A genetic approach of wine yeast fermentation capacity in nitrogen-starvation reveals the key role of nitrogen signaling

Background

In conditions of nitrogen limitation, Saccharomyces cerevisiae strains differ in their fermentation capacities, due to differences in their nitrogen requirements. The mechanisms ensuring the maintenance of glycolytic flux in these conditions are unknown. We investigated the genetic basis of these differences, by studying quantitative trait loci (QTL) in a population of 133 individuals from the F2 segregant population generated from a cross between two strains with different nitrogen requirements for efficient fermentation.

Results

By comparing two bulks of segregants with low and high nitrogen requirements, we detected four regions making a quantitative contribution to these traits. We identified four polymorphic genes, in three of these four regions, for which involvement in the phenotype was validated by hemizygote comparison. The functions of the four validated genes, GCN1, MDS3, ARG81 and BIO3, relate to key roles in nitrogen metabolism and signaling, helping to maintain fermentation performance.

Conclusions

This study reveals that differences in nitrogen requirement between yeast strains results from a complex allelic combination. The identification of three genes involved in sensing and signaling nitrogen and specially one from the TOR pathway as affecting nitrogen requirements suggests a role for this pathway in regulating the fermentation rate in starvation through unknown mechanisms linking nitrogen signaling to glycolytic flux.

Electronic supplementary material

The online version of this article (doi: 10.1186/1471-2164-15-495) contains supplementary material, which is available to authorized users.     

Crystal Structure of a Thermophilic GrpE Protein: Insight into Thermosensing Function for the DnaK Chaperone System

A homodimeric GrpE protein functions as a nucleotide exchange factor of the eubacterium DnaK molecular chaperone system. The co-chaperone GrpE accelerates ADP dissociation from, and promotes ATP binding to, DnaK, which cooperatively facilitates the DnaK chaperone cycle with another co-chaperone, DnaJ. GrpE characteristically undergoes two-step conformational changes in response to elevation of the environmental temperature. In the first transition at heat-shock temperatures, a fully reversible and functionally deficient structural alteration takes place in GrpE, and then the higher temperatures lead to the irreversible dissociation of the GrpE dimer into monomers as the second transition. GrpE is also thought to be a thermosensor of the DnaK system, since it is the only member of the DnaK system that changes its structure reversibly and loses its function at heat-shock temperatures of various organisms. We here report the crystal structure of GrpE from Thermus thermophilus HB8 (GrpE_Tth) at 3.23 Å resolution. The resolved structure is compared with that of GrpE from mesophilic Escherichia coli (GrpE_Eco), revealing structural similarities, particularly in the DnaK interaction regions, and structural characteristics for the thermal stability of GrpE_Tth. In addition, the structure analysis raised the possibility that the polypeptide chain in the reported GrpE_Eco structure was misinterpreted. Comparison of these two GrpE structures combined with the results of limited proteolysis experiments provides insight into the protein dynamics of GrpE_Tth correlated with the shift of temperature, and also suggests that the localized and partial unfolding at the plausible DnaK interaction sites of GrpE_Tth causes functional deficiency of nucleotide exchange factor in response to the heat shock.     

Multifaceted roles of miR-1s in repressing the fetal gene program in the heart

miRNAs are an important class of regulators that play roles in cellular homeostasis and disease. Muscle-specific miRNAs, miR-1-1 and miR-1-2, have been found to play important roles in regulating cell proliferation and cardiac function. Redundancy between miR-1-1 and miR-1-2 has previously impeded a full understanding of their roles in vivo. To determine how miR-1s regulate cardiac function in vivo, we generated mice lacking miR-1-1 and miR-1-2 without affecting nearby genes. miR-1 double knockout (miR-1 dKO) mice were viable and not significantly different from wild-type controls at postnatal day 2.5. Thereafter, all miR-1 dKO mice developed dilated cardiomyopathy (DCM) and died before P17. Massively parallel sequencing showed that a large portion of upregulated genes after deletion of miR-1s is associated with the cardiac fetal gene program including cell proliferation, glycolysis, glycogenesis, and fetal sarcomere-associated genes. Consistent with gene profiling, glycogen content and glycolytic rates were significantly increased in miR-1 dKO mice. Estrogen-related Receptor β (Errβ) was identified as a direct target of miR-1, which can regulate glycolysis, glycogenesis, and the expression of sarcomeric proteins. Cardiac-specific overexpression of Errβ led to glycogen storage, cardiac dilation, and sudden cardiac death around 3-4 weeks of age. We conclude that miR-1 and its primary target Errβ act together to regulate the transition from prenatal to neonatal stages by repressing the cardiac fetal gene program. Loss of this regulation leads to a neonatal DCM.     

