Natalia Chiquete-Félix

A glycolytic metabolon in Saccharomyces cerevisiae is stabilized by F‐actin

In the Saccharomyces cerevisiae glycolytic pathway, 11 enzymes catalyze the stepwise conversion of glucose to two molecules of ethanol plus two CO2 molecules. In the highly crowded cytoplasm, this pathway would be very inefficient if it were dependent on substrate/enzyme diffusion. Therefore, the existence of a multi‐enzymatic glycolytic complex has been suggested. This complex probably uses the cytoskeleton to stabilize the interaction of the various enzymes. Here, the role of filamentous actin (F‐actin) in stabilization of a putative glycolytic metabolon is reported. Experiments were performed in isolated enzyme/actin mixtures, cytoplasmic extracts and permeabilized yeast cells. Polymerization of actin was promoted using phalloidin or inhibited using cytochalasin D or latrunculin. The polymeric filamentous F‐actin, but not the monomeric globular G‐actin, stabilized both the interaction of isolated glycolytic pathway enzyme mixtures and the whole fermentation pathway, leading to higher fermentation activity. The associated complexes were resistant against inhibition as a result of viscosity (promoted by the disaccharide trehalose) or inactivation (using specific enzyme antibodies). In S. cerevisiae, a glycolytic metabolon appear to assemble in association with F‐actin. In this complex, fermentation activity is enhanced and enzymes are partially protected against inhibition by trehalose or by antibodies.

The branched mitochondrial respiratory chain from Debaryomyces hansenii: components and supramolecular organization.

The branched respiratory chain in mitochondria from the halotolerant yeast Debaryomyces hansenii contains the classical complexes I, II, III and IV plus a cyanide-insensitive, AMP-activated, alternative-oxidase (AOX). Two additional alternative oxidoreductases were found in this organism: an alternative NADH dehydrogenase (NDH2e) and a mitochondrial isoform of glycerol-phosphate dehydrogenase (MitGPDH). These monomeric enzymes lack proton pump activity. They are located on the outer face of the inner mitochondrial membrane. NDH2e oxidizes exogenous NADH in a rotenone-insensitive, flavone-sensitive, process. AOX seems to be constitutive; nonetheless, most electrons are transferred to the cytochromic pathway. Respiratory supercomplexes containing complexes I, III and IV in different stoichiometries were detected. Dimeric complex V was also detected. In-gel activity of NADH dehydrogenase, mass spectrometry, and cytochrome c oxidase and ATPase activities led to determine the composition of the putative supercomplexes. Molecular weights were estimated by comparison with those from the yeast Y. lipolytica and they were IV2, I-IV, III2-IV4, V2, I-III2, I-III2-IV, I-III2-IV2, I-III2-IV3 and I-III2-IV4. Binding of the alternative enzymes to supercomplexes was not detected. This is the first report on the structure and organization of the mitochondrial respiratory chain from D. hansenii.

The Acrosomal Matrix From Guinea Pig Sperm Contains Structural Proteins, Suggesting the Presence of an Actin Skeleton

The mammalian sperm acrosome contains a large number of hydrolytic enzymes. When the acrosomal reaction and fertilization occur, these enzymes are released in an orderly fashion, suggesting that the acrosomal matrix is highly organized. It was decided to determine the identity of the structural scaffold underlying the organization of the acrosome. In permeabilized acrosomes and in the Triton X-100-extracted acrosomal matrices from guinea pig sperm, we used indirect immunofluorescence, immunogold labeling, and Western blotting to identify F-actin, spectrin, myosin, calmodulin, and gelsolin. These proteins were detected in the acrosomal matrix for the first time. In noncapacitated, intact spermatozoa the addition of the F-actin monomerizing agent cytochalasin D resulted in loss of the acrosome, suggesting that F-actin is needed to preserve an intact acrosome. Our results suggest that the acrosomal architecture is supported by a dynamic F-actin skeleton, which probably regulates the differential rate of release of the acrosomal enzymes during acrosomal reaction and fertilization.

