding with mito L347P PINK1 compared to 151 or mito 151 PINK1.

Full length L347P PINK1 also interacted less with Hsp90 compared to WT PINK1, and none of the GFP fusion proteins associated with Hsp90. These data suggest that the Hsp90 chaperone interac tion on the cytosolic side can prevent PINK1 from further mitochondrial entry, consequentially leading to the release of PINK1 from the mitochondria once pro teolysis removes PINK1 from the transmembrane anchor. Discussion As mentioned in the Introduction, both cytosolic and mitochondrial functions of PINK1 have been suggested. Elucidating the exact PINK1 subcellular localization will help us to understand these reported functions. The dis tribution of PINK1 in cells suggests that while a small percentage of PINK1 can be fully imported or associated with the mitochondria, the majority of PINK1 is believed to reside in the cytosol.

The demonstration that PINK1 contains a functional MLS and localizes within the mitochondria supports the hypothesis that PINK1 has a functional role in the mitochondria. While this functional Anacetrapib role is unclear, several studies suggest a role of PINK1 in the mitochondrial fission fusion pathway and in mitophagy of damaged mitochondria. Other compelling scientific data supports the hypothesis that PINK1 is also a cytosolic kinase.