Insight into the structure of Mesorhizobium loti arylamine N-acetyltransferase 2 (MLNAT2): a biochemical and computational study

The arylamine N-acetyltransferases (NAT; EC 2.3.1.5) are xenobiotic-metabolizing enzymes (XME) that catalyze the transfer of an acetyl group from acetylCoA (Ac-CoA) to arylamine, hydrazines and their N-hydroxylated metabolites. Eukaryotes may have up to three NAT isoforms, but Mesorhizobium loti is the only prokaryote with two functional NAT isoforms (MLNAT1 and MLNAT2). The three-dimensional structure of MLNAT1 has been determined (Holton, S.J., Dairou, J., Sandy, J., Rodrigues-Lima, F., Dupret, J.M., Noble, M.E.M. and Sim, E. (2005) Structure of Mesorhizobium loti arylamine N-acetyltransferase 1. Acta Cryst, F61, 14-16). No MLNAT2 crystals have yet been produced, despite the production of sufficient quantities of pure protein. Using purified recombinant MLNAT1 and MLNAT2, we showed here that MLNAT1 was intrinsically more stable than MLNAT2. To test whether different structural features could explain these differences in intrinsic stability, we constructed a high-quality homology model for MLNAT2 based on far UV-CD data. Despite low levels of sequence identity with other prokaryotic NAT enzymes ( approximately 28% identity), this model suggests that MLNAT2 adopts the characteristic three-domain NAT fold. More importantly, molecular dynamics simulations on the structures of MLNAT1 and MLNAT2 suggested that MLNAT2 was less stable than MLNAT1 due to differences in amino-acid sequence/structure features in the alpha/beta lid domain.     

The Paf1 Complex Subunit Rtf1 Buffers Cells Against the Toxic Effects of [PSI+] and Defects in Rkr1-Dependent Protein Quality Control in Saccharomyces cerevisiae

The Rtf1 subunit of the Paf1 complex is required for specific histone modifications, including histone H2B lysine 123 monoubiquitylation. In Saccharomyces cerevisiae, deletion of RTF1 is lethal in the absence of Rkr1, a ubiquitin-protein ligase involved in the destruction of nonstop proteins, which arise from mRNAs lacking stop codons or translational readthrough into the poly(A) tail. We performed a transposon-based mutagenesis screen to identify suppressors of rtf1Δ rkr1Δ lethality and found that a mutation in the gene encoding the protein chaperone Hsp104 rescued viability. Hsp104 plays a role in prion propagation, including the maintenance of [PSI(+)], which contributes to the synthesis of nonstop proteins. We demonstrate that rtf1Δ and rkr1Δ are synthetically lethal only in the presence of [PSI(+)]. The deletion, inactivation, and overexpression of HSP104 or the overexpression of prion-encoding genes URE2 and LSM4 clear [PSI(+)] and rescue rtf1Δ rkr1Δ lethality. In addition, the presence of [PSI(+)] decreases the fitness of rkr1Δ strains. We investigated whether the loss of RTF1 exacerbates an overload in nonstop proteins in rkr1Δ [PSI(+)] strains but, using reporter plasmids, found that rtf1Δ decreases nonstop protein levels, indicating that excess nonstop proteins may not be the cause of synthetic lethality. Instead, our data suggest that the loss of Rtf1-dependent histone modifications increases the burden on quality control pathways in cells lacking Rkr1 and containing [PSI(+)].     

