in yeast, mitochondrial-fission is regulated by the cytosolic dynamin-like GTPase (E)nm I p) in conjunction with a peripheral protein, Mdvlp, and a C-tail-anchored outer membrane protein, Fislp. In mammals, a dynamin-related protein (Drpl) and Fisl are involved in the mitochondrial-fission reaction as Dnml and Fisl orthologues, respectively. The involvement of other component(s), such as the Mdvl homologue, and the mechanisms regulating mitochondrial-fission remain unclear. Here, we identified rat Fisl (rFisl) and analyzed its structure-function relationship. Blue-native-polyacrylamide gel electrophoresis revealed that rFisl formed a similar to 200-kDa complex in the outer mitochondrial membrane. Its expression in HeLa cells promoted extensive mitochondrial fragmentation, and gene knock-down by RNAi induced extension of the mitochondrial networks. Taking advantage of these properties, we analyzed functional domains of rFisl These experiments revealed that the N-terminal and C-terminal segments are both essential for oligomeric rFisl interaction, and the middle TPR-like domains regulate proper oligomer assembly. Any mutations that disturb the proper oligomeric assembly compromise mitochondrial division-stimulating activity of rFisl. (c) 2005 Elsevier Inc. All rights reserved.