The Deletion of the Succinate Dehydrogenase Gene KlSDH1 in Kluyveromyces lactis Does Not Lead to Respiratory Deficiency

Abstract

We have isolated a Kluyveromyces lactis mutant unable to grow on all respiratory carbon sources with the exception of lactate. Functional complementation of this mutant led to the isolation of KlSDH1, the gene encoding the flavoprotein subunit of the succinate dehydrogenase (SDH) complex, which is essential for the aerobic utilization of carbon sources. Despite the high sequence conservation of the SDH genes in Saccharomyces cerevisiae and K. lactis, they do not have the same relevance in the metabolism of the two yeasts. In fact, unlike SDH1, KlSDH1 was highly expressed under both fermentative and nonfermentative conditions. In addition to this, but in contrast with S. cerevisiae, K. lactis strains lacking KlSDH1 were still able to grow in the presence of lactate. In these mutants, oxygen consumption was one-eighth that of the wild type in the presence of lactate and was normal with glucose and ethanol, indicating that the respiratory chain was fully functional. Northern analysis suggested that alternative pathway(s), which involves pyruvate decarboxylase and the glyoxylate cycle, could overcome the absence of SDH and allow (i) lactate utilization and (ii) the accumulation of succinate instead of ethanol during growth on glucose.Succinate dehydrogenase (SDH) is a component of complex II of the respiratory chain that catalyses the oxidation of succinate to fumarate in the Krebs cycle and feeds electrons to the ubiquinone pool. The complex, which is highly conserved through evolution, is located in the inner mitochondrial membrane and consists of two catalytic and two structural subunits, all encoded by nuclear genes (38). In Saccharomyces cerevisiae, the four genes (SDH1 to SDH4) coding for SDH have been isolated and characterized (26,27,45,47). The flavoprotein subunit (11, 42) responsible for the oxidation of succinate to fumarate is encoded by two paralogous genes, SDH1 and SDH1b, although only SDH1 is necessary for growth on respiratory carbon sources (11). SDH2 codes for the iron-protein subunit (31) that contains three different iron-sulfur centers (22) and, together with the protein Sdh1p, constitutes the catalytic core of the SDH complex, which conveys electrons from the covalently attached flavin adenine dinucleotide (FAD) of Sdh1p first to the iron-sulfur centers and then to ubiquinone. SDH3 and SDH4 code for two small hydrophobic peptides, which anchor the complex to the inner mitochondrial membrane (10, 15). In humans, the mutations in the SDH genes have been associated to several mitochondrial-related pathologies suggesting, beside the enzymatic activity of the complex in the Krebs cycle, its involvement in superoxide handling (39,43).In S. cerevisiae, the expression of the SDH genes is repressed by glucose and derepressed on respiratory carbon sources (31, 45), and the loss of SDH functions results in the inability of cells to grow on any respiratory carbon sources (12,47).In this paper we report the isolation of the KlSDH1 gene (EMBL accession number AJ555233) encoding the Kluyveromyces lactis fla...

Deletion of the Glucose-6-Phosphate Dehydrogenase Gene Kl ZWF1 Affects both Fermentative and Respiratory Metabolism in Kluyveromyces lactis

“…8A). We have previously shown that K. lactis Klsdh1⌬ mutants devoid of succinate dehydrogenase activity were still able to grow in lactate (31). The requirement of G6PDH for growth on lactate is also supported by the observation that the capability of Klsdh1⌬ mutants to grow on this substrate (31) was lost in Klzwf1⌬ Klsdh1⌬ double mutants (not shown).…”

“…The following K. lactis strains were used in this work: MW179-1D (MAT␣ metA1 ade leu2 trip ura3) (31), CBS2359/3 (MATa hisA1) (provided by M. Wésolowsky), GG1996 (MATa rag2::LoxP) (34), and MS16-21B (MATa metA1 ade leu2 Klsdh1::kanMX4URA3 (this work).…”

“…The respiration rate was measured at 30°C, using a Clarktype electrode according to the method of Ferrero et al (10) under the conditions described previously (31).…”

Intracellular NADPH Levels Affect the Oligomeric State of the Glucose 6-Phosphate Dehydrogenase

“…2A, lanes 6 and 10). Since Klcox14⌬, differently from Klsdh1⌬ (32), is a respiratorydeficient mutant (11) and Klndi1⌬ is devoid of the major NADH reoxidation activity (35), one can conclude that the expression of the second G6PDH band in the presence of ethanol requires respiration and efficient coenzyme reoxidation.…”

“…1). The Klsdh1⌬ and Klcox14⌬ mutants are unable to assemble complex II and IV (11,32), respectively. Both parental and mutant strains showed a single band of G6PDH activity when grown in glucose ( Fig.…”