Adaptive basis of geographic variation: genetic,phenotypic and environmental differences among beach mouse populations

A major goal in evolutionary biology is to understand how and why populations differentiate, both genetically and phenotypically, as they invade a novel habitat. A classical example of adaptation is the pale colour of beach mice, relative to their dark mainland ancestors, which colonized the isolated sandy dunes and barrier islands on Florida''s Gulf Coast. However, much less is known about differentiation among the Gulf Coast beach mice, which comprise five subspecies linearly arrayed on Florida''s shoreline. Here, we test the role of selection in maintaining variation among these beach mouse subspecies at multiple levels—phenotype, genotype and the environments they inhabit. While all beach subspecies have light pelage, they differ significantly in colour pattern. These subspecies are also genetically distinct: pair-wise F_st-values range from 0.23 to 0.63 and levels of gene flow are low. However, we did not find a correlation between phenotypic and genetic distance. Instead, we find a significant association between the average ‘lightness’ of each subspecies and the brightness of the substrate it inhabits: the two most genetically divergent subspecies occupy the most similar habitats and have converged on phenotype, whereas the most genetically similar subspecies occupy the most different environments and have divergent phenotypes. Moreover, allelic variation at the pigmentation gene, Mc1r, is statistically correlated with these colour differences but not with variation at other genetic loci. Together, these results suggest that natural selection for camouflage—via changes in Mc1r allele frequency—contributes to pigment differentiation among beach mouse subspecies.     

Genome-wide screening of alpha-tocopherol sensitive genes in heart tissue from alpha-tocopherol transfer protein null mice (ATTP(-/-))

Alpha-tocopherol transfer protein (ATTP) null mice (ATTP(-/-)) have a systemic deficiency of alpha-tocopherol (AT). The heart AT levels of ATTP(-/-) are <10% of those in ATTP(+/+) mice. The genomic responses of heart to AT deficiency were determined in 3 months old male ATTP(-/-) mice and compared with their ATTP(+/+) littermate controls using Affymetrix 430A 2.0 high density oligonucleotide arrays. Differential analysis of approximately 13000 genes identified repression of genes related to immune system and activation of genes related to lipid metabolism and inflammation with no significant change in the expression of classical antioxidant genes (catalase, superoxide dismutase, glutathione peroxidase) in ATTP(-/-) as compared to ATTP(+/+) mice. The present data identifies novel classes of AT sensitive genes in heart tissue.     

A Conserved Region between the TPR and Activation Domains of p67phox Participates in Activation of the Phagocyte NADPH Oxidase

The phagocyte NADPH oxidase, dormant in resting cells, is activated during phagocytosis to produce superoxide, a precursor of microbicidal oxidants. The membrane-integrated protein gp91^phox serves as the catalytic core, because it contains a complete electron-transporting apparatus from NADPH to molecular oxygen for superoxide production. Activation of gp91^phox requires the cytosolic proteins p67^phox, p47^phox, and Rac (a small GTPase). p67^phox, comprising 526 amino acids, moves upon cell stimulation to the membrane together with p47^phox and there interacts with Rac; these processes are prerequisite for gp91^phox activation. Here we show that a region of p67^phox (amino acids 190–200) C-terminal to the Rac-binding domain is evolutionarily well conserved and participates in oxidase activation at a later stage in conjunction with an activation domain. Alanine substitution for Tyr-198, Leu-199, or Val-204 abrogates the ability of p67^phox to support superoxide production by gp91^phox-based oxidase as well as its related oxidases Nox1 and Nox3; the activation also involves other invariant residues such as Leu-193, Asp-197, and Gly-200. Intriguingly, replacement of Gln-192 by alanine or that of Tyr-198 by phenylalanine or tryptophan rather enhances superoxide production by gp91^phox-based oxidase, suggesting a tuning role for these residues. Furthermore, the Y198A/V204A or L199A/V204A substitution leads to not only a complete loss of the activity of the reconstituted oxidase system but also a significant decrease in p67^phox interaction with the gp91^phox NADPH-binding domain, although these mutations affect neither the protein integrity nor the Rac binding activity. Thus the extended activation domain of p67^phox (amino acids 190–210) containing the D(Y/F)LGK motif plays an essential role in oxidase activation probably by interacting with gp91^phox.     

