Comparative Analysis of PvPAP Gene Family and Their Functions in Response to Phosphorus Deficiency in Common Bean

BACKGROUND: Purple acid phosphatases (PAPs) play a vital role in adaptive strategies of plants to phosphorus (P) deficiency. However, their functions in relation to P efficiency are fragmentary in common bean. PRINCIPAL FINDINGS: Five PvPAPs were isolated and sequenced in common bean. Phylogenetic analysis showed that PvPAPs could be classified into two groups, including a small group with low molecular mass, and a large group with high molecular mass. Among them, PvPAP3, PvPAP4 and PvPAP5 belong to the small group, while the other two belong to the large group. Transient expression of 35S:PvPAPs-GFP on onion epidermal cells verified the variations of subcellular localization among PvPAPs, suggesting functional diversities of PvPAPs in common bean. Quantitative PCR results showed that most PvPAPs were up-regulated by phosphate (Pi) starvation. Among them, the expression of the small group PvPAPs responded more to Pi starvation, especially in the roots of G19833, the P-efficient genotype. However, only overexpressing PvPAP1 and PvPAP3 could result in significantly increased utilization of extracellular dNTPs in the transgenic bean hairy roots. Furthermore, overexpressing PvPAP3 in Arabidopsis enhanced both plant growth and total P content when dNTPs were supplied as the sole external P source. CONCLUSIONS: The results suggest that PvPAPs in bean varied in protein structure, response to P deficiency and subcellular localization. Among them, both PvPAP1 and PvPAP3 might function as utilization of extracellular dNTPs

Identification of a Phytase Gene in Barley (Hordeum vulgare L.)

Background: Endogenous phytase plays a crucial role in phytate degradation and is thus closely related to nutrient efficiency in barley products. The understanding of genetic information of phytase in barley can provide a useful tool for breeding new barley varieties with high phytase activity. Methodology/Principal Findings: Quantitative trait loci (QTL) analysis for phytase activity was conducted using a doubled haploid population. Phytase protein was purified and identified by the LC-ESI MS/MS Shotgun method. Purple acid phosphatase (PAP) gene was sequenced and the position was compared with the QTL controlling phytase activity. A major QTL for phytase activity was mapped to chromosome 5 H in barley. The gene controlling phytase activity in the region was named as mqPhy. The gene HvPAP a was mapped to the same position as mqPhy, supporting the colinearity between HvPAP a and mqPhy. Conclusions/Significance: It is the first report on QTLs for phytase activity and the results showed that HvPAP a, which shares a same position with the QTL, is a major phytase gene in barley grains

Direct Ubiquitin Independent Recognition and Degradation of a Folded Protein by the Eukaryotic Proteasomes-Origin of Intrinsic Degradation Signals

Eukaryotic 26S proteasomes are structurally organized to recognize, unfold and degrade globular proteins. However, all existing model substrates of the 26S proteasome in addition to ubiquitin or adaptor proteins require unstructured regions in the form of fusion tags for efficient degradation. We report for the first time that purified 26S proteasome can directly recognize and degrade apomyoglobin, a globular protein, in the absence of ubiquitin, extrinsic degradation tags or adaptor proteins. Despite a high affinity interaction, absence of a ligand and presence of only helices/loops that follow the degradation signal, apomyoglobin is degraded slowly by the proteasome. A short floppy F-helix exposed upon ligand removal and in conformational equilibrium with a disordered structure is mandatory for recognition and initiation of degradation. Holomyoglobin, in which the helix is buried, is neither recognized nor degraded. Exposure of the floppy F-helix seems to sensitize the proteasome and primes the substrate for degradation. Using peptide panning and competition experiments we speculate that initial encounters through the floppy helix and additional strong interactions with N-terminal helices anchors apomyoglobin to the proteasome. Stabilizing helical structure in the floppy F-helix slows down degradation. Destabilization of adjacent helices accelerates degradation. Unfolding seems to follow the mechanism of helix unraveling rather than global unfolding. Our findings while confirming the requirement for unstructured regions in degradation offers the following new insights: a) origin and identification of an intrinsic degradation signal in the substrate, b) identification of sequences in the native substrate that are likely to be responsible for direct interactions with the proteasome, and c) identification of critical rate limiting steps like exposure of the intrinsic degron and destabilization of an unfolding intermediate that are presumably catalyzed by the ATPases. Apomyoglobin emerges as a new model substrate to further explore the role of ATPases and protein structure in proteasomal degradatio

