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Mitochondrial fission requires recruitment of the GTPase Drp1 to mitochondria, but the molecules that mediate this recruitment have been disputed. Fis1, Mff, MiD49, and MiD51 can all recruit Drp1 to mitochondria and promote fission. MiD49 and MiD51 can promote mitochondrial fission, but their activity depends on cellular context.

OPA1, a dynamin-related guanosine triphosphatase mutated in dominant optic atrophy, is required for the fusion of mitochondria. Proteolytic cleavage by the mitochondrial processing peptidase generates long isoforms from eight messenger RNA (mRNA) splice forms, whereas further cleavages at protease sites S1 and S2 generate short forms. Using OPA1-null cells, we developed a cellular system to study how individual OPA1 splice forms function in mitochondrial fusion. Only mRNA splice forms that generate a long isoform in addition to one or more short isoforms support substantial mitochondrial fusion activity. On their own, long and short OPA1 isoforms have little activity, but, when coexpressed, they functionally complement each other. Loss of mitochondrial membrane potential destabilizes the long isoforms and enhances the cleavage of OPA1 at S1 but not S2. Cleavage at S2 is regulated by the i-AAA protease Yme1L. Our results suggest that mammalian cells have multiple pathways to control mitochondrial fusion through regulation of the spectrum of OPA1 isoforms.

Mitochondrial fusion requires the coordinated fusion of the outer and inner membranes. Three large GTPases-OPA1 and the mitofusins Mfn1 and Mfn2-are essential for the fusion of mammalian mitochondria. OPA1 is mutated in dominant optic atrophy, a neurodegenerative disease of the optic nerve. In yeast, the OPA1 ortholog Mgm1 is required for inner membrane fusion in vitro; nevertheless, yeast lacking Mgm1 show neither outer nor inner membrane fusion in vivo, because of the tight coupling between these two processes. We find that outer membrane fusion can be readily visualized in OPA1-null mouse cells in vivo, but these events do not progress to inner membrane fusion. Similar defects are found in cells lacking prohibitins, which are required for proper OPA1 processing. In contrast, double Mfn-null cells show neither outer nor inner membrane fusion. Mitochondria in OPA1-null cells often contain multiple matrix compartments bounded together by a single outer membrane, consistent with uncoupling of outer versus inner membrane fusion. In addition, unlike mitofusins and yeast Mgm1, OPA1 is not required on adjacent mitochondria to mediate membrane fusion. These results indicate that mammalian mitofusins and OPA1 mediate distinct sequential fusion steps that are readily uncoupled, in contrast to the situation in yeast. INTRODUCTIONMitochondria are dynamic organelles that continually exchange contents through membrane fusion. Mitochondrial fusion controls the morphology of the organelle and is critically important for maintaining the function of the mitochondrial network. Loss of fusion has been linked to reduced respiratory activity, embryonic lethality, apoptosis, and neurodegeneration (Okamoto and Shaw, 2005;Detmer and Chan, 2007;Suen et al., 2008). In addition, mutations in Mfn2 and OPA1, two genes involved in mitochondrial fusion, cause the human neurodegenerative diseases CharcotMarie-Tooth type 2A (CMT2A) (Zuchner et al., 2004) and dominant optic atrophy (DOA) (Alexander et al., 2000;Delettre et al., 2000), respectively.Mitochondrial fusion is a multi-step process requiring the coordinated fusion of both the outer and inner membranes, ultimately resulting in mixing of matrix contents. The coordination of outer and inner membrane fusion has been studied in budding yeast (Sesaki et al., 2003;Wong et al., 2003;Meeusen et al., 2006;Hoppins et al., 2007). In yeast, the outer membrane proteins Fzo1p and Ugo1p and the inner membrane protein Mgm1p are essential for mitochondrial fusion. In an in vitro mitochondrial fusion assay, Mgm1p is required for fusion of the inner but not outer mitochondrial membranes (Meeusen et al., 2006). However, in vivo, yeast lacking Mgm1 have no outer membrane fusion (Sesaki et al., 2003). Ugo1p forms a complex with both Fzo1p and Mgm1p and may coordinate the activity of both proteins (Wong et al., 2003;Sesaki and Jensen, 2004).In mammals, mitochondrial fusion requires the action of three large GTPases: the mitofusins (Mfns) Mfn1 and Mfn2, which are orthologues of Fzo1p, and the dynamin-related ...