An extensive allelic series of Drosophila kae1 mutants reveals diverse and tissue-specific requirements for t6A biogenesis

N⁶-threonylcarbamoyl-adenosine (t6A) is one of the few RNA modifications that is universally present in life. This modification occurs at high frequency at position 37 of most tRNAs that decode ANN codons, and stabilizes cognate anticodon–codon interactions. Nearly all genetic studies of the t6A pathway have focused on single-celled organisms. In this study, we report the isolation of an extensive allelic series in the Drosophila ortholog of the core t6A biosynthesis factor Kae1. kae1 hemizygous larvae exhibit decreases in t6A that correlate with allele strength; however, we still detect substantial t6A-modified tRNAs even during the extended larval phase of null alleles. Nevertheless, complementation of Drosophila Kae1 and other t6A factors in corresponding yeast null mutants demonstrates that these metazoan genes execute t6A synthesis. Turning to the biological consequences of t6A loss, we characterize prominent kae1 melanotic masses and show that they are associated with lymph gland overgrowth and ectopic generation of lamellocytes. On the other hand, kae1 mutants exhibit other phenotypes that reflect insufficient tissue growth. Interestingly, whole-tissue and clonal analyses show that strongly mitotic tissues such as imaginal discs are exquisitely sensitive to loss of kae1, whereas nonproliferating tissues are less affected. Indeed, despite overt requirements of t6A for growth of many tissues, certain strong kae1 alleles achieve and sustain enlarged body size during their extended larval phase. Our studies highlight tissue-specific requirements of the t6A pathway in a metazoan context and provide insights into the diverse biological roles of this fundamental RNA modification during animal development and disease.     

Insight into the molecular switch mechanism of human Rab5a from molecular dynamics simulations

Rab5a is currently a most interesting target because it is responsible for regulating the early endosome fusion in endocytosis and possibly the budding process. We utilized longtime-scale molecular dynamics simulations to investigate the internal motion of the wild-type Rab5a and its A30P mutant. It was observed that, after binding with GTP, the global flexibility of the two proteins is increasing, while the local flexibility in their sensitive sites (P-loop, switch I and II regions) is decreasing. Also, the mutation of Ala30 to Pro30 can cause notable flexibility variations in the sensitive sites. However, this kind of variations is dramatically reduced after binding with GTP. Such a remarkable feature is mainly caused by the water network rearrangements in the sensitive sites. These findings might be of use for revealing the profound mechanism of the displacements of Rab5a switch regions, as well as the mechanism of the GDP dissociation and GTP association.     

Binding of LL-37 to model biomembranes: Insight into target vs host cell recognition

Pursuing the molecular mechanisms of the concentration dependent cytotoxic and hemolytic effects of the human antimicrobial peptide LL-37 on cells, we investigated the interactions of this peptide with lipids using different model membranes, together with fluorescence spectroscopy for the Trp-containing mutant LL-37(F27W). Minimum concentrations inhibiting bacterial growth and lipid interactions assessed by dynamic light scattering and monolayer penetration revealed the mutant to retain the characteristics of native LL-37. Although both LL-37 and the mutant intercalated effectively into zwitterionic phosphatidylcholine membranes the presence of acidic phospholipids caused augmented membrane binding. Interestingly, strongly attenuated intercalation of LL-37 into membranes containing both cholesterol and sphingomyelin (both at X = 0.3) was observed. Accordingly, the distinction between target and host cells by LL-37 is likely to derive from i) acidic phospholipids causing enhanced association with the former cells as well as ii) from attenuated interactions with the outer surface of the plasma membrane of the peptide secreting host, imposed by its high content of cholesterol and sphingomyelin. Our results further suggest that LL-37 may exert its antimicrobial effects by compromising the membrane barrier properties of the target microbes by a mechanism involving cytotoxic oligomers, similarly to other peptides forming amyloid-like fibers in the presence of acidic phospholipids.     

