These studies proved that PINK1 MLS is sufficient for mitochondrial targeting. The submitochondrial localization of PINK1, by bio chemical fractionation, selleck products shows that all forms of PINK1 are found at the outer membrane, intermembrane space, and inner membrane, but not the matrix. How ever, the subcellular Inhibitors,Modulators,Libraries localization of endogenous and overexpressed Inhibitors,Modulators,Libraries PINK1 in cell culture models show that PINK1 does not solely localize to the mitochondrial fraction, as cytosolic and microsomal fractions are found to contain all cleaved forms of PINK1. Overex pression of cytosolic PINK1, one that lacks the MLS, exhibits protective function against MPTP toxicity in mice and in cell culture. Also, proteins found to associate with PINK1 are either cytosolic or cytosolically exposed.

Only HtrA2 and TRAP1 are found to associate with PINK1 in the mitochondria. Currently no studies have examined the func tion of the mitochondrial form of PINK1 in the absence of the cytosolic PINK1. Several important questions arise from PINK1 dual Dacomitinib localization, what purpose does the PINK1 MLS serve if a functional PINK1 protein is also found in the cytosol How does PINK1 redistribute after mitochondrial pro cessing Is the function of PINK1 different in mitochon Inhibitors,Modulators,Libraries dria as compared to the cytosol We are very interested to understand the mechanism behind PINK1 dual distri bution, especially given the evidence that the mitochon drial pool of PINK1 is tethered to the OMM and removal of the PINK1 transmembrane domain mislocalizes Inhibitors,Modulators,Libraries PINK1 inside the mitochondria.

We previously showed that PINK1 cleaved forms are generated from the mito chondrial processing of PINK1 precursor, thus suggest ing that PINK1 cytosolic redistribution occurs after cleavage. We hypothesize that while the PINK1 MLS can direct proteins to the mitochondria, thoroughly the required interaction between the PINK1 kinase domain and Hsp90 chaperone favors a retrograde movement, thus resulting in a cytosolic localization. To test our hypothesis, we fused wildtype PINK1 as well as PINK1 mutant that lacks Hsp90 chaperone interaction with other known MLS and examined the cytosolic and mito chondrial distribution of these proteins when expressed in a cell culture model. Results PINK1 N terminal cleavages occur before and after PINK1 transmembrane domain At first glance, PINK1 MLS is similar either to those of inner membrane or intermembrane space proteins. The difference between these two signals is the cleavage site after the transmembrane domain, which would determine whether or not the protein is anchored. Overexpression of WT PINK1 in cell lines leads to the generation of three or more PINK1 forms, suggesting the presence of multiple cleavage sites.