Claudina Rodrigues-Pousada

Yap, a novel family of eight bZIP proteins in Saccharomyces cerevisiae with distinct biological functions.

Saccharomyces cerevisiae contains eight members of a novel and fungus-specific family of bZIP proteins that is defined by four atypical residues on the DNA-binding surface. Two of these proteins, Yap1 and Yap2, are transcriptional activators involved in pleiotropic drug resistance. Although initially described as AP-1 factors, at least four Yap proteins bind most efficiently to TTACTAA, a sequence that differs at position +/-2 from the optimal AP-1 site (TGACTCA); further, a Yap-like derivative of the AP-1 factor Gcn4 (A239Q S242F) binds efficiently to the Yap recognition sequence. Molecular modeling suggests that the Yap-specific residues make novel contacts and cause physical constraints at the +/-2 position that may account for the distinct DNA-binding specificities of Yap and AP-1 proteins. To various extents, Yap1, Yap2, Yap3, and Yap5 activate transcription from a promoter containing a Yap recognition site. Yap-dependent transcription is abolished in strains containing high levels of protein kinase A; in contrast, Gcn4 transcriptional activity is stimulated by protein kinase A. Interestingly, Yap1 transcriptional activity is stimulated by hydrogen peroxide, whereas Yap2 activity is stimulated by aminotriazole and cadmium. In addition, unlike other yap mutations tested, yap4 (cin5) mutations affect chromosome stability, and they suppress the cold-sensitive phenotype of yap1 mutant strains. Thus, members of the Yap family carry out overlapping but distinct biological functions.

The ABC transporter proteins Pat1 and Pat2 are required for import of long-chain fatty acids into peroxisomes of Saccharomyces cerevisiae.

Peroxisomes of Saccharomyces cerevisiae are the exclusive site of fatty acid beta‐oxidation. We have found that fatty acids reach the peroxisomal matrix via two independent pathways. The subcellular site of fatty acid activation varies with chain length of the substrate and dictates the pathway of substrate entry into peroxisomes. Medium‐chain fatty acids are activated inside peroxisomes hby the acyl‐CoA synthetase Faa2p. On the other hand, long‐chain fatty acids are imported from the cytosolic pool of activated long‐chain fatty acids via Pat1p and Pat2p, peroxisomal membrane proteins belonging to the ATP binding cassette transporter superfamily. Pat1p and Pat2p are the first examples of membrane proteins involved in metabolite transport across the peroxisomal membrane.

Structure of a Dioxygen Reduction Enzyme from Desulfovibrio Gigas

Desulfovibrio gigas is a strict anaerobe that contains a well-characterized metabolic pathway that enables it to survive transient contacts with oxygen. The terminal enzyme in this pathway, rubredoxin:oxygen oxidoreductase (ROO) reduces oxygen to water in a direct and safe way. The 2.5 Å resolution crystal structure of ROO shows that each monomer of this homodimeric enzyme consists of a novel combination of two domains, a flavodoxin-like domain and a Zn-β-lactamase-like domain that contains a di-iron center for dioxygen reduction. This is the first structure of a member of a superfamily of enzymes widespread in strict and facultative anaerobes, indicating its broad physiological significance.

STUDIES ON THE REDOX CENTERS OF THE TERMINAL OXIDASE FROM DESULFOVIBRIO GIGAS AND EVIDENCE FOR ITS INTERACTION WITH RUBREDOXIN