Staphylococcus epidermidis: metabolic adaptation and biofilm formation in response to different oxygen concentrations

Staphylococcus epidermidis has become a major health hazard. It is necessary to study its metabolism and hopefully uncover therapeutic targets. Cultivating S. epidermidis at increasing oxygen concentration [O2] enhanced growth, while inhibiting biofilm formation. Respiratory oxidoreductases were differentially expressed, probably to prevent reactive oxygen species formation. Under aerobiosis, S. epidermidis expressed high oxidoreductase activities, including glycerol-3-phosphate dehydrogenase, pyruvate dehydrogenase, ethanol dehydrogenase and succinate dehydrogenase, as well as cytochromes bo and aa3; while little tendency to form biofilms was observed. Under microaerobiosis, pyruvate dehydrogenase and ethanol dehydrogenase decreased while glycerol-3-phosphate dehydrogenase and succinate dehydrogenase nearly disappeared; cytochrome bo was present; anaerobic nitrate reductase activity was observed; biofilm formation increased slightly. Under anaerobiosis, biofilms grew; low ethanol dehydrogenase, pyruvate dehydrogenase and cytochrome bo were still present; nitrate dehydrogenase was the main terminal electron acceptor. KCN inhibited the aerobic respiratory chain and increased biofilm formation. In contrast, methylamine inhibited both nitrate reductase and biofilm formation. The correlation between the expression and/or activity or redox enzymes and biofilm-formation activities suggests that these are possible therapeutic targets to erradicate S. epidermidis.

Newly synthesized cAMP is integrated at a membrane protein complex signalosome to ensure receptor response specificity.

Spatiotemporal regulation of cAMP within the cell is required to achieve receptor‐specific responses. The mechanism through which the cell selects a specific response to newly synthesized cAMP is not fully understood. In hepatocyte plasma membranes, we identified two functional and independent cAMP‐responsive signaling protein macrocomplexes that produce, use, degrade, and regulate their own nondiffusible (sequestered) cAMP pool to achieve their specific responses. Each complex responds to the stimulation of an adenosine G protein‐coupled receptor (Ado‐GPCR), bound to either A2A or A2B, but not simultaneously to both. Each isoprotein involved in each signaling cascade was identified by measuring changes in cAMP levels after receptor activation, and its participation was confirmed by antibody‐mediated inactivation. A2A‐Ado‐GPCR selective stimulation activates adenylyl cyclase 6 (AC6), which is bound to AKAP79/150, to synthesize cAMP which is used by two other AKAP79/150‐tethered proteins: protein kinase A (PKA) and phosphodiesterase 3A (PDE3A). In contrast, A2B‐Ado‐GPCR stimulation activates D‐AKAP2‐attached AC5 to generate cAMP, which is channeled to two other D‐AKAP2‐tethered proteins: guanine‐nucleotide exchange factor 2 (Epac2) and PDE3B. In both cases, prior activation of PKA or Epac2 with selective cAMP analogs prevents de novo cAMP synthesis. In addition, we show that cAMP does not diffuse between these protein macrocomplexes or ‘signalosomes’. Evidence of coimmunoprecipitation and colocalization of some proteins belonging to each signalosome is presented. Each signalosome constitutes a minimal functional signaling unit with its own machinery to synthesize and regulate a sequestered cAMP pool. Thus, each signalosome is devoted to ensure the transmission of a unique and unequivocal message through the cell.

Effects of ubiquinone derivatives on the mitochondrial unselective channel of Saccharomyces cerevisiae

Ubiquinone derivatives modulate the mammalian mitochondrial Permeability Transition Pore (PTP). Yeast mitochondria harbor a similar structure: the respiration- and ATP-induced Saccharomyces cerevisiae Mitochondrial Unselective Channel (ScMUC). Here we show that decylubiquinone, a well-characterized inhibitor of the PTP, suppresses ScMUC opening in diverse strains and independently of respiratory chain modulation or redox-state. We also found that naturally occurring derivatives such as hexaprenyl and decaprenyl ubiquinones lacked effects on the ScMUC. The PTP-inactive ubiquinone 5 (Ub5) promoted the ScMUC-independent activation of the respiratory chain in most strains tested. In an industrial strain however, Ub5 blocked the protection elicited by dUb. The results indicate the presence of a ubiquinone-binding site in the ScMUC.

A critical tyrosine residue determines the uncoupling protein-like activity of the yeast mitochondrial oxaloacetate carrier

The mitochondrial Oac (oxaloacetate carrier) found in some fungi and plants catalyses the uptake of oxaloacetate, malonate and sulfate. Despite their sequence similarity, transport specificity varies considerably between Oacs. Indeed, whereas ScOac (Saccharomyces cerevisiae Oac) is a specific anion-proton symporter, the YlOac (Yarrowia lipolytica Oac) has the added ability to transport protons, behaving as a UCP (uncoupling protein). Significantly, we identified two amino acid changes at the matrix gate of YlOac and ScOac, tyrosine to phenylalanine and methionine to leucine. We studied the role of these amino acids by expressing both wild-type and specifically mutated Oacs in an Oac-null S. cerevisiae strain. No phenotype could be associated with the methionine to leucine substitution, whereas UCP-like activity was dependent on the presence of the tyrosine residue normally expressed in the YlOac, i.e. Tyr-ScOac mediated proton transport, whereas Phe-YlOac lost its protonophoric activity. These findings indicate that the UCP-like activity of YlOac is determined by the tyrosine residue at position 146.

