Excessive Tpk3p activity leads to loss of mitochondrial respiration. Cells were grown for 24 hours to diauxic shift in minimal medium in the presence or absence of 4 mM cAMP. Mitochondrial respiration was measured using an Oroboros high resolution respirometer. Typical traces from two biological replicates (red and green lines) wild type and Δpde2 cells grown in the presence of 4 mM cAMP are shown (A). Routine respiration (designated by R) represents oxygen consumption from cells placed in the respirometer immediately from culture and without drugs. The addition of TET, an ATPsynthase inhibitor, causes proton build up on the outside of the inner mitochondrial membrane, inhibiting respiration. Respiration that does occur happens as a result of the small LEAK of protons across the inner membrane (designated by L). Dissipation of this proton gradient by the proton ionophore FCCP restores mitochondrial respiration (designated by ETS) but removes the major control point, being the proton gradient which is controlled by the rate of oxidative phosphorylation. The final addition of Antimycin A blocks activity of complex III, preventing electrons moving to complex IV and therefore oxygen consumption is halted and non-mitochondrial oxygen consumption (designated by NMT) is accounted for. The oxygen consumption under these conditions was determined for strains wild type, Δpde2, Δpde2 Δtpk3 and Δpde2Δtpk3 + TPK3 grown in the absence (B) and presence (C) of 4 mM cAMP. Error bars are the standard error of 3 independent experiments.
Leadsham and Gourlay BMC Cell Biology 2010 11:92 doi:10.1186/1471-2121-11-92