Book of Abstracts: Albany 2007
June 19-23 2007
Peculiarities of Succinate Transport in the Yeast Saccharomyces Cerevisiae
Succinate is one of the important metabolites which can be accumulated in the yeast growth medium during fermentation. This dicarboxylic acid is an important compound used in pharmaceutical and food industries as well as for making dyes, detergents, surfactants, et cetera (1). Despite the fact that this phenomenon is known for more than half a century (2), mechanism of succinate excretion is not yet well understood whereas data on efflux of other organic acids are controversial: in experiments done in vivo, active excretion of malic acid was suggested for Saccharomyces cerevisiae (3), while plasmalemma H+-ATPase was not involved in producing of citric acid by the yeast Yarrowia lipolytica (4).
To study succinate transport, we used plasma membrane vesicles of the yeast S. cerevisiae, housing primary H+-pump, H+-ATPase. These vesicles were suitable for transport studies and had low nonspecific ion permeability (5, 6). About half of the vesicles were inverted (Table I: see control activities in the absence and presence of osmotic stabilizer); upon addition of MgATP, H+-ATPase of such vesicles generated and maintained both components of electrochemical proton gradient (ΔμH+), chemical (ΔpH), and electrical (Em), which formations were monitored by ACMA or oxonol V fluorescence quenching, respectively. In these in vitro experiments, intravesicular space (acid pH and positive Em) corresponded to the yeast growth medium and transport of succinate into these vesicles, to its excretion in vivo.
ACMA or oxonol V quenching was prevented by orthovanadate when added before MgATP or the fluorescence signals were recovered when orthovanadate was added at the steady state. When studying succinate influence on the formation of ΔμH+, nitrate and sulfate were used as controls. First is a permeant anion which stimulates ΔpH formation, preventing the membrane potential generation if added before MgATP (Table II) and collapsing Em, if added at the steady state. The action of sulfate was similar to this of nitrate; however, the increase of ΔpH was less significant, while this anion decreased membrane potential without preventing Em formation completely. By contrast, succinate at much lesser concentration decreased (if added before MgATP) or dissipated (if added at the steady state) both components of ΔμH+. Decrease of Em indicated that succinate was transported as an anion. At the concentrations which effectively decreased ΔpH and Em, succinate did not inhibit ATPase (Table I); at higher concentrations it slightly increased its activity similar to sodium sulfate, probably due to non-specific activation of the enzyme by sodium and partial release of the H+-pump from under proton control (Tables I, II). Decrease of ΔμH+ by succinate could not be explained by inhibition of ATPase. H+-ATPase, by pumping protons, lowered intravesicular pH to 3.5 (5) while constants of dissociation of succinic acid are 4.21 and 5.63. As a result succinate binds H+; thus, decreasing ΔμH+.
Inhibition and dissipation of ΔpH, studied in greater detail, were characterized by saturation kinetics, allowing measurement of K1/2. For the inhibition of the formation of ΔpH, it was calculated as 0.40 mM succinate; this value was close to K1/2 of ΔpH dissipation by succinate (0.54 mM). These data suggest that succinate transport (excretion) occurs via a carrier in the plasma membrane of S. cerevisiae.
Direct uptake of [14C]succinate into plasma membrane vesicles in the presence of MgATP also followed Michaelis-Menten kinetics; Km of such transport was 0.47 mM succinate, being in good agreement to the values reported above. However, the dependence of succinate transport rate was biphasic with the second constant equal to 0.90 mM succinate. Interestingly, succinate influx was also found in the absence of MgATP with Lineweaver-Burk plot for initial rates of [14C]succinic acid uptake being linear and Km of 0.99 mM, almost equal to the second constant for the transport in the presence of MgATP.
Therefore, these data, obtained by two independent techniques, pointed to the conclusion that succinate uptake in vitro (that corresponds to its excretion in vivo) occurs via specific succinate or non-specific dicarboxylic carrier similar to general organic acid permease suggested earlier for in vivo uptake of mono-, di-, and tricarboxylic acids (7). It seems possible that such influx may occur both via specific (ATP-dependent) and non-specific (ATP-independent) carriers. The latter may be the same which carries out efflux of organic acids in vivo.
This research was supported by the Support Fund of Leading Scientific Schools of Russia NS-4318.2006.4
References and Footnotes
Valery V. Petrov
Institute of Biochemistry and Physiology of Microorganisms