Functional reconstitution and characterization of the Arabidopsis Mg2+ transporter AtMRS2-10 in proteoliposomes

Magnesium (Mg²⁺) plays critical role in many physiological processes. The mechanism of Mg²⁺ transport has been well documented in bacteria; however, less is known about Mg²⁺ transporters in eukaryotes. The AtMRS2 family, which consists of 10 Arabidopsis genes, belongs to a eukaryotic subset of the CorA superfamily proteins. Proteins in this superfamily have been identified by a universally conserved GlyMetAsn motif and have been characterized as Mg²⁺ transporters. Some members of the AtMRS2 family, including AtMRS2-10, may complement bacterial mutants or yeast mutants that lack Mg²⁺ transport capabilities. Here, we report the purification and functional reconstitution of AtMRS2-10 into liposomes. AtMRS2-10, which contains an N-terminal His-tag, was expressed in Escherichia coli and solubilized with sarcosyl. The purified AtMRS2-10 protein was reconstituted into liposomes. AtMRS2-10 was inserted into liposomes in a unidirectional orientation. Direct measurement of Mg²⁺ uptake into proteoliposomes revealed that reconstituted AtMRS2-10 transported Mg²⁺ without any accessory proteins. Mutation in the GMN motif, M400 to I, inactivated Mg²⁺ uptake. The AtMRS2-10-mediated Mg²⁺ influx was blocked by Co(III)hexamine, and was independent of the external pH from 5 to 9. The activity of AtMRS2-10 was inhibited by Co²⁺ and Ni²⁺; however, it was not inhibited by Ca²⁺, Fe²⁺, or Fe³⁺. While these results indicate that AtMRS2-10 has similar properties to the bacterial CorA proteins, unlike bacterial CorA proteins, AtMRS2-10 was potently inhibited by Al³⁺. These studies demonstrate the functional capability of the AtMRS2 proteins in proteoliposomes to study structure–function relationships.     

Large-scale development of expressed sequence tag-derived simple sequence repeat markers and diversity analysis in Arachis spp.

Large-scale development of expressed sequence tag simple sequence repeat (EST-SSR) markers was performed in peanut (Arachis hypogaea L.) to obtain more informative genetic markers. A total of 10,102 potential non-redundant EST sequences, including 3,445 contigs and 6,657 singletons, were generated from cDNA libraries of the gynophore, roots, leaves and seedlings. A total of 3,187 primer pairs were designed on flanking regions of SSRs, some of which allowed one and two base mismatches. Among the 3,187 markers generated, 2,540 (80%) were trinucleotide repeats, 302 (9%) were dinucleotide repeats, and 345 (11%) were tetranucleotide repeats. Pre-polymorphic analyses of 24 Arachis accessions were performed using 10% polyacrylamide gels. A total of 1,571 EST-SSR markers showing clear polymorphisms were selected for further polymorphic analysis with a Fluoro-fragment Analyzer. The 16 Arachis accessions examined included cultivated peanut varieties as well as diploid species with the A or B genome. Altogether 1,281 (81.5%) of the 1,571 markers were polymorphic among the 16 accessions, and 366 (23.3%) were polymorphic among the 12 cultivated varieties. Diversity analysis was performed and the genotypes of all 16 Arachis accessions showed similarity coefficients ranging from 0.37 to 0.97. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1007/s11032-011-9604-8) contains supplementary material, which is available to authorized users.     

Autophagy-dependent senescence in response to DNA damage and chronic apoptotic stress

Autophagy regulates cell survival and cell death upon various cellular stresses, yet the molecular signaling events involved are not well defined. Here, we established the function of a proteolytic Cyclin E fragment (p18-CycE) in DNA damage-induced autophagy, apoptosis, and senescence. p18-CycE was identified in hematopoietic cells undergoing DNA damage-induced apoptosis. In epithelial cells exposed to DNA damage, chronic but not transient expression of p18-CycE leads to higher turnover of LC3 I/II and increased emergence of autophagosomes and autolysosomes. Levels of p18-CycE, which was generated by proteolytic cleavage of endogenous Cyclin E, were greatly increased by chloroquine and correlated with LC 3II conversion. Preventing p18-CycE genesis blocked conversion of LC3 I to LC3 II. Upon DNA damage, cytoplasmic ataxia-telangiectasia-mutated (ATM) was phosphorylated in p18-CycE-expressing cells resulting in sustained activation of the adenosine-mono-phosphate-dependent kinase (AMPK). These lead to sustained activation of mammalian autophagy-initiating kinase ULK1, which was abrogated upon inhibiting ATM and AMPK phosphorylation. Moreover, p18-CycE was degraded via autophagy followed by induction of senescence. Both autophagy and senescence were prevented by inhibiting autophagy, which leads to increased apoptosis in p18-CycE-expressing cells by stabilizing p18-CycE expression. Senescence was further associated with cytoplasmic co-localization and degradation of p18-CycE and Ku70. In brief, chronic p18-CycE expression-induced autophagy leads to clearance of p18-CycE following DNA damage and induction of senescence. Autophagy inhibition stabilized the cytoplasmic p18-CycE-Ku70 complex leading to apoptosis. Thus, our findings define how chronic apoptotic stress and DNA damage initiate autophagy and regulate cell survival through senescence and/or apoptosis.     

