1.N.6. The Mitochondrial Inner/Outer Membrane Fusion (MMF) Family
The dynamic nature of mitochondria is critical for function. Chen and Chan (2010) have discussed the molecular basis of mitochondrial fusion, its protective role in neurodegeneration, and its importance in cellular function. The mammalian mitofusins Mfn1 and Mfn2, GTPases localized to the outer membrane, mediate outer-membrane fusion. OPA1, a GTPase associated with the inner membrane, mediates subsequent inner-membrane fusion. Mutations in Mfn2 or OPA1 cause neurodegenerative diseases. Mitochondrial fusion enables content mixing within a mitochondrial population, thereby preventing permanent loss of essential components. Cells with reduced mitochondrial fusion show a subpopulation of mitochondria that lack mtDNA nucleoids. Such mtDNA defects lead to respiration-deficient mitochondria, and their accumulation in neurons leads to impaired outgrowth of cellular processes and consequent neurodegeneration.
In yeast, three proteins are essential for mitochondrial fusion. Fzo1 and Mgm1 are conserved guanosine triphosphatases that reside in the outer and inner membranes, respectively. At each membrane, these conserved proteins are required for the distinct steps of membrane tethering and lipid mixing. The third essential component is Ugo1, an outer membrane protein with a region homologous to but distantly related to a region in the Mitochondrial Carrier (MC) family. Hoppins et al., 2009 showed that Ugo1 is a modified member of this family, containing three transmembrane domains and existing as a dimer, a structure that is critical for the fusion function of Ugo1. Their analyses of Ugo1 indicate that it is required for both outer and inner membrane fusion after membrane tethering, indicating that it operates at the lipid-mixing step of fusion. This role is distinct from the fusion dynamin-related proteins and thus demonstrates that at each membrane, a single fusion protein is not sufficient to drive the lipid-mixing step. Instead, this step requires a more complex assembly of proteins. The formation of a fusion pore has not yet been demonstrated (Hoppins and Nunnari, 2009). The Ugo1 protein is a member of the MC superfamily.
The fundamental function of the large GTPase dynamin-related protein (DRP) family is to regulate membrane dynamics in a variety of different cellular processes (Praefcke and McMahon, 2004). The canonical member of the DRP family, dynamin, and the mitochondrial division dynamin, Dnm1, have been the most extensively characterized, and each likely promotes membrane scission by the use of forces generated by GTPase cycle dependent self-assembly (Niemann et al., 2001; Hinshaw, 2000). Other members of the dynamin family are involved in different types of membrane remodeling events (Danino and Hinshaw, 2001). Two of these are Fzo1 and Mgm1, highly conserved mitochondrial DRPs in yeast that are essential for outer and inner mitochondrial fusion respectively. As proteins that mediate mitochondrial membrane fusion events, Fzo1 and Mgm1 represent a novel membrane remodeling function for DRPs (Hoppins et al., 2009).
Mitochondrial outer- and inner-membrane fusion events are coupled in vivo but separable and mechanistically distinct in vitro, indicating that separate fusion machines exist in each membrane. Outer-membrane fusion requires trans interactions of the dynamin-related GTPase Fzo1, GTP hydrolysis, and an intact inner-membrane proton gradient. Inner-membrane fusion also requires GTP hydrolysis but distinctly requires an inner-membrane electrical potential. The conserved intermembrane-space dynamin-related GTPase Mgm1 is required to tether and fuse mitochondrial inner membranes. Mgm1 also plays a role in inner-membrane dynamics, specifically in the maintenance of crista structures (Meeusen et al., 2006). Trans Mgm1 interactions on opposing inner membranes function similarly to tether and fuse inner membranes as well as maintain crista structures.
Mfn1 and Mfn2, in mammalian cells are required for mitochondrial fusion, Mfn1 and Mfn2 possess functional distinctions. For instance, the formation of tethered structures in vitro occurs more readily when mitochondria are isolated from cells overexpressing Mfn1 than Mfn2 (Ishihara et al. 2004). In addition, Mfn2 specifically has been shown to associate with Bax and Bak (TC#1.A.21), resulting in altered Mfn2 activity, indicating that the mitofusins possess unique functional characteristics. Lipidic holes may open on opposing bilayers as intermediates, and fusion in cardiac myocytes is coupled with outer mitochondrial membrane destabilization that is opportunistically employed during the mitochondrial permeability transition (Papanicolaou et al. 2012).
