Identification of a novel protein that regulates mitochondrial fusion by modulating mitofusin (Mfn) protein function

Yuka Eura 0 Naotada Ishihara 0 Toshihiko Oka 0 Katsuyoshi Mihara ) 0 0 Department of Molecular Biology, Graduate School of Medical Science, Kyushu University , Fukuoka 812-8582 , Japan - Mitofusin proteins 1 and 2 (Mfn1 and Mfn2, respectively) of the mammalian mitochondrial outer membrane are homologues of Drosophila FZO and yeast Fzo1, and both are essential for GTP-dependent mitochondrial fusion. We identified a 55-kDa Mfn-binding protein named MIB. It is a member of the medium-chain dehydrogenase/reductase protein superfamily, and has a conserved coenzymebinding domain (CBD). The majority of MIB is localized ce in the cytoplasm but a small amount is associated with en mitochondria. Exogenous expression of MIB in HeLa ic cells induced mitochondrial fragmentation, which was lS prevented by coexpression of Mfn1, suggesting a functional le interaction of MIB with Mfn proteins; the GGVG sequence C in the CBD of MIB is essential for its function. By contrast, fo MIB knockdown resulted in growth arrest of the cells, l a n r u o J Introduction Mitochondria are extremely dynamic organelles, and they frequently change their morphology depending upon the cell environment or pathological conditions (Shaw and Nunnari, 2002; Karbowski and Youle, 2003; Chen and Chan, 2004; Okamoto and Shaw, 2005). The dynamic behavior of mitochondria is crucial for many cellular functions (Karbowski and Youle, 2003; Chan, 2006). Mitochondrial shape is maintained under a balance of mitochondrial fusion and division (Sesaki and Jensen, 1999), and GTPases of large molecular size are involved in these processes. Drosophila and yeast Fzo1 proteins and their mammalian homologues mitofusin proteins 1 and 2 (Mfn1 and Mfn2, respectively) are mitochondrial outer membrane proteins involved in the mitochondrial fusion reaction; they are anchored to the outer membrane through the C-terminal segment, with both an Nterminal GTPase domain and a C-terminal coiled-coil segment in the cytoplasm. Mfn1 and Mfn2 are both essential for mammalian mitochondrial fusion (Chen et al., 2003; Eura et al., 2003), and knockout of either gene is embryonic lethal (Chen et al., 2003). Cultured cells lacking either of the Mfn proteins exhibit a distinct type of fragmented mitochondria and similar results are obtained for Mfn1- or Mfn2knockdown cells (Chen et al., 2003; Eura et al., 2003). The mammalian dynamin-related protein Drp1 (also named Dlp1) localizes mainly, like its yeast orthologue Dnm1, to the although apoptotic sensitivity was not affected by either its knockdown or its overexpression. Furthermore, MIB knockdown induced a large extension of mitochondrial network structures. By contrast, a double knockdown of MIB and Mfn1 resulted in mitochondrial fragmentation and reversal of the growth arrest, the morphology and growth phenotype induced by knockdown of Mfn1 alone, again suggesting that MIB modulates Mfn1 function. Together, these findings suggest that MIB is essential for cellular function by regulating mitochondrial membrane dynamics in cooperation with Mfn proteins. cytoplasm, partially associates with mitochondria or peroxisomes, and is involved in mitochondrial and peroxisomal fission (Shaw and Nunnari, 2002; Karbowski and Youle, 2003; Koch et al., 2003a; Chan, 2006). Upon induction of apoptosis, Drp1 colocalizes to the mitochondrial fission foci with Bax (Frank et al., 2001; Lee et al., 2004; Youle and Karbowski, 2005). Drp1K38A, the dominant-negative form of Drp1 with a mutated GTPase domain induces, when exogenously expressed, the growth of mitochondrial tubularnetwork structures and simultaneously inhibits the progression of apoptosis (Frank et al., 2001). In yeast, the mitochondrial outer membrane C-tail anchor-protein Fis1 functions as the receptor of cytoplasmic Dnm1 via the peripheral adaptor proteins Mdv1 and Caf4, and mediates mitochondrial fission (Shaw and Nunnari, 2002; Okamoto and Shaw, 2005). Fis1 and Dnm1 have orthologues in many other eukaryotes including humans, although Mdv1 and Caf4 have only been identified in yeast. Human Fis1 (hFis1) is a C-tail anchor protein with two TPR segments in the N-terminal cytoplasmic domain. Whether hFis1 functions as the Drp1 receptor and interacts with any peripheral adaptor proteins, such as Mdv1 and Caf4, remains to be clarified. Yeast Mgm1 and the mammalian counterpart OPA1, are dynamin-like proteins of the mitochondrial inner membrane both of which have a C-terminal GTPase domain in the intermembrane space (Shaw and Nunnari, 2002; Olichon et al., 2002). They are involved in both mitochondrial membrane fusion and inner-membrane remodeling (Olichon et al., 2003; Okamoto and Shaw, 2005). Mgm1 is present as a large (high molecular size) and a small (processed, small molecular size) isoform (l-Mgm1 and s-Mgm1, respectively) (Herlan et al., 2004). The processing is mediated by Pcp1 (also named Mdm37, Ugo2 or Rbd1), and the presence of both Mgm1 isoforms is essential for mitochondrial function (Herlan et al., 2003; McQuibban et al., 2003; Sesaki et al., 2003). Mammalian OPA1 is the gene product of OPA1, mutations of which cause the optic atrophy type I (Alexander et al., 2000; Delettre et al., 2000). Cipolat et al. (Cipolat et al., 2004) demonstrated that OPA1 requires Mfn1 to promote mitochondrial fusion. To date, genetic or genomic approaches in yeast and mammals have identified several proteins distinct from the large molecular size GTPases described above, such as Ugo1, Mdm30 (mitochondrial distribution and morphology 30) and Mdm33 in yeast, and Dap3 (death-associated protein 3), MTP18 and endophilin B1 (also named Bif-1 or SH3GLB) in mammals. Ugo1 is a mitochondrial bi-topic outer-membrane protein involved in mitochondrial fusion (Sesaki and Jensen, 2004). Ugo1 and Fzo1 exhibit distinct localization on the outer membrane and they probably mediate distinct steps of the ce mitochondrial fusion reaction (Sesaki and Jensen, 2004). en Mdm33 is a 54-kDa inner-membrane protein with two ic transmembrane domains and at least three coiled-coil S structures, and is involved in inner-membrane fission lle (Messerschmitt et al., 2003). Mdm30 is an ~70-kDa protein C localized mainly in the cytoplasm but also in mitochondria fo that is required for maintaining fusion-competent l mitochondria (Fritz et al., 2003). It contains an F-box motif a n that is commonly found in the Skp1-cullin-F-box (SCF) ru ubiquitin ligase and modulates mitochondrial morphology Jo through regulation of the steady-state level of Fzo1 (Fritz et al., 2003; Escobar-Henriques et al., 2006). Dap3 is a 46-kDa protein that contains a GTP-binding domain, is localized in the mitochondrial matrix and is involved in the apoptosisinduced fission process (Mukamel and Kimchi, 2004). MTP18 (mitochondrial protein 18-kDa) is an integral inner-membrane protein that is a transcriptional downstream target of PI3kinase signaling. Its overexpression induces mitochondrial fragmentation and its knockdown induces highl (...truncated)