Testis-specific ATP synthase subunits associated with shaping
mitochondrial membranes in the nebenkern
During fruit fly sperm formation, the mitochondria fuse into a spherical nebenkern with mitochondrial membranes forming concentric layers that enclose two topologically distinct compartments. Previously we identified Knon as a gene required for nebenkern shaping and sperm motility. Knon encodes a testis-specific large paralog of the D subunit of F1Fo-ATP synthase, a protein complex that shapes cristae by forming dimers on the inner mitochondrial membrane. Since nebenkern lack cristae, we hypothesize that Knon can replace subunit D on the peripheral stalk of ATP synthase, where it sterically hinders dimerization, enabling ATP synthase-harboring membranes to curve more freely in the nebenkern. I tried to demonstrate Knon’s inhibition of testis ATP synthase dimerization by resolving ATP synthase complexes on a blue-native protein gel, but without positive results. We believe three other testis-specific paralogs of ATP synthase subunits play a role in nebenkern shaping, but previously we had a limited set of genetic tools for characterizing them. When we used the CRISPR/Cas system to mutate two of the respective genes, ATPsynCF6L and ATPsynGL, we found that the mutants exhibited defective sperm. Further experiments are needed to show that the roles of Knon, the CF6-like, and the G-like subunits in mediating mitochondrial dynamics is through inhibition of ATP synthase dimerization.