Mutations in Mfn2 (but not Mfn1) result in the neurological disorder Charcot-Marie-Tooth syndrome. These mutations can be complemented by the formation of Mfn1–Mfn2CMT2A hetero-oligomers but not homo-oligomers of Mfn2+–Mfn2CMT2A (Detmer and Chan, 2007). This suggests that within the Mfn1–Mfn2 hetero-oligomeric complex, each molecule is functionally distinct. This suggests that control of the expression levels of each protein likely represents the most basic form of regulation to alter mitochondrial dynamics in mammalian tissues. Indeed, the expression levels of Mfn1 and Mfn2 vary according to cell or tissue type as does the mitochondrial morphology (Eura et al. 2003).
Mgm1 in yeast is a highly conserved protein essential for mitochondrial fusion. The mammalian ortholog is OPA1, and mutations in this protein lead to the neurological disorder Dominant Optic Atrophy. Multiple isoforms of Mgm1 and OPA1 exist at steady state in yeast and mammalian cells respectively. While alternative splicing is uniquely involved in the generation of these isoforms in mammalian cells, generation of isoforms by proteolytic processing is common to both proteins.
While outer and inner membrane fusion events are separable in vitro, they are temporally linked in vivo, indicating that a mechanism exists to synchronize the outer and inner membrane fusion machines. Ugo1 is an essential fusion component. Sequence analysis of Ugo1 suggests that it may contain as many as six transmembrane domains (Belenkiy et al. 2000) but experimental evidence shows that the N-terminus faces the cytosol while the C-terminus is localized to the intermembrane space, requiring an odd number of transmembrane domains (Sesaki and Jensen, 2001).
The mitochondrial inner/outer membrane fusion complex, Fzo/Mgm1/Ugo1. Only the Ugo1 protein is a member of the MC superfamily, and PCD2 is a functional domain required for mitochondrial fusion. It has a single carrier domain (Coonrod et al. 2007).
The Fzo/Mgm1/Ugo1 complex of Saccharomyces cerevisiae
The mammalian mitochondrial membrane fusion complex, Mitofusin 1/2 (Mfn1)/Mfn2/Optical Atrophy Protein 1 (OPA1) complex (the equivalent of the yeast Ugo1 protein)/dynamin-related protein 1 Drp1 (Chandhok et al. 2018). Mfn1 and Mfn2 are two very similar (60% identity) GTPase dynamin-like proteins in the outer mitochondrial membrane (members of the CDD P-loop[ NTPase Family) while OPA1 is a sequence divergent GTPase in the inner membrane (Chen and Chan, 2010). Mfn2 plays roles in mitochondrial fusion and mitochondrial endoplasmic reticulum interactions (Ranieri et al. 2013; Schneeberger et al. 2013). Mfn2, when defective can give rise to Charcot-Marie-Tooth disease, diabetes, neurodegenerative diseases, obesity and vascular diseases (Chandhok et al. 2018). It may also function in insulin-dependent myogenesis (Pawlikowska et al. 2007). Drp1 (DLP1, DNM1L) mediates membrane fusion and fission through
oligomerization into membrane-associated tubular structures that wrap
around the scission site to constrict and sever the mitochondrial
membrane in a GTP hydrolysis-dependent mechanism (Smirnova et al. 2001; Taguchi et al. 2007). Sequences flanking the TMSs facilitate membrane fusion by mitofusin (Huang et al. 2017). Opa1 is a mitochondrial remodeling protein with a dual role in maintaining mitochondrial morphology and energetics by mediating inner membrane fusion and maintaining the cristae structure. This and the fusion/fission process by dynamins is described by Lee and Yoon 2018.
Mfn1/Mfn2/OPA1/Drp1 complex of Homo sapiens
Mfn1 or Fzo1B (Q8IWA4)
Mfn2, Hsg or Fzo1A (O95140)