main then releases PINK1 for ret rograde movement while PINK1 kinase interacts with the Hsp90 chaperone. As demonstrated by Zhou et al, we find that PINK1 TM is required for kinase domain facing the cyto sol. In addition, PINK1 kinase domain facing the cytosol also inhibitor Navitoclax requires Hsp90 interaction and we believe it is the combined effects of TM and chaperone interaction that give mitochondrial PINK1 its proper topology. We have demonstrated that PINK1 2 lacks the TM domain and thus its association with mitochondria must be through another mechanism. The question turns to whether or not PINK1 1 is tethered to the mitochondrial mem brane We already know that this PINK1 cleaved form is rapidly degraded by the proteasome.

Given the evidence that the first cleavage site might reside within the TM region, this suggests that PINK1 1 might be loosely anchored or not anchored at all in its transient half life. Conclusions In conclusion, the interaction of the kinase domain with Hsp90 plays a significant role in PINK1 topology and cytosolic redistribution. It is conceivable that Hsp90 binding to the PINK1 kinase domain is preventing the vectorial movement of PINK1 precursor protein during the entire import process. While PINK1 is targeted to the mitochondria, PINK1 function in the mitochondria is unclear. Published results show that loss of PINK1 can lead to mitochondrial dysfunction, but it is not clear that this is the result of losing mitochondrial PINK1 or cytosolic PINK1.

Echoing a concern previously raised by Beilina et al, the possibility that the cytosol con tains mature PINK1 kinase challenges researchers to delineate how exactly PINK1 function links directly to mitochondrial functions. Embedded in this dual subcel lular localization model is the proposal that PINK1 has compartment AV-951 specific functions, as was found for yeast fumarase. We believe that functional studies of PINK1 need to implement the experimental design of examin ing PINK1 function when it resides in only one subcel lular compartment in order to tease apart PINK1 functional roles. Hela CCL 2 cells were purchased from ATCC and cul tured in DMEM complete media supplemented with 10% FBS and 1% penicillin streptomycin. Transient transfection method with Lipofectamine 2000 was per formed according to manufacturers protocol. Briefly, Hela CCL 2 cells were plated onto 60 mm2 tissue culture dishes at 90% confluency at the time of transfection.

Rapamycin AY-22989 2 ug of cDNA was diluted in 250 uL OPTI MEM. 5 uL of Lipofectamine 2000 was diluted in 250 uL of OPTI MEM. The mixture of cDNA and Lipo fectamine 2000 was added to cells in OPTI MEM. The transfection media was replaced by DMEM growth media six hours after transfection. Cells were subjected to experiments 48 hours following transfection. Co Immunoprecipitation Co IP experiments followed the methods described pre viously. Briefly, cells were lysed in 1% Triton X 100 buffer. Lysates were rotated for 1 hr at 4 C then cleared by centrifugation at 13,000 g for 10