Decreased expression of cytosolic pyruvate kinase in potato tubers leads to a decline in pyruvate resulting in an in vivo repression of the alternative oxidase

The aim of this work was to investigate the effect of decreased cytosolic pyruvate kinase (PKc) on potato (Solanum tuberosum) tuber metabolism. Transgenic potato plants with strongly reduced levels of PKc were generated by RNA interference gene silencing under the control of a tuber-specific promoter. Metabolite profiling showed that decreased PKc activity led to a decrease in the levels of pyruvate and some other organic acids involved in the tricarboxylic acid cycle. Flux analysis showed that this was accompanied by changes in carbon partitioning, with carbon flux being diverted from glycolysis toward starch synthesis. However, this metabolic shift was relatively small and hence did not result in enhanced starch levels in the tubers. Although total respiration rates and the ATP to ADP ratio were largely unchanged, transgenic tubers showed a strong decrease in the levels of alternative oxidase (AOX) protein and a corresponding decrease in the capacity of the alternative pathway of respiration. External feeding of pyruvate to tuber tissue or isolated mitochondria resulted in activation of the AOX pathway, both in the wild type and the PKc transgenic lines, providing direct evidence for the regulation of AOX by changes in pyruvate levels. Overall, these results provide evidence for a crucial role of PKc in the regulation of pyruvate levels as well as the level of the AOX in heterotrophic plant tissue, and furthermore reveal that these parameters are interlinked in vivo.     

Melatonin controls cell proliferation and modulates mitochondrial physiology in pancreatic stellate cells

Journal of Physiology and Biochemistry - We have investigated the effects of melatonin on major pathways related with cellular proliferation and energetic metabolism in pancreatic stellate cells....     

Biochemical properties of a novel cell wall protein associated with elongation growth in higher plants

A monoclonal antibody of IgM-type (TIM-11B2) was screened froma hybridoma library. The antibody recognizes a 40 kDa glycoprotein,p40, with high specificity. This protein was detected in allplant species examined so far and was found to be located bothsolubly and ionically-bound within the primary cell wall. The strongest immunobiochemical signals of p40 were found intissues undergoing elongation growth, whereas in other tissuesonly a faint signal could be detected. Those included the non-elongatingparts of different seedlings, such as the apical part of monocotprimary leaves or the leaves of dicots grown in light. Inhibitionof pea epicotyl growth by white light irradiation resulted ina strong decrease of the immunostain signal. On the other hand,induction of rapid coleoptile growth in rice seedlings inducedby submergence resulted in a strong increase of the immunobiochemicalsignal of p40. Time-course studies on the expression of p40during protoplast regeneration revealed that p40 is apparentlynot involved in cell wall formation. The hypothesis that p40is characteristic for tissues with the ability for elongationgrowth is discussed. Comparison of biochemical data and location of p40 with proteinsdescribed up to now indicate that this glycoprotein has notbeen characterized before. Key words: Cell wall protein, elongation growth, monoclonal antibody     

Paraphyly and budding speciation in the hairy snail (Pulmonata,Hygromiidae)

Delimitation of species is often complicated by discordance of morphological and genetic data. This may be caused by the existence of cryptic or polymorphic species. The latter case is particularly true for certain snail species showing an exceptionally high intraspecific genetic diversity. The present investigation deals with the Trochulus hispidus complex, which has a complicated taxonomy. Our analyses of the COI sequence revealed that individuals showing a T. hispidus phenotype are distributed in nine highly differentiated mitochondrial clades (showing p‐distances up to 19%). The results of a parallel morphometric investigation did not reveal any differentiation between these clades, although the overall variability is quite high. The phylogenetic analyses based on 12S, 16S and COI sequences show that the T. hispidus complex is paraphyletic with respect to several other morphologically well‐defined Trochulus species (T. clandestinus, T. villosus, T. villosulus and T. striolatus) which form well‐supported monophyletic groups. The nc marker sequence (5.8S–ITS2–28S) shows only a clear separation of T. o. oreinos and T. o. scheerpeltzi, and a weakly supported separation of T. clandestinus, whereas all other species and the clades of the T. hispidus complex appear within one homogeneous group. The paraphyly of the T. hispidus complex reflects its complicated history, which was probably driven by geographic isolation in different glacial refugia and budding speciation. At our present state of knowledge, it cannot be excluded that several cryptic species are embedded within the T. hispidus complex. However, the lack of morphological differentiation of the T. hispidus mitochondrial clades does not provide any hints in this direction. Thus, we currently do not recommend any taxonomic changes. The results of the current investigation exemplify the limitations of barcoding attempts in highly diverse species such as T. hispidus.     