Rubredoxin-oxygen oxidoreductase (ROO) is the final component of a soluble electron transfer chain that couples NADH oxidation to oxygen consumption in the anaerobic sulfate reducerDesulfovibrio gigas. It is an 86-kDa homodimeric flavohemeprotein containing two FAD molecules, one mesoheme IX, and one Fe-uroporphyrin I per monomer, capable of fully reducing oxygen to water. EPR studies on the native enzyme reveal two components with g values at ∼2.46, 2.29, and 1.89, which are assigned to low spin hemes and are similar to the EPR features of P-450 hemes, suggesting that ROO hemes have a cysteinyl axial ligation. At pH 7.6, the flavin redox transitions occur at 0 ± 15 mV for the quinone/semiquinone couple and at −130 ± 15 mV for the semiquinone/hydroquinone couple; the hemes reduction potential is −350 ± 15 mV. Spectroscopic studies provided unequivocal evidence that the flavins are the electron acceptor centers from rubredoxin, and that their reduction proceed through an anionic semiquinone radical. The reaction with oxygen occurs in the flavin moiety. These data are strongly corroborated by the finding that rubredoxin and ROO are located in the same polycistronic unit of D. gigas genome. For the first time, a clear role for a rubredoxin in a sulfate-reducing bacterium is presented.

Two redox centers within Yap1 for H2O2 and thiol-reactive chemicals signaling

The Yap1 transcription factor regulates yeast responses to H2O2 and to several unrelated chemicals and metals. Activation by H2O2 involves Yap1 Cys303-Cys598 intra-molecular disulfide bond formation directed by the H2O2 sensor Orp1/Gpx3. We show here that the electrophile N-ethylmaleimide activates Yap1 by covalent modification of Yap1 C-terminal Cys598, Cys620, and Cys629, in an Orp1 and Yap1-oxidation-independent way, thus establishing an alternate and distinct mode of Yap1 activation. We also show that menadione, a superoxide anion generator and a highly reactive electrophile, operates both modes of Yap1 activation. Further, the Yap1 C-terminal domain reactivity towards other electrophiles (4-hydroxynonenal, iodoacetamide) and metals (cadmium, selenium) suggests a common mechanism for sensing thiol reactive chemicals, involving thiol chemical modification. We propose that Yap1 has two distinct molecular redox centers, one triggered by ROS (hydroperoxides and the superoxide anion) and the other by chemicals with thiol reactivity (electrophiles and divalent heavy metals cations). These data indicate that yeast cells cannot sense these compounds through the same molecular devices, albeit they are all electrophilic.

Yeast activator proteins and stress response: an overview

Yeast, and especially Saccharomyces cerevisiae, are continuously exposed to rapid and drastic changes in their external milieu. Therefore, cells must maintain their homeostasis, which is achieved through a highly coordinated gene expression involving a plethora of transcription factors, each of them performing specific functions. Here, we discuss recent advances in our understanding of the function of the yeast activator protein family of eight basic‐leucine zipper trans‐activators that have been implicated in various forms of stress response.

The Yap family and its role in stress response

The budding yeast Saccharomyces cerevisiae possesses a very flexible and complex programme of gene expression when exposed to several environmental challenges. Homeostasis is achieved through a highly coordinated mechanism of transcription regulation involving several transcription factors, each one acting singly or in combination to perform specific functions. Here, we review our current knowledge of the function of the Yap transcription factors in stress response. They belong to b‐ZIP proteins comprising eight members with specificity at the DNA‐binding domain distinct from that of the conventional yeast AP‐1 factor, Gcn4. We finish with new insights into the links of transcriptional networks controlling several cellular processes. The data reviewed in this article illustrate how much our comprehension of the biology of Yap family involved in stress response has advanced, and how much research is still needed to unravel the complexity of the role of these transcriptional factors. The complexities of these regulatory interactions, as well as the dynamics of these processes, are important to understand in order to elucidate the control of stress response, a highly conserved process in eukaryotes. Copyright

Thermostabilization of Proteins by Diglycerol Phosphate, a New Compatible Solute from the Hyperthermophile Archaeoglobus fulgidus

