Functional characterization of mitofusin‐like protein from Dictyostelium discoideum

The balance between mitochondrial fission and fusion maintains mitochondrial morphology and function. In mammals, three large GTPases ‐ Opa1 and the mitofusins Mfn1 and Mfn2 ‐ are required for the fusion of inner and outer mitochondrial membranes. The Dictyostelium mitofusin MfnA GTPase domain shares only 24% identity with the corresponding region of human Mfn1 and lacks a typical Fzo‐mitofusin family region. To define the cellular function of the Dictyostelium protein, we used molecular genetic approaches to manipulate the production level of MfnA and to follow the localization of the fluorophore‐tagged protein. In cells producing MfnA with a C‐terminal eYFP‐tag, the protein is targeted to the outer mitochondrial membrane. Overproduction of wild type MfnA and GTPase deficient mutants K132A or S133N leads to peri‐nuclear mitochondrial clustering and the phenotype is not due to any cytoskeletal defect. Unlike mammalian Mfn1/2, both N‐terminal and C‐terminal MfnA domains are required for mitochondrial targeting. N‐terminal deleted construct shows partial co‐localization with the ER, while double deletion of N‐terminal and transmembrane domain induces association with plasma membrane and contractile vacuoles. MfnA null cells have fewer and more dispersed mitochondria but appear otherwise normal. Thus our results reveal that MfnA alters mitochondrial morphology by fusing outer mitochondrial membranes.