Interaction of the K(+)-channel KAT1 with the coat protein complex II coat component Sec24 depends on a di-acidic endoplasmic reticulum export motif

The correct functioning of ion channels depends not only on the control of their activity but also on the regulation of the number of channels in the membrane. For example, it has been proposed that the density of the plant K(+)-channel KAT1 may be adjusted by controlling its export from its site of synthesis, the endoplasmic reticulum (ER). Efficient transport of the channel to the plasma membrane was found to depend on a di-acidic ER export signal in the C-terminus of the protein. Studies in yeast and mammals indicate that di-acidic ER export motifs are essential for enrichment of proteins into ER-derived coat protein complex II (COPII) vesicles and are recognized by Sec24 a component of the COPII coat. To investigate whether similar mechanisms also exist in plants we have analysed the interaction of KAT1 with Sec24 in vivo using fluorescence resonance energy transfer (FRET) measurements in Vicia faba guard cells. These measurements revealed a FRET signal between KAT1 and Sec24 fused to the cyan fluorescent protein and the yellow fluorescent protein, respectively, indicating an interaction between KAT1 and Sec24. The FRET signal only occurred in the perinuclear region of the ER and was dependent on the di-acidic ER export motif of KAT1. Together, the results point to a highly conserved mechanism for ER export of KAT1 whereby the channel is recruited into COPII vesicles via binding of the di-acidic motif to Sec24.     

S1P-lyase independent clearance of extracellular sphingosine 1-phosphate after dephosphorylation and cellular uptake

Sphingosine 1-phosphate (S1P) is the natural ligand for a specific family of G protein-coupled receptors (-Rs). The type 1 S1P-R (S1P(1)) is important for lymphocyte egress, and blood-borne S1P as the natural ligand for S1P(1) is involved in the maintenance of lymphocyte circulation. This report reveals that extracellular S1P was cleared by all tested primary cells and cell lines with exponential progression. Clearance of S1P, but not sphingosine (Sph) was inhibited with the protein phosphatase inhibitor sodium orthovanadate. Fluorescence microscopy and flow cytometry using fluorescently labeled S1P and Sph showed a major cellular uptake of Sph, but not S1P. HPLC-analyses with C17-Sph demonstrated that cellular Sph accumulation was transient in tested cell lines, but enduring in mouse splenocytes. Sub cellular fractionation resulted in dephosphorylation of S1P to Sph by nuclear, membrane, and cytosolic fractions. Degradation of Sph however only occurred in combined membrane and cytosolic fractions. Inhibitors for Sph kinases 1/2, ceramide synthase, and S1P-lyase, as well as S1P-lyase deficiency did not block clearance of extracellular S1P. In vivo experiments revealed a transient increase in plasma S1P levels after single intravenous injection into C57BL/6 mice. This exogenously added S1P was cleared within 15-30 min in contrast to ex vivo incubation of whole blood which required more than 8 h for comparable clearance from plasma. Our data thus show that extracellular S1P is dephosphorylated and subsequently converted by cells, which appears to be important for clearance of the signaling molecule S1P in the local tissue environment after infections or injuries.     

Secretory cargo composition affects polarized secretion in MDCK epithelial cells

Polarized epithelial cells secrete proteins at either the apical or basolateral cell surface. A number of non-epithelial secretory proteins also exhibit polarized secretion when they are expressed in polarized epithelial cells but it is difficult to predict where foreign proteins will be secreted in epithelial cells. The question is of interest since secretory epithelia are considered as target tissues for gene therapy protocols that aim to express therapeutic secretory proteins. In the parathyroid gland, parathyroid hormone is processed by furin and co-stored with chromogranin A in secretory granules. To test the secretion of these proteins in epithelial cells, they were expressed in MDCK cells. Chromogranin A and a secreted form of furin were secreted apically while parathyroid hormone was secreted 60% basolaterally. However, in the presence of chromogranin A, the secretion of parathyroid hormone was 65% apical, suggesting that chromogranin can act as a “sorting escort” (sorting chaperone) for parathyroid hormone. Conversely, apically secreted furin did not affect the sorting of parathyroid hormone. The apical secretion of chromogranin A was dependent on cholesterol, suggesting that this protein uses an established cellular sorting mechanism for apical secretion. However, this sorting does not involve the N-terminal membrane-binding domain of chromogranin A. These results suggest that foreign secretory proteins can be used as “sorting escorts” to direct secretory proteins to the apical secretory pathway without altering the primary structure of the secreted protein. Such a system may be of use in the targeted expression of secretory proteins from epithelial cells. David V. Cohn—Deceased.     

Soil-to-plant and soil-to-grain transfer of 137Cs in field-grown maize hybrids during two contrasting seasons: assessing the phenotypic variability and its genetic component

Field-grown maize hybrids were assessed for variability in ¹³⁷Cs accumulation in vegetative parts of young and mature maize shoots and grains during 2 years with contrasting climatic conditions. Trials were carried out at different sites in the Tula region of Russia, which is characterized by a highly homogenous soil classified as Luvic Chernozem according to FAO/UNESCO, and average contamination levels of about 509–564 Bq ¹³⁷Cs kg⁻¹ soil. In the first year, 19 hybrids were tested. The two hybrids with the highest and the two with the lowest ¹³⁷Cs concentration ratios (C _r) were also tested in the second year, together with another 11 hybrids. All samples were additionally assessed for their potassium content. In both investigation periods ¹³⁷Cs accumulation in vegetative shoots and grains was found to vary up to more than twofold between hybrids. However, C _r values of those hybrids that showed a relatively low ¹³⁷Cs accumulation in the first year were not necessarily low in the second year, and the ratio between the ¹³⁷Cs C _r of low- and high-accumulating hybrids was much smaller than in the year before. In both vegetative shoots and grains the variance caused by the different years was larger than the genotypic variance, thus indicating the limits of genotype selection for this trait. Significant correlations were determined between the ⁴⁰K and ¹³⁷Cs C _r values in the same tissue, but for one hybrid indications for uncoupling of the two traits were found. Average Cs/K ratios in young shoots, mature shoots and grains were 0.06, 0.05 and 0.02, respectively, indicating tissue- and stage-specific regulation of accumulation within each plant. The findings are discussed with respect to new approaches towards a better understanding of ¹³⁷Cs accumulation and its potential reduction in plants. Katharina Schneider was deceased.     