ABSTRACT Diglycerol phosphate accumulates under salt stress in the archaeonArchaeoglobus fulgidus (L. O. Martins, R. Huber, H. Huber, K. O. Stetter, M. S. da Costa, and H. Santos, Appl. Environ. Microbiol. 63:896–902, 1997). This solute was purified after extraction from the cell biomass. In addition, the optically active and the optically inactive (racemic) forms of the compound were synthesized, and the ability of the solute to act as a protecting agent against heating was tested on several proteins derived from mesophilic or hyperthermophilic sources. Diglycerol phosphate exerted a considerable stabilizing effect against heat inactivation of rabbit muscle lactate dehydrogenase, bakers yeast alcohol dehydrogenase, andThermococcus litoralis glutamate dehydrogenase. Highly homologous and structurally well-characterized rubredoxins fromDesulfovibrio gigas, Desulfovibrio desulfuricans (ATCC 27774), and Clostridium pasteurianum were also examined for their thermal stabilities in the presence or absence of diglycerol phosphate, glycerol, and inorganic phosphate. These proteins showed different intrinsic thermostabilities, with half-lives in the range of 30 to 100 min. Diglycerol phosphate exerted a strong protecting effect, with approximately a fourfold increase in the half-lives for the loss of the visible spectra of D. gigas and C. pasteurianumrubredoxins. In contrast, the stability of D. desulfuricansrubredoxin was not affected. These different behaviors are discussed in the light of the known structural features of rubredoxins. The data show that diglycerol phosphate is a potentially useful protein stabilizer in biotechnological applications.

POST-TERMINATION RIBOSOME INTERACTIONS WITH THE 5'UTR MODULATE YEAST MRNA STABILITY

A novel form of post‐transcriptional control is described. The 5′ untranslated region (5′UTR) of the Saccharomyces cerevisiae gene encoding the AP1‐like transcription factor Yap2 contains two upstream open reading frames (uORF1 and uORF2). The YAP2‐type of uORF functions as a cis‐acting element that attenuates gene expression at the level of mRNA turnover via termination‐dependent decay. Release of post‐termination ribosomes from the YAP2 5′UTR causes accelerated decay which is largely independent of the termination modulator gene UPF1. Both of the YAP2 uORFs contribute to the destabilization effect. A G/C‐rich stop codon context, which seems to promote ribosome release, allows an uORF to act as a transferable 5′UTR‐destabilizing element. Moreover, termination‐dependent destabilization is potentiated by stable secondary structure 3′ of the uORF stop codon. The potentiation of uORF‐mediated destabilization is eliminated if the secondary structure is located further downstream of the uORF, and is also influenced by a modulatory mechanism involving eIF2. Destabilization is therefore linked to the kinetics of acquisition of reinitiation‐competence by post‐termination ribosomes in the 5′UTR. Our data explain the destabilizing properties of YAP2‐type uORFs and also support a more general model for the mode of action of other known uORFs, such as those in the GCN4 mRNA.

Association of African swine fever virus with the cytoskeleton.

The association of African swine fever virus (ASFV) with the cytoskeleton was investigated. Immunofluorescent studies of ASFV infected cells with anti-ASFV serum showed a temporal and spatial development of viral inclusions which moved from a peripheral to a perinuclear location and fused to give a single large perinuclear factory. The migration and fusion of viral inclusions was inhibited by colchicine suggesting a function for microtubules in assembly site organization not previously described. Accumulation of virions outside the inclusions and inhibition of viral release was also observed in colchicine treated cells. Viral antigens and structural elements were retained on the cytoskeleton fraction of Triton X-100 extracted cells. Reorganization of cytoskeletal elements around the assembly sites was demonstrated by transmission electronmicroscopy and by immunofluorescent studies using monoclonal antibodies against actin, tubulin and vimentin. Intermediate filaments accumulated around the viral factories, microtubules were greatly decreased in number and microfilaments were reorganized in association with the plasma membrane. Bundles of 15 nm tubules of unknown origin were also observed around the assembly sites. The distribution of viral proteins in soluble, cytoskeleton and detergent insoluble nuclear fractions was studied by pulse-chase experiments with [35S]methionine. SDS-PAGE analysis showed the presence in the cytoskeletal and nuclear fractions of 150, 72, 38, 28, 19 and 15 kDa virus structural proteins which increased after a 5 h chase. Our results indicate a close association of ASFV replication with the cytoskeleton similar to events described during FV3 replication but which differ from those occurring in poxvirus-infected cells.