Molecular Cloning, Characterization and Expression Analysis of Two Members of the Pht1 Family of Phosphate Transporters in Glycine max

BACKGROUND: Phosphorus is one of the macronutrients essential for plant growth and development. The acquisition and translocation of phosphate are pivotal processes of plant growth. In a large number of plants, phosphate uptake by roots and translocation within the plant are presumed to occur via a phosphate/proton cotransport mechanism. PRINCIPAL FINDINGS: We cloned two cDNAs from soybean (Glycine max), GmPT1 and GmPT2, which show homology to the phosphate/proton cotransporter PHO84 from the budding yeast Saccharomyces cerevisiae. The amino acid sequence of the products predicted from GmPT1 and GmPT2 share 61% and 63% identity, respectively, with the PHO84 in amino acid sequence. The deduced structure of the encoded proteins revealed 12 membrane-spanning domains with a central hydrophilic region. The molecular mass values are ∼58.7 kDa for GmPT1 and ∼58.6 kDa for GmPT2. Transiently expressed GFP-protein fusions provide direct evidence that the two Pi transporters are located in the plasma membrane. Uptake of radioactive orthophosphate by the yeast mutant MB192 showed that GmPT1 and GmPT2 are dependent on pH and uptake is reduced by the addition of uncouplers of oxidative phosphorylation. The K(m) for phosphate uptake by GmPT1 and GmPT2 is 6.65 mM and 6.63 mM, respectively. A quantitative real time RT-PCR assay indicated that these two genes are expressed in the roots and shoots of seedlings whether they are phosphate-deficient or not. Deficiency of phosphorus caused a slight change of the expression levels of GmPT1 and GmPT2. CONCLUSIONS: The results of our experiments show that the two phosphate transporters have low affinity and the corresponding genes are constitutively expressed. Thereby, the two phosphate transporters can perform translocation of phosphate within the plant

MHO1, an Evolutionarily Conserved Gene, Is Synthetic Lethal with PLC1; Mho1p Has a Role in Invasive Growth

The novel protein Memo (Mediator of ErbB2 driven cell motility) was identified in a screen for ErbB2 interacting proteins and found to have an essential function in cell motility. Memo is evolutionarily conserved with homologs found in all branches of life; the human and yeast proteins have a similarity of >50%. In the present study we used the model organism S. cerevisiae to characterize the Memo-homologue Mho1 (Yjr008wp) and to investigate its function in yeast. In a synthetic lethal screen we found MHO1 as a novel synthetic lethal partner of PLC1, which encodes the single phospholipase C in yeast. Double-deleted cells lacking MHO1 and PLC1, proliferate for up to ten generations. Introduction of human Memo into the memoΔplc1Δ strain rescued the synthetic lethal phenotype suggesting that yeast and human proteins have similar functions. Mho1 is present in the cytoplasm and the nucleus of yeast cells; the same distribution of Memo was found in mammalian cells. None of the Memo homologues have a characteristic nuclear localization sequence, however, a conserved nuclear export sequence is found in all. In mammalian cells, blocking nuclear export with Leptomycin B led to nuclear Memo accumulation, suggesting that it is actively exported from the nucleus. In yeast MHO1 expression is induced by stress conditions. Since invasive growth in S. cerevisiea is also stress-induced, we tested Mho1's role in this response. MHO1 deletion had no effect on invasion induced by nutrient deprivation, however, Mho1 overexpression blocked the invasive ability of yeast cells, suggesting that Mho1 might be acting in a dominant negative manner. Taken together, our results show that MHO1 is a novel synthetic lethal interactor with PLC1, and that both gene products are required for proliferation. Moreover, a role for Memo in cell motility/invasion appears to be conserved across species

Perturbation of the yeast N-acetyltransferase NatB induces elevation of protein phosphorylation levels

