Raúl García

The Sequential Activation of the Yeast HOG and SLT2 Pathways Is Required for Cell Survival to Cell Wall Stress

Yeast mitogen-activated protein kinase (MAPK) signaling pathways transduce external stimuli into cellular responses very precisely. The MAPKs Slt2/Mpk1 and Hog1 regulate transcriptional responses of adaptation to cell wall and osmotic stresses, respectively. Unexpectedly, we observe that the activation of a cell wall integrity (CWI) response to the cell wall damage caused by zymolyase (beta-1,3 glucanase) requires both the HOG and SLT2 pathways. Zymolyase activates both MAPKs and Slt2 activation depends on the Sho1 branch of the HOG pathway under these conditions. Moreover, adaptation to zymolyase requires essential components of the CWI pathway, namely the redundant MAPKKs Mkk1/Mkk2, the MAPKKK Bck1, and Pkc1, but it does not require upstream elements, including the sensors and the guanine nucleotide exchange factors of this pathway. In addition, the transcriptional activation of genes involved in adaptation to cell wall stress, like CRH1, depends on the transcriptional factor Rlm1 regulated by Slt2, but not on the transcription factors regulated by Hog1. Consistent with these findings, both MAPK pathways are essential for cell survival under these circumstances because mutant strains deficient in different components of both pathways are hypersensitive to zymolyase. Thus, a sequential activation of two MAPK pathways is required for cellular adaptation to cell wall damage.

The High Osmotic Response and Cell Wall Integrity Pathways Cooperate to Regulate Transcriptional Responses to Zymolyase-induced Cell Wall Stress in Saccharomyces cerevisiae

The adaptation of Saccharomyces cerevisiae to situations in which cell wall integrity is seriously compromised mainly involves the cell wall integrity (CWI) pathway. However, in a recent work ( Bermejo, C., Rodriguez, E., García, R., Rodríguez-Peña, J. M., Rodríguez de la Concepción, M. L., Rivas, C., Arias, P., Nombela, C., Posas, F., and Arroyo, J. (2008) Mol. Biol. Cell 19, 1113-1124 ) we have demonstrated the co-participation of the high osmotic response (HOG) pathway to ensure yeast survival to cell wall stress mediated by zymolyase, which hydrolyzes the β-1,3 glucan network. Here we have characterized the role of both pathways in the regulation of the overall yeast transcriptional responses to zymolyase treatment using whole genome expression profiling. A main group of yeast genes is dependent on both MAPKs, Slt2 and Hog1, for their induction. The transcriptional activation of these genes depends on the MAPKKK Bck1, the transcription factor Rlm1, and elements of the sho1 branch of the HOG pathway, but not on the sensors of the CWI pathway. A second group of genes is dependent on Slt2 but not Hog1 or Pbs2. However, the induction of these genes is dependent on upstream elements of the HOG pathway such as Sho1, Ste50, and Ste11, in accordance with a sequential activation of the HOG and CWI pathways. Zymolyase also promotes an osmotic-like transcriptional response with the activation of a group of genes dependent on elements of the Sho1 branch of HOG pathway but not on Slt2, with the induction of many of them dependent on Msn2/4. Additionally, in the absence of Hog1, zymolyase induces an alternative response related to mating and filamentation as a consequence of the cross-talk between these pathways and the HOG pathway. Finally, in the absence of Slt2, zymolyase increases the induction of genes associated with osmotic adaptation with respect to the wild type, suggesting an inhibitory effect of the CWI pathway over the HOG pathway. These studies clearly reveal the complexity of the signal transduction machinery responsible for regulating yeast adaptation responses to cell wall stress.