Role of Wasp and the small GTPases RhoA, RhoB, and Cdc42 during capacitation and acrosome reaction in spermatozoa of english‐guinea pigs

Cytoskeleton remodeling is necessary for capacitation and the acrosome reaction in spermatozoa. F‐actin is located in the acrosome and equatorial region during capacitation, but is relocated in the post‐acrosomal region during the acrosome reaction in spermatozoa from bull, rat, mice, and guinea pig. Actin polymerization and relocalization are generally regulated by small GTPases that activate Wasp protein, which coordinates with Arp2/3, profilin I, and profilin II to complete cytoskeletal remodeling. This sequence of events is not completely described in spermatozoa, though. Therefore, the aim of this study was to determine if Wasp interacts with small GTPases (RhoA, RhoB, and Cdc42) and proteins (Arp2/3, profilin I, and profilin II) that co‐localize with F‐actin during capacitation and the acrosome reaction in English guinea pig spermatozoa obtained from the vas deferens. The spermatozoa were capacitated in calcium‐free medium, incubated with an activator or an inhibitor of GTPases, and then induced to acrosome react using calcium. The distribution patterns of F‐actin were compared to the patterns of Wasp and its putative interaction partners: Wasp and RhoB, but not RhoA or Cdc42, localization overlap with F‐actin during capacitation and the acrosome reaction. Activation of small GTPases localized RhoB to the post‐acrosomal region whereas their inhibition prevented acrosome exocytosis. Arp2/3 and profilin II appear to interact with Wasp in the post‐acrosomal region and flagellum, while profilin I and Wasp could be found in the equatorial region. Thus, Wasp and F‐actin distribution overlap during capacitation and acrosome reaction, and small GTPases play an important role in cytoskeleton remodeling during these processes in spermatozoa. Mol. Reprod. Dev. 83: 927–937, 2016

Metabolic Optimization by Enzyme-Enzyme and Enzyme-Cytoskeleton Associations

Probably enzymes are not dispersed in the cytoplasm, but are bound to each other and to specific cytoskeleton proteins. Associations result in substrate channeling from one enzyme to another. Multienzymatic complexes, or metabolons have been detected in glycolysis, the Krebs cycle and oxidative phosphorylation. Also, some glycolytic enzymes interact with mitochondria. Metabolons may associate with actin or tubulin, gaining stability. Metabolons resist inhibition mediated by the accumulation of compatible solutes observed during the stress response. Compatible solutes protect membranes and proteins against stress. However, when stress is over, compatible solutes inhibit growth, probably due to the high viscosity they promote. Viscosity inhibits protein movements. Enzymes that undergo large conformational changes during catalysis are more sensitive to viscosity. Enzyme association seems to protect the more sensitive enzymes from viscosity-mediated inhibition. The association-mediated protection of the enzymes in a given metabolic pathway would constitute a new property of metabolons: that is, to enhance survival during stress. It is proposed that resistance to inhibition is due to elimination of non-productive conformations in highly motile enzymes.

Wolbachia pipientis grows in Saccharomyces cerevisiae evoking early death of the host and deregulation of mitochondrial metabolism

Wolbachia sp. has colonized over 70% of insect species, successfully manipulating host fertility, protein expression, lifespan, and metabolism. Understanding and engineering the biochemistry and physiology of Wolbachia holds great promise for insect vector‐borne disease eradication. Wolbachia is cultured in cell lines, which have long duplication times and are difficult to manipulate and study. The yeast strain Saccharomyces cerevisiae W303 was used successfully as an artificial host for Wolbachia wAlbB. As compared to controls, infected yeast lost viability early, probably as a result of an abnormally high mitochondrial oxidative phosphorylation activity observed at late stages of growth. No respiratory chain proteins from Wolbachia were detected, while several Wolbachia F1F0‐ATPase subunits were revealed. After 5 days outside the cell, Wolbachia remained fully infective against insect cells.