RESUMO
Metabolons are dynamic associations of enzymes catalyzing consecutive reactions within a given pathway. Association results in enzyme stabilization and increased metabolic efficiency. Metabolons may use cytoskeletal elements, membranes and membrane proteins as scaffolds. The effects of glucose withdrawal on a putative glycolytic metabolon/F-actin system were evaluated in three Saccharomyces cerevisiae strains: a WT and two different obligate fermentative (OxPhos-deficient) strains, which obtained most ATP from glycolysis. Carbon source withdrawal led to inhibition of fermentation, decrease in ATP concentration and dissociation of glycolytic enzymes from F-actin. Depending on the strain, inactivation/reactivation transitions of fermentation took place in seconds. In addition, when ATP was very low, green fluorescent protein-labeled F-actin reorganized from highly dynamic patches to large, non-motile actin bodies containing proteins and enzymes. Glucose addition restored fermentation and cytoskeleton dynamics, suggesting that in addition to ATP concentration, at least in one of the tested strains, metabolon assembly/disassembly is a factor in the control of the rate of fermentation.
Assuntos
Citoesqueleto de Actina/ultraestrutura , Actinas/metabolismo , Citoesqueleto/enzimologia , Glicólise , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Citoesqueleto/ultraestrutura , Fermentação , Glucose/metabolismo , Gliceraldeído-3-Fosfato Desidrogenases/metabolismo , Proteínas dos Microfilamentos/metabolismo , Fosforilação Oxidativa , Fosfoglicerato Quinase/metabolismo , Saccharomyces cerevisiae/enzimologia , Saccharomyces cerevisiae/ultraestruturaRESUMO
Polytomella spp. is a colorless alga of the family Chlamydomonadaceae that lacks chloroplasts and cell wall. A highly active ubiquinol-cytochrome c oxidoreductase (bc1 complex), sensitive to antimycin and myxothiazol, has been purified and characterized from this alga (Gutiérrez-Cirlos et al., 1994, J. Biol. Chem. 269, 9147-9154). Both in mitochondrial membranes and in the isolated complex, the visible spectrum of cytochrome b from Polytomella spp. exhibits an atypical alpha-band with a maximum at 567 nm. This maximum is shifted 3-4 nm to the red when compared with b-type cytochromes from other organisms. Analysis of the b hemes of the bc1 complex by high performance liquid chromatography revealed no differences in the retention time and in the absorption spectra of the b-type hemes from Polytomella spp. and hemin, indicating that the prosthetic group in this alga is protoheme and thus ruling out the possibility that the red-shift could be due to different chemical substitutions in the porphyrin rings of the bL or bH hemes. The two b hemes were characterized by electrochemical redox titration; at pH 7.8-8.0, the midpoint potential for bL was-143 mV and for bH +25 mV. The spectra of the two b-type hemes were recorded in the presence of different reductants, at selected electrochemical potentials, and in the presence of antimycin A, to distinguish between the contribution of bL and bH to the visible spectrum. Both hemes bL and bH of the algal cytochrome b contribute to the observed bathochromic absorption maximum in the alpha-band of the spectrum. The data also show that the low potential bL heme from Polytomella spp. is spectroscopically similar to that of other organisms, with two transitions in the alpha-peak at 558.7 and 568.4 nm. The high-potential heme bH also exhibits a spectrum with two transitions at 557.2 and 568.9 nm, which surprisingly differs from the spectra of cytochrome bH of mammals, plants, yeasts, and bacteria, which all exhibit a single transition centered around 560 nm.