Abstract
Mitofusins (Mfns) are dynamin-related GTPases that mediate mitochondrial outer-membrane fusion, a process that is required for mitochondrial and cellular health. In Mfn1 and Mfn2 paralogs, a conserved phenylalanine (Phe-202 (Mfn1) and Phe-223 (Mfn2)) located in the GTPase domain on a conserved β strand is part of an aromatic network in the core of this domain. To gain insight into the poorly understood mechanism of Mfn-mediated membrane fusion, here we characterize a Mitofusin mutant variant etiologically linked to Charcot-Marie-Tooth syndrome. From analysis of mitochondrial structure in cells and mitochondrial fusion in vitro, we found that conversion of Phe-202 to leucine in either Mfn1 or Mfn2 diminishes the fusion activity of heterotypic complexes with both Mfn1 and Mfn2 and abolishes fusion activity of homotypic complexes. Using coimmunoprecipitation and native gel analysis, we further dissect the steps of mitochondrial fusion and demonstrate that the mutant variant has normal tethering activity but impaired higher-order nucleotide-dependent assembly. The defective coupling of tethering to membrane fusion observed here suggests that nucleotide-dependent self-assembly of Mitofusin is required after tethering to promote membrane fusion.
Highlights
Mitofusins (Mfns) are dynamin-related GTPases that mediate mitochondrial outer-membrane fusion, a process that is required for mitochondrial and cellular health
To gain insight into the molecular mechanism of mitochondrial outer-membrane tethering and fusion, we performed a functional screen of mutant variants of Mfn1 at highly conserved positions in the GTPase domain
Mfn1-null mouse embryonic fibroblasts (MEFs) have a fragmented mitochondrial network characterized by short, rod-shaped mitochondria due to low rates of mitochondrial fusion [14, 33]
Summary
A catalytic domain variant of mitofusin requiring a wildtype paralog for function uncouples mitochondrial outermembrane tethering and fusion. Mitofusins (Mfns) are dynamin-related GTPases that mediate mitochondrial outer-membrane fusion, a process that is required for mitochondrial and cellular health. Mitochondria are highly dynamic organelles that form an interconnected network through ongoing fusion, division, and movement [1] These activities are required to maintain both the structure and function of the mitochondrial network. This is crucial for the health of cells as aberrant mitochondrial structures have been observed in many pathological states and neurodegenerative disorders [1,2,3,4,5,6,7] Both mitochondrial fusion and division are mediated by large GTPase proteins of the dynamin-related protein (DRP) family. Mouse embryonic fibroblast; eGFP, enhanced GFP; mt-paGFP, mitochondrial matrix–targeted photoactivatable GFP; ROI, region of interest; BN, blue native; GMPPNP, 5Ј-guanylyl imidodiphosphate; RFP, red fluorescent protein; CFP, cyan fluorescent protein; MIB, mitochondrial isolation buffer; NA, numerical aperture; -ME, -mercaptoethanol; NiNTA, nickel-nitrilotriacetic acid; Bis-Tris, 2-[bis(2-hydroxyethyl)amino]2-(hydroxymethyl)propane-1,3-diol; F, forward; R, reverse
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