Glucose-induced Ubiquitylation and Endocytosis of the Yeast Jen1 Transporter: ROLE OF LYSINE 63-LINKED UBIQUITIN CHAINS*

Protein ubiquitylation is essential for many events linked to intracellular protein trafficking. Despite the significance of this process, the molecular mechanisms that govern the regulation of ubiquitylation remain largely unknown. Plasma membrane transporters are subjected to tightly regulated endocytosis, and ubiquitylation is a key signal at several stages of the endocytic pathway. The yeast monocarboxylate transporter Jen1 displays glucose-regulated endocytosis. We show here that casein kinase 1-dependent phosphorylation and HECT-ubiquitin ligase Rsp5-dependent ubiquitylation are required for Jen1 endocytosis. Ubiquitylation and endocytosis of Jen1 are induced within minutes in response to glucose addition. Jen1 is modified at the cell surface by oligo-ubiquitylation with ubiquitin-Lys⁶³ linked chain(s), and Jen1-Lys³³⁸ is one of the target residues. Ubiquitin-Lys⁶³-linked chain(s) are also required directly or indirectly to sort Jen1 into multivesicular bodies. Jen1 is one of the few examples for which ubiquitin-Lys⁶³-linked chain(s) was shown to be required for correct trafficking at two stages of endocytosis: endocytic internalization and sorting at multivesicular bodies.Ubiquitylation is one of the most prevalent protein post-translational modifications in eukaryotes. In addition to its role in promoting proteasomal degradation of target proteins, ubiquitylation has been shown to regulate multiple processes, including DNA repair, signaling, and intracellular trafficking. Ubiquitylation serves as a key signal mediating the internalization of plasma membrane receptors and transporters, followed by their intracellular transport and subsequent recycling or lysosomal/vacuolar degradation (1, 2). In Saccharomyces cerevisiae, transporters usually display both constitutive and accelerated endocytosis regulated by factors such as excess substrate, changes in nutrient availability, and stress conditions. Ubiquitylation of these cell surface proteins acts as a signal triggering their internalization (1). A single essential E3⁴ ubiquitin ligase, Rsp5, has been implicated in the internalization of most, if not all, endocytosed proteins (3). Rsp5 is the unique member in S. cerevisiae of the HECT (homologous to E6AP COOH terminus)-ubiquitin ligases of the Nedd4/Rsp5 family (4). In a few cases, Rsp5-dependent cell surface ubiquitylation was shown to involve PY-containing adapters that bind to Rsp5 (5–7). Rsp5-mediated ubiquitylation is also required for sorting into multivesicular bodies (MVBs) of endosomal membrane proteins that come from either the plasma membrane (through endocytosis) or the Golgi (through vacuolar protein sorting (VPS) pathway) (8). Although much progress has been made in elucidating the mechanistic basis of various steps in protein trafficking, the precise requirement for a specific type and length of Ub chains at various stages of the endocytic pathway remains to be addressed.The ubiquitin profile needed for proper internalization has been established for some yeast membrane proteins (1). The α-factor receptor Ste2 was described as undergoing monoubiquitylation on several lysines (multimonoubiquitylation). The a-factor receptor, Ste3p; the general transporter of amino acids, Gap1; the zinc transporter, Ztr1; and the uracil transporter, Fur4, have been shown to be modified by short chains of two to three ubiquitins, each attached to one, two, or more target lysine residues (oligo-ubiquitylation). Among them, Fur4 and Gap1 were the only transporters demonstrated to undergo plasma membrane oligo-ubiquitylation with ubiquitin residues linked via ubiquitin-Lys⁶³ (9, 10). In addition, the two siderophore transporters Arn1 and Sit1 were also shown to undergo Lys⁶³-linked cell surface ubiquitylation (11, 12). Whether these four transporters are representative of a larger class of plasma membrane substrates remains to be determined. Little is known about the type of ubiquitylation involved and/or required for sorting to MVBs. Some MVB cargoes appear to undergo monoubiquitylation (8), whereas Sna3, an MVB cargo of unknown function, undergoes Lys⁶³-linked ubiquitylation (13). Lys⁶³-linked ubiquitin chains were also recently reported to be required, directly or indirectly, for MVB sorting of the siderophore transporter, Sit1, when trafficking through the VPS pathway in the absence of its external substrate (11). In agreement with the possibility that additional membrane-bound proteins might undergo Lys⁶³-linked ubiquitylation, a proteomic study aiming to uncover ubiquitylated yeast proteins showed that Lys⁶³-ubiquitin chains are far more abundant than previously thought (14).The transport of monocarboxylates, such as lactate and pyruvate, as well as ketone bodies across the plasma membrane is essential for the metabolism of cells of various organisms. A family of monocarboxylate transporters has been reported that includes mainly mammalian members (15). In S. cerevisiae, two monocarboxylate-proton symporters have been described, Jen1 and Ady2 (16, 17). These transporters exhibit differences in their mechanisms of regulation and specificity. Jen1 is a lactate-pyruvate-acetate-propionate transporter induced in lactic or pyruvic acid-grown cells (18). Ady2, which accepts acetate, propionate, or formate, is present in cells grown in non-fermentable carbon sources (19). Jen1 has unique regulatory characteristics and has been extensively studied. It was the first secondary porter of S. cerevisiae characterized by heterologous expression in Pichia pastoris at both the cell and the membrane vesicle levels (20). The addition of glucose to lactic acid-grown cells very rapidly triggers loss of Jen1 activity and repression of JEN1 gene expression (21, 22). Newly synthesized Jen1-GFP fusion protein is sorted to the plasma membrane in an active and stable form, and loss of Jen1-GFP activity upon glucose addition is the result of its endocytosis followed by vacuolar degradation (23). Data from large scale analyses based on mass spectrometry approaches led to the detection of two sites of ubiquitylation for Jen1, one located in the N terminus of the protein and the second in the central loop (14), and several sites of phosphorylation in the N terminus, central loop, and C terminus of the protein (14, 24). In the present study, we aimed at further characterizing the internalization step of endocytosis of the transporter Jen1 and the potential role of the phosphorylation and ubiquitylation events required for its correct endocytic trafficking.     