Genetic and transcriptomic analysis of transcription factor genes in the model halophilic Archaeon: coordinate action of TbpD and TfbA

<p>Abstract</p> <p>Background</p> <p>Archaea are prokaryotic organisms with simplified versions of eukaryotic transcription systems. Genes coding for the general transcription factors TBP and TFB are present in multiple copies in several Archaea, including <it>Halobacterium </it>sp. NRC-1. Multiple TBP and TFBs have been proposed to participate in transcription of genes via recognition and recruitment of RNA polymerase to different classes of promoters.</p> <p>Results</p> <p>We attempted to knock out all six TBP and seven TFB genes in <it>Halobacterium </it>sp. NRC-1 using the <it>ura</it>3-based gene deletion system. Knockouts were obtained for six out of thirteen genes, <it>tbp</it>CDF and <it>tfb</it>ACG, indicating that they are not essential for cell viability under standard conditions. Screening of a population of 1,000 candidate mutants showed that genes which did not yield mutants contained less that 0.1% knockouts, strongly suggesting that they are essential. The transcriptomes of two mutants, Δ<it>tbp</it>D and Δ<it>tfb</it>A, were compared to the parental strain and showed coordinate down regulation of many genes. Over 500 out of 2,677 total genes were regulated in the Δ<it>tbp</it>D and Δ<it>tfb</it>A mutants with 363 regulated in both, indicating that over 10% of genes in both strains require the action of both TbpD and TfbA for normal transcription. Culturing studies on the Δ<it>tbp</it>D and Δ<it>tfb</it>A mutant strains showed them to grow more slowly than the wild-type at an elevated temperature, 49°C, and they showed reduced viability at 56°C, suggesting TbpD and TfbA are involved in the heat shock response. Alignment of TBP and TFB protein sequences suggested the expansion of the TBP gene family, especially in <it>Halobacterium </it>sp. NRC-1, and TFB gene family in representatives of five different genera of haloarchaea in which genome sequences are available.</p> <p>Conclusion</p> <p>Six of thirteen TBP and TFB genes of <it>Halobacterium </it>sp. NRC-1 are non-essential under standard growth conditions. TbpD and TfbA coordinate the expression of over 10% of the genes in the NRC-1 genome. The Δ<it>tbp</it>D and Δ<it>tfb</it>A mutant strains are temperature sensitive, possibly as a result of down regulation of heat shock genes. Sequence alignments suggest the existence of several families of TBP and TFB transcription factors in <it>Halobacterium </it>which may function in transcription of different classes of genes.</p

Modulation of Aβ(42 )low-n oligomerization using a novel yeast reporter system

BACKGROUND: While traditional models of Alzheimer's disease focused on large fibrillar deposits of the Aβ(42 )amyloid peptide in the brain, recent work suggests that the major pathogenic effects may be attributed to SDS-stable oligomers of Aβ(42). These Aβ(42 )oligomers represent a rational target for therapeutic intervention, yet factors governing their assembly are poorly understood. RESULTS: We describe a new yeast model system focused on the initial stages of Aβ(42 )oligomerization. We show that the activity of a fusion of Aβ(42 )to a reporter protein is compromised in yeast by the formation of SDS-stable low-n oligomers. These oligomers are reminiscent of the low-n oligomers formed by the Aβ(42 )peptide in vitro, in mammalian cell culture, and in the human brain. Point mutations previously shown to inhibit Aβ(42 )aggregation in vitro, were made in the Aβ(42 )portion of the fusion protein. These mutations both inhibited oligomerization and restored activity to the fusion protein. Using this model system, we found that oligomerization of the fusion protein is stimulated by millimolar concentrations of the yeast prion curing agent guanidine. Surprisingly, deletion of the chaperone Hsp104 (a known target for guanidine) inhibited oligomerization of the fusion protein. Furthermore, we demonstrate that Hsp104 interacts with the Aβ(42)-fusion protein and appears to protect it from disaggregation and degradation. CONCLUSION: Previous models of Alzheimer's disease focused on unravelling compounds that inhibit fibrillization of Aβ(42), i.e. the last step of Aβ(42 )assembly. However, inhibition of fibrillization may lead to the accumulation of toxic oligomers of Aβ(42). The model described here can be used to search for and test proteinacious or chemical compounds for their ability to interfere with the initial steps of Aβ(42 )oligomerization. Our findings suggest that yeast contain guanidine-sensitive factor(s) that reduce the amount of low-n oligomers of Aβ(42). As many yeast proteins have human homologs, identification of these factors may help to uncover homologous proteins that affect Aβ(42 )oligomerization in mammals

