Thus, we conclude that Tollip, via an association with Parkin, is an essential coordinator to sort damaged mitochondrial\derived cargo to the lysosomes. (Malik gene on a fragile site within chromosome 6 (Denison for 10?min at 4C and then supernatant removed. role of the endosomal adaptor Tollip during the mitochondrial stress response and identify its interaction and colocalisation with the Parkinson’s disease\associated E3 ubiquitin ligase Parkin. The interaction between Tollip and Parkin is dependent on the ubiquitin\binding CUE domain of Tollip, but independent of Tom1 and mitophagy. Interestingly, this interaction is independent of Parkin mitochondrial recruitment and ligase activity but requires an intact ubiquitin\like (UBL) domain. Importantly, Tollip regulates Parkin\dependent endosomal trafficking of a discrete subset of mitochondrial\derived vesicles (MDVs) to facilitate delivery to lysosomes. Retromer function and an interaction with Tom1 allow Tollip to facilitate late endosome/lysosome trafficking in response to mitochondrial stress. We find that upregulation of TOM20\positive MDVs upon mitochondrial stress requires Tollip interaction with ubiquitin, endosomal membranes and Tom1 to ensure their trafficking to the lysosomes. Thus, we conclude that Tollip, via an association with Parkin, is an essential coordinator to sort damaged mitochondrial\derived cargo to the lysosomes. (Malik gene on a fragile site within chromosome 6 (Denison for 10?min at 4C and then supernatant removed. Protein concentrations were then calculated using a Pierce? BCA Protein Assay Kit (Thermo Scientific), according to manufacturer’s instructions. Samples were then diluted and mixed with an equal amount of 2 SDS loading buffer. For Western blot analyses, samples were heated to 95C for 5?min, and an equal amount of protein loaded per well and then separated on SDSCPAGE denaturing gels and transferred onto PVDF membranes. Membranes were blocked in 5% milk for 1?h, prior to incubation overnight at 4C with primary antibodies. Membranes were washed in TBS\T 3 times then incubated for 1?h with fluorescently labelled LI\COR secondary antibodies and visualised using a LI\COR Rabbit Polyclonal to MAP3KL4 imaging system. Quantification was performed by densitometry using Image Studio Lite software and samples normalised to loading controls. ATP assay SH\SY5Y cells were plated in 96\well plates at a density of 40,000 cells/well in replicate wells for each condition. Replicate plates were utilised to allow for measurement of both ATP production and total protein. Following an overnight recovery, cells were washed and replenished with DMEM without glucose (Thermo Fisher Scientific, 11966025) containing 10?mM galactose or with normal growth media containing glucose, in the absence or presence of 10?M oligomycin. Cells were incubated for 2?h at 37C prior to harvesting for ATP production using the Mitochondrial ToxGlo Assay (Promega, G8000) or for protein using a BCA assay. For measuring ATP, luminescence readings were captured on a GloMax Microplate Reader (Promega). A background subtraction was performed (media only) on these values and normalised against the total protein content as measured by a BCA protein assay. Results represent replicate readings, across 3C4 independent experiments for each condition. Mitochondrial isolation HEK293 cells were CGP 37157 grown in 100\mm dishes to approximately 80% confluence, with 3 dishes used per condition. Cells were treated with AO for 2?h, then media removed and cells gently washed in PBS twice. Mitochondria were then purified using a Mitochondrial Isolation Kit (Abcam, ab110170) according to manufacturer’s instructions. Samples were then analysed by SDSCPAGE and Western blotting, as described. BioID assays HeLa WT or knockout cell lines were transfected with a Myc\tagged BioID\Tollip construct, or empty vector, then selected CGP 37157 using 500?g/ml Geneticin? and clonal colonies isolated and screened by Western blot analysis for expression of the construct. Clonal lines were then transfected with a HA\Parkin construct and selected using 1.5?g/ml puromycin. BioID cell lines expressing HA\Parkin were then seeded in 100\mm dishes in DMEM 24?h prior to treatment, when fresh DMEM containing 50?M biotin and stressor (or vehicle) was added for 6 or 24?h. A biotin\free condition was used to assess background. Media were then removed, cells washed twice in ice\cold PBS and 500?l of cold lysis buffer (500?mM NaCl, 0.4% SDS, 2% Triton X\100, 5?mM EDTA, 1?mM DTT in 50?mM TrisCHCl pH 7.4 and 1 cOmplete? protease inhibitor cocktail) added before scraping cells. Lysates were mixed with an equal amount of 50?mM TrisCHCl pH 7.4, then centrifuged at 11,000?for 15?min at 4C and supernatant transferred onto streptavidinCagarose beads. A small amount of supernatant was kept for whole\cell lysates. Pulldowns were performed on streptavidinCagarose beads on a rotator overnight at 4C. For washing, beads CGP 37157 were pelleted at 11,000?for 1?min at 4C and resuspended in wash buffer (50?mM Tris pH 7.4, 250?mM NaCl, 0.2% SDS, 1% Triton X\100, 2.5?mM EDTA, 0.5?mM DTT) four times..

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