Core 2 GlcNAc modification and megalin ligand-binding activity

Megalin, a receptor-like transporter glycoprotein, is expressed on kidney proximal tubular cells and reabsorbs small-molecular-weight proteins from the glomerular filtrate. Here, we report that mouse megalins differently modified with core 2 beta6GlcNAc transferase had different kinetic properties to a fluorescence-labeled ligand, retinol-binding protein (RBP). BALB/c mice, a wild-type strain in terms of the expression of kidney-specific core 2 beta6GlcNAc transferase, express megalin carrying the core 2 extended Le(x) epitope, while DBA/2 mice, a mutant-strain of the core 2 beta6GlcNAc transferase, express megalin lacking the epitope. We purified these two types of megalin using lentil lectin chromatography and measured the ligand-binding activities of the megalins using Cy5-labeled RBP by applying gel permeation chromatography (GPC) and fluorescence correlation spectroscopy (FCS). The analysis by GPC indicated that the apparent V(max) of the interaction between Cy5-labeled RBP and the megalins of BALB/c and DBA/2 mice was 60 microM and 30 microM, respectively, and the apparent K(m) was 11 microM and 17 microM, respectively. Scatchard analysis demonstrated the presence of two binding sites. Linear regression analysis resulted in a two-binding-site model characterized by a high-affinity site (K(dBALB)=12.0 microM; K(dDBA)=20.9 microM) and a low-affinity site (K(dBALB)=36.2 microM; K(dDBA)=58.8 microM). FCS analysis exhibited quite different K(m) and V(max) values from those obtained by GPC, but similar K(m) values for the two types of megalin, and a lower V(max) value for DBA/2 megalin than BALB/c megalin. These results suggest that the core 2 GlcNAc extended glycan chains on megalin can change the ligand-binding affinity and capacity.     

Silencing of NtMPK4 impairs CO-induced stomatal closure, activation of anion channels and cytosolic Casignals in Nicotiana tabacum guard cells

Light-induced stomatal opening in C3 and C4 plants is mediated by two signalling pathways. One pathway is specific for blue light and involves phototropins, while the second pathway depends on photosyntheticaly active radiation (PAR). Here, the role of Nt MPK4 in light-induced stomatal opening was studied, as silencing of this MAP kinase stimulates stomatal opening. Stomata of Nt MPK4-silenced plants do not close in elevated atmospheric CO₂, and show a reduced response to PAR. However, stomatal closure can still be induced by abscisic acid. Measurements using multi-barrelled intracellular micro-electrodes showed that CO₂ activates plasma membrane anion channels in wild-type Nicotiana tabacum guard cells, but not in Nt MPK4-silenced cells. Anion channels were also activated in wild-type guard cells after switching off PAR. In approximately half of these cells, activation of anion channels was accompanied by an increase in the cytosolic free Ca²⁺ concentration. The activity of anion channels was higher in cells showing a parallel increase in cytosolic Ca²⁺ than in those with steady Ca²⁺ levels. Both the darkness-induced anion channel activation and Ca²⁺ signals were repressed in Nt MPK4-silenced guard cells. These data show that CO₂ and darkness can activate anion channels in a Ca²⁺-independent manner, but the anion channel activity is enhanced by parallel increases in the cytosolic Ca²⁺ concentration. Nt MPK4 plays an essential role in CO₂- and darkness-induced activation of guard-cell anion channels, through Ca²⁺-independent as well as Ca²⁺-dependent signalling pathways.     

Pollen UDP-glucose pyrophosphorylase showing polymorphism well-correlated to the S genotype of Pyrus pyrifolia

A polymorphic protein well-correlated to the diploid S genotypes of the pollen parent was detected by two-dimensional gel electrophoresis in Pyrus pyrifolia (Japanese pear). Its molecular weight was about 50 kDa, and it was expressed primarily in pollen. Partial amino acid sequences of the polymorphic protein from 'Nijisseiki' (S2S4), a cultivar of P. pyrifolia, were determined. Based on these sequences, two cDNA sequences associated with the S2 and S4 genotypes were identified by PCR-based methods. Both encode a protein of 458 amino acids whose sequence has high similarity to eukaryotic UDP-glucose pyrophosphorylases (UGPases) (EC 2.7.7.9), so they were named UGPases PA and PC. As there are only three amino acid substitutions between UGPases PA and PC, it is unlikely that they are pollen factors that recognize self and non-self S-RNases. Although this UGPase had more than 75% sequence identity to the known plant UGPases, its C-terminal sequences differed markedly. This unique C-terminal region of UGPases PA and PC may act in their subcellular localization in the pollen or interact with some other factor(s).     

Separation and quantitative determination of dolichol and dolichyl phosphate in rat and trout liver

The dolichol concentrations in rat and trout liver were found respectively to be 50-59 and 16-21 micrograms/g using three experimental methods: densitometric scanning of thin-layer plates, colorimetric assay and HPLC analysis. By HPLC of benzoylated dolichols, the distribution of the dolichols according to the number of their isoprene residues, was determined in rat and trout liver. The major component was dolichol -18 in rat and dolichol -19 in trout liver. Dolichyl phosphate concentrations were found to be 6-7 micrograms/g of rat liver and 8-9 micrograms/g of trout liver by densitometric scanning of thin-layer plates.