Disease-associated mutations in Drp1 have fundamentally different effects on the mitochondrial fission machinery.

Abstract

Patient mutations have been identified throughout dynamin-related protein 1 (Drp1), the key mediator of mitochondrial fission. These mutations generally impact children and often result in severe neurological defects and, in some cases, death. Previously, the underlying functional defect leading to patient phenotypes has been speculated based on comparisons with synthetic mutations at similar sites. Drp1 is comprised by four domains, including a GTPase domain (G-domain) that regulates GTP binding and hydrolysis and a middle domain (MD) that mediates Drp1 self-assembly. We analyzed six mutations, four in the MD and two in the G-domain. All the MD mutants studied were found to be dimer-limited in solution, and three of these MD mutants were found to be impaired in self-assembly. However, one of the MD mutants retains oligomerization capability despite being located within this self-assembly region. Further, this mutant retains its ability to interact with a pre-formed lipid template but is unable to re-shape large, unilamellar vesicles (LUVs). This indicates that Drp1 dimers alone are sufficient to form a helical polymer on lipid template. Differences were also observed between the two G-domain mutations. One of the mutants is in a nucleotide-binding loop that is critical for functional hydrolysis. This mutant exhibited impaired GTP hydrolysis but can still oligomerize in solution and with a lipid template. In contrast, the other G-domain mutation is located relatively distant from the nucleotide binding site. This mutant poorly tubulates LUVs, highlighting the role of G-domain interactions in driving membrane curvature. Overall, the functional defects caused by mutations in Drp1 are highly variable even when the mutations occur within the same functional domain. This study provides a framework for characterizing additional Drp1 mutations to provide a comprehensive understanding of functional sites within this essential protein.