The cytoplasmic dynamin-related guanosine triphosphatase Drp1 is recruited to mitochondria and
The cytoplasmic dynamin-related guanosine triphosphatase Drp1 is recruited to mitochondria and mediates mitochondrial fission. not Fis1; and (f) conditional knockout of Fis1 in colon carcinoma cells revealed that it is dispensable for mitochondrial fission. Thus Mff functions as an essential factor in mitochondrial recruitment of Drp1. Introduction Mitochondrial morphology is usually dynamically changed by continuous fission and fusion to form small models or interconnected mitochondrial networks and this dynamic morphology is essential for normal mitochondrial and cellular functions (Karbowski and Youle 2003 Okamoto and Shaw 2005 Chan 2006 McBride et al. 2006 Cerveny et al. 2007 Hoppins et al. 2007 Benard and Karbowski 2009 These morphological changes are closely associated with apoptosis: apoptotic stimuli trigger considerable mitochondrial fission accompanied by cristae disorganization permeabilization of the mitochondrial outer membrane (MOM) and release of apoptosis regulatory proteins including cytochrome (Scorrano et al. 2002 Frezza et al. 2006 High molecular excess weight GTPases are key regulators of these morphological dynamics. In mammals mitofusin proteins (Mfn1 and Mfn2) of MOM and the inner membrane protein Opa1 are essential for mitochondrial fusion (Alexander et al. 2000 Delettre et al. 2000 Santel and Fuller 2001 Opa1 is also involved in cristae remodeling (Olichon et al. 2003 Cristae are thought to Rabbit polyclonal to PELI1. trap large pools of cytochrome induces mitochondrial fragmentation and down-regulation of induces a perinuclear accumulation of elongated mitochondria. Furthermore Drp1 and hFis1 coimmunoprecipitate after cross-linking in vitrosuggesting that mitochondrial fission mechanisms are somewhat conserved throughout eukaryotes (Yoon et al. 2003 Mitochondria undergo considerable fragmentation early during apoptosis and Drp1 is essential for the normal progression of apoptosis (Youle and Karbowski 2005 Parone and Martinou 2006 Arnoult 2007; Suen et al. 2008 Ishihara et al. 2009 In this context overexpression induces Drp1-dependent mitochondrial fission and apoptosis and conversely knockdown inhibits the progression of apoptosis (Lee et al. 2004 However JI-101 you will find conflicting observations: hFis1 localizes throughout the MOM in contrast to the punctate localization of Drp1 and mitochondrial recruitment of Drp1 is not affected by knockdown (Lee et al. 2004 Stojanovski et al. 2004 Wasiak et al. 2007 Similarly neither mitochondria-associated Drp1 nor mitochondrial fission is usually affected by overexpression (Suzuki et al. 2003 These contradictory observations on hFis1 may suggest that although Fis1 is required for the mitochondrial fission the Fis1 level is not a limiting factor in the mitochondrial fission process and mitochondrial recruitment of Drp1 is usually regulated by other JI-101 elements. In addition to Fis1 MOM-anchored proteins ganglioside-induced differentiation-associated protein 1 (GDAP1; Niemann et al. 2005 and RING (really interesting new gene)-type E3-ubiquitin ligase March5/MITOL (Karbowski et al. 2007 are involved in mitochondrial fission. A recent study recognized another tail-anchored MOM protein mitochondrial fission factor (Mff; Gandre-Babbe and van der Bliek 2008 However their specific functions in Drp1-dependent mitochondrial fission are not known. Here we study the requirement of mitochondrial proteins for mitochondrial targeting of Drp1 by manipulating the expression of mitochondrial fission and fusion proteins including hFis1 Mff March5/MITOL GDAP1 and Opa1 and found that Mff clearly limited Drp1 function in mitochondrial fission and apoptosis. In contrast these effects were not observed for the other proteins including hFis1. In JI-101 this context Drp1 JI-101 and Mff actually interacted both in vivo and in vitro. Furthermore conditional knockout (KO; CKO) of hFis1 in human colon carcinoma cells revealed that hFis1 is usually dispensable for mitochondrial fission. We thus concluded that Mff but not hFis1 is an essential factor for mitochondrial recruitment of Drp1 during mitochondrial fission in mammalian cells. Results Mff down-regulation inhibits mitochondrial recruitment of Drp1 and induces mitochondria elongation We first examined the effect of knockdown on mitochondrial localization of Drp1 and mitochondrial morphology using three impartial pairs of.