High-throughput RNAi screening to dissect cellular pathways: A how-to guide

RNA interference (RNAi) has become a powerful tool to dissect cellular pathways and characterize gene functions. The availability of genome-wide RNAi libraries for various model organisms and mammalian cells has enabled high-throughput RNAi screenings. These RNAi screens successfully identified key components that had previously been missed in classical forward genetic screening approaches and allowed the assessment of combined loss-of-function phenotypes. Crucially, the quality of RNAi screening results depends on quantitative assays and the choice of the right biological context. In this review, we provide an overview on the design and application of high-throughput RNAi screens as well as data analysis and candidate validation strategies.     

Production and purification of human indoleamine 2,3-dioxygenase (HuIDO) protein in a baculovirus expression system and production and characterization of egg yolk antibody against the purified HuIDO

The human indoleamine 2,3-dioxygenase (HuIDO) baculoviral construct, for expression of HuIDO protein with a hexa-histidine and FLAG (DYKDDDDK) tag, was produced using the BacPAK Baculovirus Expression System. HuIDO baculovirus was used to infect Sf21 insect cells to produce functionally active protein in large amounts. Conditions for protein purification by metal affinity chromatography were determined and optimized. Addition of haemin ensured optimal activity of the purified heme-containing oxygenase. The soluble purified protein was used to immunize a chicken to produce large quantities of polyclonal IgY against HuIDO. The anti-HuIDO IgY antibody specifically detected HuIDO produced by a range of cell types including transfectants and native HuIDO expression induced in IFN-gamma-stimulated cells. The antibody detected HuIDO in cell lysates by western blotting and in the cytoplasm of cells by microscopy. The antibody was unable to block the function of the enzyme, indicating that this antibody binds outside the active site of HuIDO.     

Large impact of the apoplast on somatic embryogenesis in <Emphasis Type="Italic">Cyclamen persicum</Emphasis> offers possibilities for improved developmental control <Emphasis Type="Italic">in vitro</Emphasis>

Background  

Clonal propagation is highly desired especially for valuable horticultural crops. The method with the potentially highest multiplication rate is regeneration via somatic embryogenesis. However, this mode of propagation is often hampered by the occurrence of developmental aberrations and non-embryogenic callus. Therefore, the developmental process of somatic embryogenesis was analysed in the ornamental crop Cyclamen persicum by expression profiling, comparing different developmental stages of embryogenic cell cultures, zygotic vs. somatic embryos and embryogenic vs. non-embryogenic cell cultures.     

Proteomic and functional analysis of the mitotic Drosophila centrosome

Regulation of centrosome structure, duplication and segregation is integrated into cellular pathways that control cell cycle progression and growth. As part of these pathways, numerous proteins with well‐established non‐centrosomal localization and function associate with the centrosome to fulfill regulatory functions. In turn, classical centrosomal components take up functional and structural roles as part of other cellular organelles and compartments. Thus, although a comprehensive inventory of centrosome components is missing, emerging evidence indicates that its molecular composition reflects the complexity of its functions. We analysed the Drosophila embryonic centrosomal proteome using immunoisolation in combination with mass spectrometry. The 251 identified components were functionally characterized by RNA interference. Among those, a core group of 11 proteins was critical for centrosome structure maintenance. Depletion of any of these proteins in Drosophila SL2 cells resulted in centrosome disintegration, revealing a molecular dependency of centrosome structure on components of the protein translation machinery, actin‐ and RNA‐binding proteins. In total, we assigned novel centrosome‐related functions to 24 proteins and confirmed 13 of these in human cells.