The Schizosaccharomyces pombe Hsp104 Disaggregase Is Unable to Propagate the [PSI+] Prion

The molecular chaperone Hsp104 is a crucial factor in the acquisition of thermotolerance in yeast. Under stress conditions, the disaggregase activity of Hsp104 facilitates the reactivation of misfolded proteins. Hsp104 is also involved in the propagation of fungal prions. For instance, the well-characterized [PSI+] prion of Saccharomyces cerevisiae does not propagate in Δhsp104 cells or in cells overexpressing Hsp104. In this study, we characterized the functional homolog of Hsp104 from Schizosaccharomyces pombe (Sp_Hsp104). As its S. cerevisiae counterpart, Sp_hsp104+ is heat-inducible and required for thermotolerance in S. pombe. Sp_Hsp104 displays low disaggregase activity and cannot propagate the [PSI+] prion in S. cerevisiae. When overexpressed in S. cerevisiae, Sp_Hsp104 confers thermotolerance to Δhsp104 cells and reactivates heat-aggregated proteins. However, overexpression of Sp_Hsp104 does not propagate nor eliminate [PSI+]. Strikingly, [PSI+] was cured by overexpression of a chimeric chaperone bearing the C-terminal domain (CTD) of the S. cerevisiae Hsp104 protein. Our study demonstrates that the ability to untangle aggregated proteins is conserved between the S. pombe and S. cerevisiae Hsp104 homologs, and points to a role of the CTD in the propagation of the S. cerevisiae [PSI+] prion

The essential iron-sulfur protein Rli1 is an important target accounting for inhibition of cell growth by reactive oxygen species

Oxidative stress mediated by reactive oxygen species (ROS) is linked to degenerative conditions in humans and damage to an array of cellular components. However, it is unclear which molecular target(s) may be the primary “Achilles’ heel” of organisms, accounting for the inhibitory action of ROS. Rli1p (ABCE1) is an essential and highly conserved protein of eukaryotes and archaea that requires notoriously ROS-labile cofactors (Fe-S clusters) for its functions in protein synthesis. In this study, we tested the hypothesis that ROS toxicity is caused by Rli1p dysfunction. In addition to being essential, Rli1p activity (in nuclear ribosomal-subunit export) was shown to be impaired by mild oxidative stress in yeast. Furthermore, prooxidant resistance was decreased by RLI1 repression and increased by RLI1 overexpression. This Rlip1 dependency was abolished during anaerobicity and accentuated in cells expressing a FeS cluster–defective Rli1p construct. The protein’s FeS clusters appeared ROS labile during in vitro incubations, but less so in vivo. Instead, it was primarily 55FeS-cluster supply to Rli1p that was defective in prooxidant-exposed cells. The data indicate that, owing to its essential nature but dependency on ROS-labile FeS clusters, Rli1p function is a primary target of ROS action. Such insight could help inform new approaches for combating oxidative stress–related disease

Bridging high-throughput genetic and transcriptional data reveals cellular responses to alpha-synuclein toxicity

Cells respond to stimuli by changes in various processes, including signaling pathways and gene expression. Efforts to identify components of these responses increasingly depend on mRNA profiling and genetic library screens, yet the functional roles of the genes identified by these assays often remain enigmatic. By comparing the results of these two assays across various cellular responses, we found that they are consistently distinct. Moreover, genetic screens tend to identify response regulators, while mRNA profiling frequently detects metabolic responses. We developed an integrative approach that bridges the gap between these data using known molecular interactions, thus highlighting major response pathways. We harnessed this approach to reveal cellular pathways related to alpha-synuclein, a small lipid-binding protein implicated in several neurodegenerative disorders including Parkinson disease. For this we screened an established yeast model for alphasynuclein toxicity to identify genes that when overexpressed alter cellular survival. Application of our algorithm to these data and data from mRNA profiling provided functional explanations for many of these genes and revealed novel relations between alpha-synuclein toxicity and basic cellular pathways.MGH/MIT Morris Udall Center of Excellence in PD Researc