Insight into the location and dynamics of the annexin A2 N-terminal domain during Ca-induced membrane bridging

Annexin A2 (AnxA2) is a Ca²⁺- and phospholipid-binding protein involved in many cellular regulatory processes. Like other annexins, it is constituted by two domains: a conserved core, containing the Ca²⁺ binding sites, and a variable N-terminal segment, containing sites for interactions with other protein partners like S100A10 (p11). A wealth of data exists on the structure and dynamics of the core, but little is known about the N-terminal domain especially in the Ca²⁺-induced membrane-bridging process. To investigate this protein region in the monomeric AnxA2 and in the heterotetramer (AnxA2-p11)₂, the reactive Cys8 residue was specifically labelled with the fluorescent probe acrylodan and the interactions with membranes were studied by steady-state and time-resolved fluorescence. In membrane junctions formed by the (AnxA2-p11)₂ heterotetramer, the flexibility of the N-terminal domain increased as compared to the protein in solution. In “homotypic” membrane junctions formed by monomeric AnxA2, acrylodan moved to a more hydrophobic environment than in the protein in solution and the flexibility of the N-terminal domain also increased. In these junctions, this domain is probably not in close contact with the membrane surface, as suggested by the weak quenching of acrylodan observed with doxyl-PCs, but pairs of N-termini likely interact, as revealed by the excimer-forming probe pyrene-maleimide bound to Cys8. We present a model of monomeric AnxA2 N-terminal domain organization in “homotypic” bridged membranes in the presence of Ca²⁺.     

NMR investigation of DNA primer-template models: structural insights into dislocation mutagenesis in DNA replication

Slipped frameshift intermediates can occur when DNA polymerase slows or stalls at sites of DNA lesions. However, this phenomenon is much less common when unmodified DNA is replicated. In order to study the effect of templating bases on the alignment of primer-templates, NMR structural investigation has been performed on primer-template oligonucleotide models which mimic the situation that dNTP has just been incorporated opposite template. NMR evidence reveals the occurrence of misalignment when dGTP is incorporated opposite template T with a downstream nucleotide C. Depending on the template sequence, further extension of the primer can lead to realignment.     

Solid-state H and N NMR studies of side-chain and backbone dynamics of phospholamban in lipid bilayers: Investigation of the N27A mutation

Phospholamban (PLB) is an integral membrane protein regulating Ca²⁺ transport through inhibitory interaction with sarco(endo)plasmic reticulum calcium ATPase (SERCA). The Asn27 to Ala (N27A) mutation of PLB has been shown to function as a superinhibitor of the affinity of SERCA for Ca²⁺ and of cardiac contractility in vivo. The effects of this N27A mutation on the side-chain and backbone dynamics of PLB were investigated with ²H and ¹⁵N solid-state NMR spectroscopy in phospholipid multilamellar vesicles (MLVs). ²H and ¹⁵N NMR spectra indicate that the N27A mutation does not significantly change the side-chain or backbone dynamics of the transmembrane and cytoplasmic domains when compared to wild-type PLB. However, dynamic changes are observed for the hinge region, in which greater mobility is observed for the CD₃-labeled Ala24 N27A-PLB. The increased dynamics in the hinge region of PLB upon N27A mutation may allow the cytoplasmic helix to more easily interact with the Ca²⁺-ATPase; thus, showing increased inhibition of Ca²⁺-ATPase.     

