Membrane topology and mitochondrial targeting of mitofusins, ubiquitous mammalian homologs of the transmembrane GTPase Fzo.

Two human Fzo-homologs, mitofusins Mfn1 and Mfn2, are shown by RT-PCR and western blot to be ubiquitous mitochondrial proteins. Protease digestion experiments reveal that Mfn2 is an outer membrane protein with N-terminal and C-terminal domains exposed towards the cytosol. The transmembrane and C-terminal domains of Mfn2 (Mfn2-TMCT) are targeted to mitochondria and deletion of these domains leads to the cytosolic localization of truncated Mfn2 (Mfn2-NT). Mfn2 is targeted to the endoplasmic reticulum or to mitochondria when the C-terminal domain is replaced by short stretches of neutral/hydrophobic (Mfn2-IYFFT) or polar/basic (Mfn2-RRD) amino acids. The coiled-coil domains of Mfn2, upstream and downstream of the transmembrane domain, are also important for mitochondrial targeting: Mfn2-mutants deleted of any of its coiled-coil domains are only partially targeted to mitochondria and significant protein amounts remain cytosolic. We show that these coiled-coil domains interact with each other: mistargeted Mfn2-NT or Mfn2-IYFFT localize to mitochondria if co-expressed with Mfn2-TMCT. This relocalization is abolished when the coiled-coil domain is deleted in any of the co-transfected molecules. We also found that Mfn2 can cluster active mitochondria in the perinuclear region independently of the cytoskeleton, bring mitochondrial membranes into close contact and modify mitochondrial structure, without disturbing the integrity of the inner and outer membrane.