The high‐osmolarity glycerol (HOG) and cell wall integrity (CWI) signalling pathways interplay: a yeast dialogue between MAPK routes

Two mitogen‐activated protein kinase (MAPK) pathways, viz. the high‐osmolarity glycerol (HOG) and the cell wall integrity (CWI) pathways, regulate stress responses in the yeast Saccharomyces cerevisiae. Whereas the former is mainly involved in adaptation of yeast cells to hyperosmotic stress, the latter is activated under conditions leading to cell wall instability. Although MAPK signalling specificity can be conceived as requiring insulation of the different pathways, it is also becoming clear that the two pathways do not compete with each other but can be positively coordinated to regulate many stress responses. This review highlights our current knowledge about the collaboration between these two MAPK pathways to counteract different kinds of environmental stress. Copyright

Tetrathiafulvalene-Based Nanotweezers—Noncovalent Binding of Carbon Nanotubes in Aqueous Media with Charge Transfer Implications

Electron donor-acceptor hybrids based on single wall carbon nanotubes (SWCNT) are one of the most promising functional structures that are currently developed in the emerging areas of energy conversion schemes and molecular electronics. As a suitable electron donor, π-extended tetrathiafulvalene (exTTF) stands out owing to its recognition of SWCNT through π-π stacking and electron donor-acceptor interactions. Herein, we explore the shape and electronic complementarity between different types of carbon nanotubes (CNT) and a tweezers-shaped molecule endowed with two exTTFs in water. The efficient electronic communication between semiconducting SWCNT/multiwall carbon nanotubes (MWCNT), on one hand, and the water-soluble exTTF nanotweezers 8, on the other hand, has been demonstrated in the ground and excited state by using steady-state as well as time-resolved spectroscopies, which were further complemented by microscopy. Importantly, appreciable electronic communication results in the electronic ground state having a shift of electron density, that is, from exTTFs to CNT, and in the electronic excited state having a full separation of electron density, that is oxidized exTTF and reduced CNT. Lifetimes in the range of several hundred picoseconds, which were observed for the corresponding electron transfer products upon light irradiation, tend to be appreciably longer in MWCNT/8 than in SWCNT/8.

Genome-wide survey of yeast mutations leading to activation of the yeast cell integrity MAPK pathway: Novel insights into diverse MAPK outcomes

BackgroundThe yeast cell wall integrity mitogen-activated protein kinase (CWI-MAPK) pathway is the main regulator of adaptation responses to cell wall stress in yeast. Here, we adopt a genomic approach to shed light on two aspects that are only partially understood, namely, the characterization of the gene functional catalog associated with CWI pathway activation and the extent to which MAPK activation correlates with transcriptional outcomes.ResultsA systematic yeast mutant deletion library was screened for constitutive transcriptional activation of the CWI-related reporter gene MLP1. Monitoring phospho-Slt2/Mpk1 levels in the identified mutants revealed sixty-four deletants with high levels of phosphorylation of this MAPK, including mainly genes related to cell wall construction and morphogenesis, signaling, and those with unknown function. Phenotypic analysis of the last group of mutants suggests their involvement in cell wall homeostasis. A good correlation between levels of Slt2 phosphorylation and the magnitude of the transcriptional response was found in most cases. However, the expression of CWI pathway-related genes was enhanced in some mutants in the absence of significant Slt2 phosphorylation, despite the fact that functional MAPK signaling through the pathway was required. CWI pathway activation was associated to increased deposition of chitin in the cell wall - a known survival compensatory mechanism - in about 30% of the mutants identified.ConclusionWe provide new insights into yeast genes related to the CWI pathway and into how the state of activation of the Slt2 MAPK leads to different outcomes, discovering the versatility of this kind of signaling pathways. These findings potentially have broad implications for understanding the functioning of other eukaryotic MAPKs.

Tuning the electronic properties of nonplanar exTTF-based push-pull chromophores by aryl substitution.

