Deletion of the ATP20 gene in Ustilago maydis produces an unstable dimer of F1FO-ATP synthase associated with a decrease in mitochondrial ATP synthesis and a high H2O2 production.

The F1FO-ATP synthase uses the energy stored in the electrochemical proton gradient to synthesize ATP. This complex is found in the inner mitochondrial membrane as a monomer and dimer. The dimer shows higher ATPase activity than the monomer and is essential for cristae folding. The monomer-monomer interface is constituted by subunits a, i/j, e, g, and k. The role of the subunit g in a strict respiratory organism is unknown. A gene knockout was generated in Ustilago maydis to study the role of subunit g on mitochondrial metabolism and cristae architecture. Deletion of the ATP20 gene, encoding the g subunit, did not affect cell growth or glucose consumption, but biomass production was lower in the mutant strain (gΔ strain). Ultrastructure observations showed that mitochondrial size and cristae shape were similar in wild-type and gΔ strains. The mitochondrial membrane potential in both strains had a similar magnitude, but oxygen consumption was higher in the WT strain. ATP synthesis was 20 % lower in the gΔ strain. Additionally, the mutant strain expressed the alternative oxidase in the early stages of growth (exponential phase), probably as a response to ROS stress. Dimer from mutant strain was unstable to digitonin solubilization, avoiding its isolation and kinetic characterization. The isolated monomeric state activated by n-dodecyl-ß-D-maltopyranoside showed similar kinetic constants to the monomer from the WT strain. A decrease in mitochondrial ATP synthesis and the presence of the AOX during the exponential growth phase suggests that deletion of the g gene induces ROS stress.

Deletion of the natural inhibitory protein Inh1 in Ustilago maydis has no effect on the dimeric state of the F1FO-ATP synthase but increases the ATPase activity and reduces the stability.

Transduction of electrochemical proton gradient into ATP synthesis is performed by F1FO-ATP synthase. The reverse reaction is prevented by the regulatory subunit Inh1. Knockout of the inh1 gene in the basidiomycete Ustilago maydis was generated in order to study the function of this protein in the mitochondrial metabolism and cristae architecture. Deletion of inh1 gen did not affect cell growth, glucose consumption, and biomass production. Ultrastructure and fluorescence analyzes showed that size, cristae shape, network, and distribution of mitochondria was similar to wild strain. Membrane potential, ATP synthesis, and oxygen consumption in wild type and mutant strains had similar values. Kinetic analysis of ATPase activity of complex V in permeabilized mitochondria showed similar values of Vmax and KM for both strains, and no effect of pH was observed. Interestingly, the dimeric state of complex V occurs in the mutant strain, indicating that this subunit is not essential for dimerization. ATPase activity of the isolated monomeric and dimeric forms of complex V indicated Vmax values 4-times higher for the mutant strain than for the WT strain, suggesting that the absence of Inh1 subunit increased ATPase activity, and supporting a regulatory role for this protein; however, no effect of pH was observed. ATPase activity of WT oligomers was stimulated several times by dodecyl-maltoside (DDM), probably by removal of ADP from F1 sector, while DDM induced an inactive form of the mutant oligomers.