Endoplasmic reticulum exit of a secretory glycoprotein in the absence of sec24p family proteins in yeast

Glycoproteins exit the endoplasmic reticulum (ER) of the yeast Saccharomyces cerevisiae in coat protein complex II (COPII) coated vesicles. The coat consists of the essential proteins Sec23p, Sec24p, Sec13p, Sec31p, Sar1p and Sec16p. Sec24p and its two nonessential homologues Sfb2p and Sfb3p have been suggested to serve in cargo selection. Using temperature-sensitive sec24-1 mutants, we showed previously that a secretory glycoprotein, Hsp150, does not require functional Sec24p for ER exit. Deletion of SFB2, SFB3 or both from wild type or the deletion of SFB2 from sec24-1 cells did not affect Hsp150 transport. SFB3 deletion has been reported to be lethal in sec24-1. However, here we constructed a sec24-1 Deltasfb3 and a sec24-1 Deltasfb2 Deltasfb3 strain and show that Hsp150 was secreted slowly in both. Turning off the SEC24 gene did not inhibit Hsp150 secretion either, and the lack of SEC24 expression in a Deltasfb2 Deltasfb3 deletant still allowed some secretion. The sec24-1 Deltasfb2 Deltasfb3 mutant grew slower than sec24-1. The cells were irregularly shaped, budded from random sites and contained proliferated ER at permissive temperature. At restrictive temperature, the ER formed carmellae-like proliferations. Our data indicate that ER exit may occur in vesicles lacking a full complement of Sec23p/24p and Sec13p/31p, demonstrating diversity in the composition of the COPII coat.     

Secretome profiling of Propionibacterium freudenreichii reveals highly variable responses even among the closely related strains

This study compared the secretomes (proteins exported out of the cell) of Propionibacterium freudenreichii of different origin to identify plausible adaptation factors. Phylosecretomics indicated strain‐specific variation in secretion of adhesins/invasins (SlpA, InlA), cell‐wall hydrolysing (NlpC60 peptidase, transglycosylase), protective (RpfB) and moonlighting (DnaK, GroEL, GaPDH, IDH, ENO, ClpB) enzymes and/or proteins. Detailed secretome comparison suggested that one of the cereal strains (JS14) released a tip fimbrillin (FimB) in to the extracellular milieu, which was in line with the electron microscopy and genomic analyses, indicating the lack of surface‐associated fimbrial‐like structures, predicting a mutated type‐2 fimbrial gene cluster (fimB‐fimA‐srtC2) and production of anchorless FimB. Instead, the cereal strain produced high amounts of SlpB that tentatively mediated adherent growth on hydrophilic surface and adherence to hydrophobic material. One of the dairy strains (JS22), producing non‐covalently bound surface‐proteins (LspA, ClpB, AraI) and releasing SlpA and InlA into the culture medium, was found to form clumps under physiological conditions. The JS22 strain lacked SlpB and displayed a non‐clumping and biofilm‐forming phenotype only under conditions of increased ionic strength (300 mM NaCl). However, this strain cultured under the same conditions was not adherent to hydrophobic support, which supports the contributory role of SlpB in mediating hydrophobic interactions. Thus, this study reports significant secretome variation in P. freudenreichii and suggests that strain‐specific differences in protein export, modification and protein–protein interactions have been the driving forces behind the adaptation of this bacterial species.     

The effect of cold and heat pretreatments on anther culture response of Avena sativa and A. sterilis

The effect of stress pretreatments on embryo induction in anther cultures of selected genotypes of Avena sativa and A. sterilis was tested. A heat pretreatment of isolated anthers at +32°C for 5 days was best for the A. sativa line WW 18019 and for A. sterilis line CAV 2648. Genotype dependency may exist since in ‘Stout’ heat pretreatment did not increase embryo production. For A. sterilis 13 green and three albino regenerants were produced, of which five plants (haploids) survived transfer to the greenhouse. For A. sativa, 30 various differentiation media/treatment combinations were used in an attempt to regenerate plants from embryos, with no success. Seven day cold treatment of cut tillers increased slightly the response level in ‘Stout’ and was routinely used in subsequent experiments. Maltose proved to be better then sucrose as a carbon source for the genotypes tested. Fourteen percent maltose promoted the highest induction in A. sterilis, but the quality of embryos was improved in the presence of 10% maltose for both species. Sub-optimal carbohydrate levels did not enhance embryo induction in oats. This revised version was published online in June 2006 with corrections to the Cover Date.     