A new family of π-extended tetrathiafulvalene (exTTF) donor-acceptor chromophores has been synthesized by [2 + 2] cycloaddition of TCNE with exTTF-substituted alkynes and subsequent cycloreversion. X-ray data and theoretical calculations, performed at the B3LYP/6-31G** level, show that the new chromophores exhibit highly distorted nonplanar molecular structures with largely twisted 1,1,4,4-tetracyanobuta-1,3-diene (TCBD) units. The electronic and optical properties, investigated by UV/vis spectroscopy and electrochemical measurements, are significantly modified when the TCBD acceptor unit is substituted with a donor phenyl group, which increases the twisting of the TCBD units and reduces the conjugation between the two dicyanovinyl subunits. The introduction of phenyl substituents hampers the oxidation and reduction processes and, at the same time, largely increases the optical band gap. An effective electronic communication between the donor and acceptor units, although limited by the distorted molecular geometry, is evidenced both in the ground and in the excited electronic states. The electronic absorption spectra are characterized by low- to medium-intense charge-transfer bands that extend to the near-infrared.

Chromatin remodeling by the SWI/SNF complex is essential for transcription mediated by the yeast cell wall integrity MAPK pathway

The SWI/SNF complex is a key element of the yeast CWI MAPK pathway, which mediates the chromatin remodeling necessary for an adequate transcriptional response to cell wall stress. The MAPK Slt2 mediates, through Rlm1, nucleosome rearrangements at cell wall stress–responsive genes by targeting the SWI/SNF complex.

An Endohedral Metallofullerene as a Pure Electron Donor: Intramolecular Electron Transfer in Donor–Acceptor Conjugates of La2@C80 and 11,11,12,12-Tetracyano-9,10-anthra-p-quinodimethane (TCAQ)

An endohedral metallofullerene, La(2)@C(80), is covalently linked to the strong electron acceptor 11,11,12,12-tetracyano-9,10-anthra-p-quinodimethane (TCAQ) by means of the Prato reaction, affording two different [5,6]-metallofulleropyrrolidines, namely 1a and 2a. 1a and 2a were isolated and fully characterized by means of MALDI-TOF mass, UV-vis-NIR absorption, and NMR spectroscopies. In addition, cyclic voltammetry (CV) and differential pulse voltammetry (DPV) corroborated the unique redox character of 2a, that is, the presence of the electron-donating La(2)@C(80) and the electron-accepting TCAQ. Although a weak electronic coupling dictates the interactions between La(2)@C(80) and TCAQ in the ground state, time-resolved transient absorption experiments reveal that in the excited state (i.e., π-π* centered at La(2)@C(80)) the unprecedented formation of the (La(2)@C(80))(•+)-(TCAQ)(•-) radical ion pair state evolves in nonpolar and polar media with a quantum efficiency of 33%.

Stable Electron Donor–Acceptor Nanohybrids by Interfacing n‐Type TCAQ with p‐Type Single‐Walled Carbon Nanotubes

Financial support from MINECO of Spain (CTQ2011-24652, PIB2010JP-00196, 2010C-07-25200, and Consolider- Ingenio CSD2007-00010), FUNMOLS (FP7-212942-1), CAM (MADRISOLAR-2 S2009/PPQ-1533), DFG (GU 517/16-1), and DFG (Excellence Cluster – Engineering of Advanced Materials) is greatly appreciated.

Stabilizing Ion and Radical Ion Pair States in a Paramagnetic Endohedral Metallofullerene/π-Extended Tetrathiafulvalene Conjugate

Electron donor-acceptor conjugates of paramagnetic endohedral metallofullerenes and π-extended tetrathiafulvalene (exTTF) were synthesized, characterized, and probed with respect to intramolecular electron transfer involving paramagnetic fullerenes. UV-vis-NIR absorption spectroscopy complemented by electrochemical measurements attested to weak electronic interactions between the electron donor, exTTF, and the electron acceptor, La@C(82), in the ground state. In the excited state, photoexcitation powers a fast intramolecular electron transfer to yield an ion and radical ion pair state consisting of one-electron-reduced La@C(82) and of one-electron-oxidized exTTF.