Cytosol–mitochondria transfer of reducing equivalents by a lactate shuttle in heterotrophic Euglena

Abstract

To assess the expression and physiological role of the mitochondrial NAD + -independent lactate dehydrogenase (iLDH) in Euglena gracilis, cells were grown with different carbon sources, and the D-and L-iLDH activities and several key metabolic intermediates were examined. iLDH activity was significant throughout the growth period, increasing by three-to fourfold from latency to the stationary phase. Intracellular levels of D-and L-lactate were high (5-40 mM) from the start of the culture and increased (20-80 mM) when the stationary phase was entered. All external carbon sources were actively consumed, reaching a minimum upon entering the stationary phase, when degradation of paramylon started. The level of ATP was essentially unchanged under all experimental conditions. Oxalate, an inhibitor of iLDH, strongly inhibited oligomycin-sensitive respiration and growth, whereas rotenone, an inhibitor of respiratory complex I, only slightly affected these parameters in lactategrown cells. Isolated mitochondria exhibited external NADH-supported respiration, which was sensitive to rotenone and flavone, and an inability to oxidize pyruvate. Addition of cytosol, NADH and pyruvate to mitochondria incubated with rotenone and flavone prompted significant O 2 uptake, which was blocked by oxalate. The data suggested that iLDH expression in Euglena is independent of substrate availability and that iLDHs play a key role in the transfer of reducing equivalents from the cytosol to the respiratory chain (lactate shuttle). Keywords: energy metabolism; lactate metabolism; NAD + -lactate dehydrogenase; NAD + -independent lactate dehydrogenase.The respiratory chain of mitochondria isolated from heterotrophic Euglena exhibits several unusual characteristics. It has a cyanide-insensitive alternative oxidase and an antimycin-insensitive, myxothiazol-sensitive, quinolcytochrome c oxidoreductase [1]. It also contains active membrane-bound NAD + -independent D-and L-lactate dehydrogenases (D-and L-iLDH) that directly transfer electrons to the quinone pool [2]. Similar enzymes that contain FAD or FMN as prosthetic groups have also been described in bacterial respiratory chains [3]. In addition, the quinone pool in Euglena mitochondria has equal concentrations of ubiquinone-9 and rhodoquinone-9 [4], which is a low redox-potential quinone also found in purple bacteria [5].We described recently that mitochondria, isolated from Euglena cultured with glutamate/malate (glu/mal) as the carbon source and harvested in the early stationary growth phase, exhibited stereospecific D-and L-iLDH activities [2]. Both enzymes were able to reduce the artificial high redoxpotential ubiquinones-1 and -2; D-iLDH showed a higher catalytic efficiency than L-iLDH, a pattern also observed in bacterial systems [6]. It was remarkable that Euglena mitochondria showed both enzyme activities because cells were grown with a carbon source different from DL-lactate or glucose. In other systems, only one of these enzymes is constitutive. In bacteria, the inducible enzyme is ex...

MrpA Functions in Energy Conversion during Acetate-Dependent Growth of Methanosarcina acetivorans

“…Valinomycin was added to a final concentration of 50 M, and after 2 min the reaction was stopped by addition of precooled perchloric acid at a 3% (vol/vol) final concentration. The ATP concentration was determined as previously described (31).…”

The Mitochondrion of Euglena gracilis

“…The presence of AOX was confirmed in silico (Perez et al 2014) and in vivo (Krnáčová et al 2015). Another pathway includes lactate dehydrogenase, which transfers electrons from lactate directly to ubiquinone (Moreno-Sánchez et al 2000) and is part of the lactate/pyruvate shuttle (Jasso-Chávez et al 2001Jasso-Chávez and Moreno-Sánchez 2003). Alternative respiratory pathways are likely involved in cellular responses to oxidative stress (Castro-Guerrero et al 2004, since E. gracilis lacks catalase, but contains ascorbate peroxidase (APX), which localizes exclusively to the cytosol (Shigeoka et al 2002).…”

“…Isolated mitochondria of E. gracilis used NADH as an external substrate for oxygen consumption (Uribe et al 1994;Jasso-Chávez and Moreno-Sánchez 2003;Krnáčová et al 2015). This seems surprising at first sight, because NADH is generally thought not to be transported across the mitochondrial inner membrane, and the enzyme or enzymes responsible for the use of NADH as an exogenous substrate during oxygen consumption in E. gracilis mitochondria have not been identified (Krnáčová et al 2015).…”

Increased synthesis of α-tocopherol, paramylon and tyrosine by Euglena gracilis under conditions of high biomass production

“…Euglena gracilis is a fresh water protist with an extensive capacity for adaptation to use a wide variety of alternative carbon sources for growth such as glucose, glutamate, malate, pyruvate, lactate and ethanol (Jasso‐Chávez and Moreno‐Sánchez 2003; Jasso‐Chávez et al. 2005; Rodríguez‐Zavala et al.…”