Seipin regulates ER–lipid droplet contacts and cargo delivery

Seipin is an endoplasmic reticulum (ER) membrane protein implicated in lipid droplet (LD) biogenesis and mutated in severe congenital lipodystrophy (BSCL2). Here, we show that seipin is stably associated with nascent ER–LD contacts in human cells, typically via one mobile focal point per LD. Seipin appears critical for such contacts since ER–LD contacts were completely missing or morphologically aberrant in seipin knockout and BSCL2 patient cells. In parallel, LD mobility was increased and protein delivery from the ER to LDs to promote LD growth was decreased. Moreover, while growing LDs normally acquire lipid and protein constituents from the ER, this process was compromised in seipin‐deficient cells. In the absence of seipin, the initial synthesis of neutral lipids from exogenous fatty acid was normal, but fatty acid incorporation into neutral lipids in cells with pre‐existing LDs was impaired. Together, our data suggest that seipin helps to connect newly formed LDs to the ER and that by stabilizing ER–LD contacts seipin facilitates the incorporation of protein and lipid cargo into growing LDs in human cells.     

Variation of RuBP carboxylase activity among anther-derived plants of Solanum phureja

The effect of genotype and ploidy on RuBP carboxylase (EC 4.1.1.39) activity, chlorophyll content, leaf area, chloroplast ultrastructure and not photosynthesis among monoploid. diploid and tetraploid anther-derived plants of Solanum phureja Juz, and Buk. was studied. Within the monoploid group, RuBP carboxylase activity and concentration displayed a significant genotypic effect. For the diploids, variation among genotypes was significant for total protein content and maximum specific activity of RuBP carboxylase, and among the tetraploids for net photosynthesis and specific leaf weight. Ploidy effect was evident regarding net photosynthesis, leaf area and chlorophyll content. The different ploidy groups among the anther-derived plants surpassed the anther donor plant for all characteristics except maximum activity of RuBP carboxylase and net photosynthesis. For the latter only the tetra-ploid group was superior to the anther source plant. However, a monoploid genotype with an increase of 9% in maximum activity of RuBP carboxylase over the anther-donor plant was identified. Segregation of trails rind differential gene expression together with possible mutations during androgenesis are discussed as sources of variation.     

Cell-based β2-adrenergic receptor-ligand binding assay using synthesized europium-labeled ligands and time-resolved fluorescence

High-sensitivity, high-throughput, and user-friendly lanthanide-based assays for receptor-ligand interactions provide an attractive alternative to the traditional radioligand displacement assays. In this study, three small-molecule pindolol ligand derivatives were synthesized and their binding properties were tested in a radioligand displacement assay. The ligand derivatives were further labeled with fluorescent europium(III) chelate for β₂-adrenergic receptor-ligand binding assay. The europium-labeled pindolol ligands having no spacer (C0) or a 12-carbon spacer (C12) arm bound to the human β₂-adrenergic receptors overexpressed in human embryonic kidney HEK293_i cells. Europium ligand with a 6-carbon spacer arm (C6) showed no binding. Competitive binding assays were developed with the functional labeled ligands. The IC₅₀ values for β₂-adrenergic antagonist propranolol were 60 and 37 nM, the Z′ values were 0.51 and 0.77, and the signal-to-background ratios were 5.5 and 16.0 for C0 and C12, respectively. This study shows that functional time-resolved fluorescent assays can be constructed using fluorescent lanthanide chelates conjugated to small-molecule ligands.     

Central boreal mire plant communities along soil nutrient potential and water content gradients

Peatlands have traditionally been exploited in forestry and agriculture over the boreal region, yet they also provide substantial source of fuel production. The large-scale exploitation of peatlands has raised a concern about the diversity of mire plant communities. We studied composition of mire plant communities along soil nutrient potential and water content gradients, to recognize the areas with the high plant diversity. Soil electrical conductivity (EC_b) was measured to characterise soil nutrient regimes and soil dielectric permittivity (DP) the soil (volumetric) water regimes. A total of 115 mire sites were studied in the central boreal region of south-western Finnish Lapland. We found that Ward’s hierarchical cluster analysis produced eight stable EC_b and DP clusters with discrete vegetation compositions. On the basis of a locally weighted regression analysis (Loess), Carex dioica L., Comarum palustre L., Equisetum fluviatile L., Menyanthes trifoliata L., and Scorpidium scorpioides (Hedw.) Limpr. were found as indicator species for nutrient-rich regimes as designated by high soil EC_b. The soil EC_b is a diagnostic measure of plant diversity as EC_b?>?7 mSm^?1 resulted in a considerable increase in species richness. Our classification method, based on electrical measurements, provides a simple way to classify mires and focus detailed research to areas with potentially high conservation value.