The SPX domain of the yeast low-affinity phosphate transporter Pho90 regulates transport activity

Yeast has two phosphate-uptake systems that complement each other: the high-affinity transporters (Pho84 and Pho89) are active under phosphate starvation, whereas Pho87 and Pho90 are low-affinity transporters that function when phosphate is abundant. Here, we report new regulatory functions of the amino-terminal SPX domain of Pho87 and Pho90. By studying truncated versions of Pho87 and Pho90, we show that the SPX domain limits the phosphate-uptake velocity, suppresses phosphate efflux and affects the regulation of the phosphate signal transduction pathway. Furthermore, split-ubiquitin assays and co-immunoprecipitation suggest that the SPX domain of both Pho90 and Pho87 interacts physically with the regulatory protein Spl2. This work suggests that the SPX domain inhibits low-affinity phosphate transport through a physical interaction with Spl2

Effects of sulfate starvation on agar polysaccharides of Gracilaria species (Gracilariaceae, Rhodophyta) from Morib, Malaysia

The effects of sulfate starvation on the agar characteristics of Gracilaria species was investigated by culturing two red algae from Morib, Malaysia, Gracilaria changii and Gracilaria salicornia in sulfate-free artificial seawater for 5 days. The seaweed samples were collected in October 2012 and March 2013, periods which have significant variation in the amount of rainfall. The agar yields were shown to be independent of sulfate availability, with only 0.60–1.20 % increment in treated G. changii and 0.31–1.40 % increment in treated G. salicornia while their gel strengths did not increase significantly (approximately 5–7 %) after sulfate starvation for both species. The gelling and melting temperatures did not vary between control and treated samples from both species, except for the treated G. changii collected in March 2013. The gel syneresis index of G. salicornia collected in March 2013 increased significantly after sulfate deprivation. Sulfate starvation introduced some variations in the content of 3, 6-anhydrogalactose and total sulfate esters, but the changes did not have a pronounced effect on the physical properties of agar

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

Lactate dehydrogenase-5 (LDH-5) overexpression in non-small-cell lung cancer tissues is linked to tumour hypoxia, angiogenic factor production and poor prognosis

Lactate dehydrogenase-5 (LDH-5) catalyses the reversible transformation of pyruvate to lactate, having a principal position in the anaerobic cellular metabolism. Induction of LDH-5 occurs during hypoxia and LDH-5 transcription is directly regulated by the hypoxia-inducible factor 1 (HIF1). Serum LDH levels have been correlated with poor prognosis and resistance to chemotherapy and radiotherapy in various neoplastic diseases. The expression, however, of LDH in tumours has never been investigated in the past. In the present study, we established an immunohistochemical method to evaluate the LDH-5 overexpression in tumours, using two novel antibodies raised against the rat muscle LDH-5 and the human LDH-5 (Abcam, UK). The subcellular patterns of expression in cancer cells were mixed nuclear and cytoplasmic. In direct contrast to cancer cells, stromal fibroblasts were reactive for LDH-5 only in a minority of cases. Serum LDH, although positively correlated with, does not reliably reflect the intratumoral LDH-5 status. Lactate dehydrogenase-5 overexpression was directly related to HIF1alpha and 2alpha, but not with the carbonic anhydrase 9 expression. Patients with tumours bearing high LDH-5 expression had a poor prognosis. Tumours with simultaneous LDH-5 and HIF1alpha (or HIF2alpha) overexpression, indicative of a functional HIF pathway, had a particularly aggressive behaviour. It is concluded that overexpression of LDH-5 is a common event in non-small-cell lung cancer, can be easily assessed in paraffin-embedded material and provides important prognostic information, particularly when combined with other endogenous markers of hypoxia and acidity

The S phase checkpoint promotes the Smc5/6 complex dependent SUMOylation of Pol2, the catalytic subunit of DNA polymerase ε

Replication fork stalling and accumulation of single-stranded DNA trigger the S phase checkpoint, a signalling cascade that, in budding yeast, leads to the activation of the Rad53 kinase. Rad53 is essential in maintaining cell viability, but its targets of regulation are still partially unknown. Here we show that Rad53 drives the hyper-SUMOylation of Pol2, the catalytic subunit of DNA polymerase ε, principally following replication forks stalling induced by nucleotide depletion. Pol2 is the main target of SUMOylation within the replisome and its modification requires the SUMO-ligase Mms21, a subunit of the Smc5/6 complex. Moreover, the Smc5/6 complex co-purifies with Pol ε, independently of other replisome components. Finally, we map Pol2 SUMOylation to a single site within the N-terminal catalytic domain and identify a SUMO-interacting motif at the C-terminus of Pol2. These data suggest that the S phase checkpoint regulate Pol ε during replication stress through Pol2 SUMOylation and SUMO-binding abilit

Analytical approaches to photobiological hydrogen production in unicellular green algae

Several species of unicellular green algae, such as the model green microalga Chlamydomonas reinhardtii, can operate under either aerobic photosynthesis or anaerobic metabolism conditions. A particularly interesting metabolic condition is that of “anaerobic oxygenic photosynthesis”, whereby photosynthetically generated oxygen is consumed by the cell’s own respiration, causing anaerobiosis in the culture in the light, and induction of the cellular “hydrogen metabolism” process. The latter entails an alternative photosynthetic electron transport pathway, through the oxygen-sensitive FeFe-hydrogenase, leading to the light-dependent generation of molecular hydrogen in the chloroplast. The FeFe-hydrogenase is coupled to the reducing site of photosystem-I via ferredoxin and is employed as an electron-pressure valve, through which electrons are dissipated, thus permitting a sustained electron transport in the thylakoid membrane of photosynthesis. This hydrogen gas generating process in the cells offers testimony to the unique photosynthetic metabolism that can be found in many species of green microalgae. Moreover, it has attracted interest by the biotechnology and bioenergy sectors, as it promises utilization of green microalgae and the process of photosynthesis in renewable energy production. This article provides an overview of the principles of photobiological hydrogen production in microalgae and addresses in detail the process of induction and analysis of the hydrogen metabolism in the cells. Furthermore, methods are discussed by which the interaction of photosynthesis, respiration, cellular metabolism, and H(2) production in Chlamydomonas can be monitored and regulated

Ion homeostasis in the Chloroplast

peer reviewedThe chloroplast is an organelle of high demand for macro- and micro-nutrient ions, which are required for the maintenance of the photosynthetic process. To avoid deficiency while preventing excess, homeostasis mechanisms must be tightly regulated. Here, we describe the needs for nutrient ions in the chloroplast and briefly highlight their functions in the chloroplastidial metabolism. We further discuss the impact of nutrient deficiency on chloroplasts and the acclimation mechanisms that evolved to preserve the photosynthetic apparatus. We finally present what is known about import and export mechanisms for these ions. Whenever possible, a comparison between cyanobacteria, algae and plants is provided to add an evolutionary perspective to the description of ion homeostasis mechanisms in photosynthesis