The Immt 151 PINK1 construct represents the first successful demon stration that we are able dilution calculator to eliminate the cytosolic pool of PINK1 while retain proper PINK1 mitochondrial topology. We then asked whether the PINK1 kinase domain itself can confer tethered topology and cytosolic distri bution. This time we deleted PINK1 MLS and fused cytochrome b2 MLS to the kinase domain. When we expressed mito 151 PINK1, which now lacks a TM but retains the C terminal kinase domain, we found this protein distributed equally to the cytosol and the mito chondria. The mitochondrial fraction of mito 151 PINK1 was protected from proteinase K digest, similar to matrix chaperone Hsp60. We also examined the subcellular distribution of 90 110 PINK1, where the PINK1 TM is deleted.

We found that 90 110 PINK1 predominantly localized to the mito chondrial fraction that is insensitive to proteinase treat ment and a small fraction of cleaved 90 110 PINK1 was found in the cytosolic fraction. Thus in the absence of a transmembrane domain, PINK1 has altered submitochondrial localization but some cytosolic redistribution remains. Taken all together, our data sug gests that 1 the TM and the kinase domain are both needed for a tethered, cytosolic facing, kinase domain topology and 2 PINK1 cytosolic redistribution requires both proteolysis after the TM and the kinase domain. It was previously shown that PINK1 lacking MLS is mostly cytosolic although it can still interact with OMM or IMS proteins.

When we expressed 151 PINK1, lacking the N terminal MLS, we found that this protein localized mostly to the cytosol, but some was still found in the mitochondrial fraction and co localized with mitochondrial markers. It is likely that 151 PINK1 contains additional internal cryptic targeting signal because mitochondrially Anacetrapib loca lized 151 PINK1 was protected from proteinase K digest. Finally, we asked whether or not PINK1 dual dis tribution is evolutionarily conserved by examining the subcellular localization of drosophila PINK1. We found drosophila PINK1 in both cytosolic and mitochondrial fractions with two cleavage sites similar to the mamma lian form. To further examine the idea that PINK1 kinase domain Hsp90 interaction modulates mitochondrial entry of PINK1, we hypothesized that destabilizing the PINK1 Hsp90 interaction will increase PINK1 import into the mitochondria. We wanted to test the idea that the Hsp90 interaction is preventing PINK1 forward movement during mitochondrial import. We chose to use the PINK1 L347P mutation, a naturally occurring PD mutation with reduced Hsp90 interaction.