<p>Abstract</p> <p>Background</p> <p>The addition of an acetyl group to protein N-termini is a widespread co-translational modification. NatB is one of the main N-acetyltransferases that targets a subset of proteins possessing an N-terminal methionine, but so far only a handful of substrates have been reported. Using a yeast <it>nat3Δ </it>strain, deficient for the catalytic subunit of NatB, we employed a quantitative proteomics strategy to identify NatB substrates and to characterize downstream effects in <it>nat3Δ</it>.</p> <p>Results</p> <p>Comparing by proteomics WT and <it>nat3Δ </it>strains, using metabolic ¹⁵N isotope labeling, we confidently identified 59 NatB substrates, out of a total of 756 detected acetylated protein N-termini. We acquired in-depth proteome wide measurements of expression levels of about 2580 proteins. Most remarkably, NatB deletion led to a very significant change in protein phosphorylation.</p> <p>Conclusions</p> <p>Protein expression levels change only marginally in between WT and <it>nat3Δ</it>. A comparison of the detected NatB substrates with their orthologous revealed remarkably little conservation throughout the phylogenetic tree. We further present evidence of post-translational N-acetylation on protein variants at non-annotated N-termini. Moreover, analysis of downstream effects in <it>nat3Δ </it>revealed elevated protein phosphorylation levels whereby the kinase Snf1p is likely a key element in this process.</p

Pleiotropic Effects of Deubiquitinating Enzyme Ubp5 on Growth and Pathogenesis of Cryptococcus neoformans

Ubiquitination is a reversible protein modification that influences various cellular processes in eukaryotic cells. Deubiquitinating enzymes remove ubiquitin, maintain ubiquitin homeostasis and regulate protein degradation via the ubiquitination pathway. Cryptococcus neoformans is an important basidiomycete pathogen that causes life-threatening meningoencephalitis primarily in the immunocompromised population. In order to understand the possible influence deubiquitinases have on growth and virulence of the model pathogenic yeast Cryptococcus neoformans, we generated deletion mutants of seven putative deubiquitinase genes. Compared to other deubiquitinating enzyme mutants, a ubp5Δ mutant exhibited severely attenuated virulence and many distinct phenotypes, including decreased capsule formation, hypomelanization, defective sporulation, and elevated sensitivity to several external stressors (such as high temperature, oxidative and nitrosative stresses, high salts, and antifungal agents). Ubp5 is likely the major deubiquitinating enzyme for stress responses in C. neoformans, which further delineates the evolutionary divergence of Cryptococcus from the model yeast S. cerevisiae, and provides an important paradigm for understanding the potential role of deubiquitination in virulence by other pathogenic fungi. Other putative deubiquitinase mutants (doa4Δ and ubp13Δ) share some phenotypes with the ubp5Δ mutant, illustrating functional overlap among deubiquitinating enzymes in C. neoformans. Therefore, deubiquitinating enzymes (especially Ubp5) are essential for the virulence composite of C. neoformans and provide an additional yeast survival and propagation advantage in the host

Stress modulation as a means to improve yeasts for lignocellulose bioconversion

The second-generation (2G) fermentation environment for lignocellulose conversion presents unique challenges to the fermentative organism that do not necessarily exist in other industrial fermentations. While extreme osmotic, heat, and nutrient starvation stresses are observed in sugar- and starch-based fermentation environments, additional pre-treatment-derived inhibitor stress, potentially exacerbated by stresses such as pH and product tolerance, exist in the 2G environment. Furthermore, in a consolidated bioprocessing (CBP) context, the organism is also challenged to secrete enzymes that may themselves lead to unfolded protein response and other stresses. This review will discuss responses of the yeast Saccharomyces cerevisiae to 2G-specific stresses and stress modulation strategies that can be followed to improve yeasts for this application. We also explore published –omics data and discuss relevant rational engineering, reverse engineering, and adaptation strategies, with the view of identifying genes or alleles that will make positive contributions to the overall robustness of 2G industrial strains