whereas the slow migrating band corresponds to a monoubiquitinated species of spartin. Then, we applied these anti-spartin antibodies in an immunofluorescence assay. As shown in . We confirmed these results using differential fractionation of cell homogenates transfected with HA-spartin or HAspartin. Immunoblotting revealed that the entire postnuclear pool of HA-spartin that encompasses the Cterminus of spartin was detected in the heavy-membrane fraction containing mitochondria. In contrast, HA-spartin was detected exclusively in the cytosolic fraction. Spartin protein has no mitochondrial targeting sequence and might associate with these organelles through the interaction of its C-terminus with proteins and/or phospholipids that reside in the mitochondria. The C-terminus of spartin encompasses the plantrelated senescence domain that is conserved in many proteins in various species including Arabidopsis thaliana suggesting of its important function. We reasoned that this domain might bind to mitochondrial phospholipids. To test this hypothesis, we expressed and purified a maltose binding protein -spartin fusion protein and MBP alone. MBP-spartin and MBP alone were applied in an in vitro protein-lipid overlay assay using nitrocellulose membranes with AM-2282 pre-spotted phospholipids. Using anti-MBP antibodies, we found that MBP-spartin interacted with cardiolipin but not with two other major mitochondrial phospholipids, namely phosphatidylethanolamine and phosphatidylcholine. The negative control interacted with neither cardiolipin nor with phosphatidylethanolamine or phosphatidylcholine. Overall, our results indicate that spartin interacts with mitochondria via its plant-related senescence domain, which binds to cardiolipin, a major phospholipid of the mitochondrial membrane. Spartin associates with outer mitochondrial membrane Cardiolipin is a major phospholipid in the inner mitochondria membrane, but it has been also found in the outer mitochondrial membrane. To determine the topology of spartin in the mitochondria, we overexpressed spartin-YFP in SK-N-SH cells and isolated mitochondrial fractions. Those fractions were either treated or not treated with proteinase K followed by immunoblotting. Overexpressed spartin-YFP, TOM20, and OPA1 were all detected in the mitochondrial fraction not treated with proteinase K. Enzymatic treatment eliminated detection of spartin-YFP and TOM20, which are anchored to the outer mitochondrial membrane. In contrast, OPA1, a resident protein of the intermembrane space, was still detected, indicating that the inner mitochondrial membrane was intact. Overall, our results suggest that spartin is associated with the outer mitochondrial membrane. Importantly, alpha-synuclein has also been reported to associate with cardiolipin and to locate to the outer mitochondria membrane. Depletion of spartin results in depolarization of the mitochondrial membrane Our previous 
studies found that in fibroblasts derived from patients with Troyer syndrome there is a lack of expression of truncated spartin protein, implying that the pathology of this disease occurs via a loss-of-function mechanism. Our present 11423396 findings show that spartin associates with mitochondria through its binding to cardiolipin, a major mitochondrial phospholipid. These results prompted us to investigate mitochondrial function after knock down of spartin’s expression. Specifically, we investigated the DYm and ATP production in cells depleted of spartin. The mito