Phytochromes from Agrobacterium tumefaciens: difference spectroscopy with extracts of wild type and knockout mutants

Phytochromes are photoreceptors that occur in plants, fungi and bacteria, among others in the phytopathogen Agrobacterium tumefaciens. We constructed single and double knockout mutants of the two A. tumefaciens phytochromes Agp1 and Agp2. In liquid culture, the double mutant revealed a reduced growth rate, whereas the growth rates of the single mutants did not differ significantly from that of the wild type. Using these mutants, we analyzed the spectral properties of native A. tumefaciens phytochromes. A wild-type A. tumefaciens cell contains about 10 molecules of Agp1 and about 19 molecules of Agp2. Dark conversion of native Agp1 and Agp2 proceeds from Pfr to Pr and from Pr to Pfr, respectively, as has already been reported for the recombinant proteins. The spectral properties of recombinant and native Agp2 were significantly different. Mixing experiments with extracts from the double mutant and recombinant Agp2 imply that the spectral properties of Agp2 are modulated by components of the extract.     

Fluorescence depolarization studies of filamentous actin analyzed with a genetic algorithm

A new method, in which a genetic algorithm was combined with Brownian dynamics and Monte Carlo simulations, was developed to analyze fluorescence depolarization data collected by the time-correlated single photon-counting technique. It was applied to studies of BODIPY-labeled filamentous actin (F-actin). The technique registered the local order and reorienting motions of the fluorophores, which were covalently coupled to cysteine 374 (C374) in actin and interacted by electronic energy migration within the actin polymers. Analyses of F-actin samples composed of different fractions of labeled actin molecules revealed the known helical organization of F-actin, demonstrating the usefulness of this technique for structure determination of complex protein polymers. The distance from the filament axis to the fluorophore was found to be considerably less than expected from the proposed position of C374 at a high filament radius. In addition, polymerization experiments with BODIPY-actin suggest a 25-fold more efficient signal for filament formation than pyrene-actin.     

Development of a set of plasmid vectors for genetic manipulations of the pathogenic yeast Candida parapsilosis

A system for genetic transformation of the yeast Candida parapsilosis, recently developed in our laboratory, opened a venue for investigation of this pathogenic species at the molecular level. In this study we extend the range of available experimental tools by construction of a genomic DNA library suitable for screening and isolation of genes by functional complementation of yeast mutants and a set of replicative plasmid vectors for genetic manipulation of C. parapsilosis cells. The plasmids are based on auxotrophic (CpGAL1, CpURA3, CpMET2, CpLYS4) and dominant (CaIMH3) selection markers. In addition, we constructed plasmid derivatives containing reporter genes yEGFP3 and KlLAC4 coding for enhanced version of the green fluorescent protein and Kluyveromyces lactis beta-galactosidase, respectively. The vectors facilitate propagation and expression of cloned genes in C. parapsilosis cells and allow intracellular localization of gene products and/or monitoring the activity of promoter sequences.     

Synthesis and evaluation of the platelet antiaggregant properties of phenolic antioxidants structurally related to rosmarinic acid

Polyphenols, such as rosmarinic acid, are widely distributed natural products with relevant antioxidant activity. Oxidative stress plays an important role in the pathogenesis of a number of disorders. Here, we report on the synthesis and biological effects of the polyphenolic esters hydroxytyrosyl gallate (1), hydroxytyrosyl protocatechuate (2) and hydroxytyrosyl caffeate (3), structurally related to rosmarinic acid. The three compounds showed a greater free radical scavenging activity than their precursors and also than rosmarinic acid. Esters 1 and 3 significantly reduced thrombin-evoked platelet aggregation, which is likely mediated to the attenuation of thrombin-stimulated Ca²⁺ release and entry. The three compounds reduced the ability of platelets to accumulate Ca²⁺ in the intracellular stores, probably by enhancing the Ca²⁺ leakage rate and reduced store-operated Ca²⁺ entry in these cells. These observations suggest that the structurally-simplified analogs to rosmarinic acid, compounds 1 and 3, might be the base of therapeutic strategies to prevent thrombotic complications associated to platelet hyperaggregability due to oxidative stress.