A different view on DNA amplifications indicates frequent, highly complex, and stable amplicons on 12q13-21 in glioma

To further understand the biological significance of amplifications for glioma development and recurrencies, we characterized amplicon frequency and size in low-grade glioma and amplicon stability in vivo in recurring glioblastoma. We developed a 12q13-21 amplicon-specific genomic microarray and a bioinformatics amplification prediction tool to analyze amplicon frequency, size, and maintenance in 40 glioma samples including 16 glioblastoma, 10 anaplastic astrocytoma, 7 astrocytoma WHO grade 2, and 7 pilocytic astrocytoma. Whereas previous studies reported two amplified subregions, we found a more complex situation with many amplified subregions. Analyzing 40 glioma, we found that all analyzed glioblastoma and the majority of pilocytic astrocytoma, grade 2 astrocytoma, and anaplastic astrocytoma showed at least one amplified subregion, indicating a much higher amplification frequency than previously suggested. Amplifications in low-grade glioma were smaller in size and displayed clearly different distribution patterns than amplifications in glioblastoma. One glioblastoma and its recurrencies revealed an amplified subregion of 5 Mb that was stable for 6 years. Expression analysis of the amplified region revealed 10 overexpressed genes (i.e., KUB3, CTDSP2, CDK4, OS-9, DCTN2, RAB3IP, FRS2, GAS41, MDM2, and RAP1B) that were consistently overexpressed in all cases that carried this amplification. Our data indicate that amplifications on 12q13-21 (a) are more frequent than previously thought and present in low-grade tumors and (b) are maintained as extended regions over long periods of time.     

Soil-Atmosphere Exchange of N<Subscript>2</Subscript>O and NO in Near-Natural Savanna and Agricultural Land in Burkina Faso (W. Africa)

In a combined field and laboratory study in the southwest of Burkina Faso, we quantified soil-atmosphere N₂O and NO exchange. N₂O emissions were measured during two field campaigns throughout the growing seasons 2005 and 2006 at five different experimental sites, that is, a natural savanna site and four agricultural sites planted with sorghum (n = 2), cotton and peanut. The agricultural fields were not irrigated and not fertilized. Although N₂O exchange mostly fluctuated between −2 and 8 μg N₂O–N m⁻² h⁻¹, peak N₂O emissions of 10–35 μg N₂O–N m⁻² h⁻¹ during the second half of June 2005, and up to 150 μg N₂O–N m⁻² h⁻¹ at the onset of the rainy season 2006, were observed at the native savanna site, whereas the effect of the first rain event on N₂O emissions at the crop sites was low or even not detectable. Additionally, a fertilizer experiment was conducted at a sorghum field that was divided into three plots receiving different amounts of N fertilizer (plot A: 140 kg N ha⁻¹; plot B: 52.5 kg N ha⁻¹; plot C: control). During the first 3 weeks after fertilization, only a minor increase in N₂O emissions at the two fertilized plots was detected. After 24 days, however, N₂O emission rates increased exponentially at plot A up to a mean of 80 μg N₂O–N m⁻² h⁻¹, whereas daily mean values at plot B reached only 19 μg N₂O–N m⁻² h⁻¹, whereas N₂O flux rates at plot C remained unchanged. The calculated annual N₂O emission of the nature reserve site amounted to 0.52 kg N₂O–N ha⁻¹ a⁻¹ in 2005 and to 0.67 kg N₂O–N ha⁻¹ a⁻¹ in 2006, whereas the calculated average annual N₂O release of the crop sites was only 0.19 kg N₂O–N ha⁻¹ a⁻¹ and 0.20 kg N₂O–N ha⁻¹ a⁻¹ in 2005 and 2006, respectively. In a laboratory study, potential N₂O and NO formation under different soil moisture regimes were determined. Single wetting of dry soil to medium soil water content with subsequent drying caused the highest increase in N₂O and NO emissions with maximum fluxes occurring 1 day after wetting. The stimulating effect lasted for 3–4 days. A weaker stimulation of N₂O and NO fluxes was detected during daily wetting of soil to medium water content, whereas no significant stimulating effect of single or daily wetting to high soil water content (>67% WHC_max) was observed. This study demonstrates that the impact of land-use change in West African savanna on N trace gas emissions is smaller—with the caveat that there could have been potentially higher N₂O and NO emissions during the initial conversion—than the effect of timing and distribution of rainfall and of the likely increase in nitrogen fertilization in the future.     

Gle1 does double duty

During nuclear export, Gle1 (the nuclear-pore-associated mRNA export factor) activates the DEAD-box protein Dbp5 to remodel exported mRNA-protein complexes on the cytoplasmic face of the nuclear pore complex. In this issue, Bolger et al. (2008) now report additional roles for Gle1 in translation initiation and termination.