Impact of the growth phase on the activity of multidrug resistance pumps and membrane potential of S. cerevisiae: effect of pump overproduction and carbon source

AbstractThe potentiometric fluorescence probe diS-C3(3) is expelled from S. cerevisiae by ABC pumps Pdr5 and Snq2 and can conveniently be used for studying their performance. The activity of these pumps in a strain with wild-type PDR1 allele was shown to drop sharply on glucose depletion from the medium and then again at the end of the diauxic shift when the cells are adapted to growth on respiratory substrates. The presence of the PDR1-3 allele causing pump overproduction prevented this second drop and the pump activity typical for diauxic cells was largely retained. Growth phase-dependent changes of membrane potential measured by the same probe in pump-free mutants included a Δψ drop in the late exponential and diauxic growth phase, indicating lowered activity of H+-ATPase. Suppression of activity of both ABC pumps and H+-ATPase obviously signifies cell transition to an energy-saving mode. Challenging respiration-adapted cells with glucose showed a novel feature of yeast ABC pumps—a strong dependence of pump activity on the type of the carbon source

Factors underlying membrane potential-dependent and -independent fluorescence responses of potentiometric dyes in stressed cells: diS-C3(3) in yeast

AbstractThe redistribution fluorescent dye diS-C3(3) responds to yeast plasma membrane depolarisation or hyperpolarisation by Δψ-dependent outflow from or uptake into the cells, reflected in changes in the fluorescence maximum λmax and fluorescence intensity. Upon membrane permeabilisation the dye redistributes between the cell and the medium in a purely concentration-dependent manner, which gives rise to Δψ-independent fluorescence responses that may mimic Δψ-dependent blue or red shift in λmax. These λmax shifts after cell permeabilisation depend on probe and ion concentrations inside and outside the cells at the moment of permeabilisation and reflect (a) permeabilisation-induced Δψ collapse, (b) changing probe binding capacity of cell constituents (inverse to the ambient ionic strength) and (c) hampering of probe equilibration by the poorly permeable cell wall. At low external ion concentrations, cell permeabilisation causes ion outflow and probe influx (hyperpolarisation-like red shift in λmax) caused by an increase in the probe-binding capacity of the cell interior and, in the case of heat shock, protein denaturation unmasking additional probe-binding sites. At high external ion levels minimising net ion efflux and at high intracellular probe concentrations at the moment of permeabilisation, the Δψ collapse causes a blue λmax shift mimicking an apparent depolarisation

Expression of Ndi1p, an alternative NADH:ubiquinone oxidoreductase, increases mitochondrial membrane potential in a C. elegans model of mitochondrial disease

AbstractThe NADH:ubiquinone oxidoreductase or complex I of the mitochondrial respiratory chain is an intricate enzyme with a vital role in energy metabolism. Mutations affecting complex I can affect at least three processes; they can impair the oxidation of NADH, reduce the enzyme's ability to pump protons for the generation of a mitochondrial membrane potential and increase the production of damaging reactive oxygen species. We have previously developed a nematode model of complex I-associated mitochondrial dysfunction that features hallmark characteristics of mitochondrial disease, such as lactic acidosis and decreased respiration. We have expressed the Saccharomyces cerevisiae NDI1 gene, which encodes a single subunit NADH dehydrogenase, in a strain of Caenorhabditis elegans with an impaired complex I. Expression of Ndi1p produces marked improvements in animal fitness and reproduction, increases respiration rates and restores mitochondrial membrane potential to wild type levels. Ndi1p functionally integrates into the nematode respiratory chain and mitigates the deleterious effects of a complex I deficit. However, we have also shown that Ndi1p cannot substitute for the absence of complex I. Nevertheless, the yeast Ndi1p should be considered as a candidate for gene therapy in human diseases involving complex I