TY - JOUR A1 - Tenenboim, Hezi A1 - Smirnova, Julia A1 - Willmitzer, Lothar A1 - Steup, Martin A1 - Brotman, Yariv T1 - VMP1-deficient Chlamydomonas exhibits severely aberrant cell morphology and disrupted cytokinesies JF - BMC plant biology N2 - Background: The versatile Vacuole Membrane Protein 1 (VMP1) has been previously investigated in six species. It has been shown to be essential in macroautophagy, where it takes part in autophagy initiation. In addition, VMP1 has been implicated in organellar biogenesis; endo-, exo- and phagocytosis, and protein secretion; apoptosis; and cell adhesion. These roles underly its proven involvement in pancreatitis, diabetes and cancer in humans. Results: In this study we analyzed a VMP1 homologue from the green alga Chlamydomonas reinhardtii. CrVMP1 knockdown lines showed severe phenotypes, mainly affecting cell division as well as the morphology of cells and organelles. We also provide several pieces of evidence for its involvement in macroautophagy. KW - VMP1 KW - Autophagy KW - Cytokinesis Y1 - 2014 U6 - https://doi.org/10.1186/1471-2229-14-121 SN - 1471-2229 VL - 14 PB - BioMed Central CY - London ER - TY - JOUR A1 - Smirnova, Julia A1 - Fernie, Alisdair R. A1 - Spahn, Christian M. T. A1 - Steup, Martin T1 - Photometric assay of maltose and maltose-forming enzyme activity by using 4-alpha-glucanotransferase (DPE2) from higher plants JF - Analytical biochemistry : methods in the biological sciences N2 - Maltose frequently occurs as intermediate of the central carbon metabolism of prokaryotic and eukaryotic cells. Various mutants possess elevated maltose levels. Maltose exists as two anomers, (alpha- and beta-form) which are rapidly interconverted without requiring enzyme-mediated catalysis. As maltose is often abundant together with other oligoglucans, selective quantification is essential. In this communication, we present a photometric maltose assay using 4-alpha-glucanotransferase (AtDPE2) from Arabidopsis thaliana. Under in vitro conditions, AtDPE2 utilizes maltose as glucosyl donor and glycogen as acceptor releasing the other hexosyl unit as free glucose which is photometrically quantified following enzymatic phosphorylation and oxidation. Under the conditions used, DPE2 does not noticeably react with other di- or oligosaccharides. Selectivity compares favorably with that of maltase frequently used in maltose assays. Reducing end interconversion of the two maltose anomers is in rapid equilibrium and, therefore, the novel assay measures total maltose contents. Furthermore, an AtDPE2-based continuous photometric assay is presented which allows to quantify beta-amylase activity and was found to be superior to a conventional test. Finally, the AtDPE2-based maltose assay was used to quantify leaf maltose contents of both Arabidopsis wild type and AtDPE2-deficient plants throughout the light-dark cycle. These data are presented together with assimilatory starch levels. (C) 2017 Published by Elsevier Inc. KW - Arabidopsis thaliana KW - beta-amylase assay KW - Disproportionating isozyme 2 (DPE2) dpe2-deficient plants KW - Maltose assay KW - Leaf maltose content Y1 - 2017 U6 - https://doi.org/10.1016/j.ab.2017.05.026 SN - 0003-2697 SN - 1096-0309 VL - 532 SP - 72 EP - 82 PB - Elsevier CY - San Diego ER - TY - JOUR A1 - Fettke, Jörg A1 - Hejazi, Mahdi A1 - Smirnova, Julia A1 - Hoechel, Erik A1 - Stage, Marion A1 - Steup, Martin T1 - Eukaryotic starch degradation : integration of plastidial and cytosolic pathways N2 - Starch is an important plant product widely used as a nutrient, as a source of renewable energy, and for many technological applications. In plants, starch is the almost ubiquitous storage carbohydrate whereas most heterotrophic prokaryotes and eukaryotes rely on glycogen. Despite close similarities in basic chemical features, starch and glycogen differ in both structural and physicochemical properties. Glycogen is a hydrosoluble macromolecule with evenly distributed branching points. Starch exists as a water-insoluble particle having a defined (and evolutionary conserved) internal structure. The biochemistry of starch requires the co-operation of up to 40 distinct (iso)enzymes whilst approximately 10 (iso)enzymes permit glycogen metabolism. The biosynthesis and degradation of native starch include the transition of carbohydrates from the soluble to the solid phase and vice versa. In this review, two novel aspects of the eukaryotic plastidial starch degradation are discussed: Firstly, biochemical reactions that take place at the surface of particulate glucans and mediate the phase transition of carbohydrates. Secondly, processes that occur downstream of the export of starch-derived sugars into the cytosol. Degradation of transitory starch mainly results in the formation of neutral sugars, such as glucose and maltose, that are transported into the cytosol via the respective translocators. The cytosolic metabolism of the neutral sugars includes the action of a hexokinase, a phosphoglucomutase, and a transglucosidase that utilizes high molecular weight glycans as a transient glucosyl acceptor or donor. Data are included on the transglucosidase (disproportionating isozyme 2) in Cyanophora paradoxa that accumulates storage carbohydrates in the cytosol rather than in the plastid. Y1 - 2009 UR - http://jxb.oxfordjournals.org/ U6 - https://doi.org/10.1093/Jxb/Erp054 SN - 0022-0957 ER - TY - THES A1 - Smirnova, Julia T1 - Carbohydrate-active enzymes metabolising maltose: kinetic and structural features Y1 - 2012 CY - Potsdam ER - TY - JOUR A1 - Ruzanski, Christian A1 - Smirnova, Julia A1 - Rejzek, Martin A1 - Cockburn, Darrell A1 - Pedersen, Henriette L. A1 - Pike, Marilyn A1 - Willats, William G. T. A1 - Svensson, Birte A1 - Steup, Martin A1 - Ebenhöh, Oliver A1 - Smith, Alison M. A1 - Field, Robert A. T1 - A bacterial glucanotransferase can replace the complex maltose metabolism required for starch to sucrose conversion in leaves at night JF - The journal of biological chemistry N2 - Controlled conversion of leaf starch to sucrose at night is essential for the normal growth of Arabidopsis. The conversion involves the cytosolic metabolism of maltose to hexose phosphates via an unusual, multidomain protein with 4-glucanotransferase activity, DPE2, believed to transfer glucosyl moieties to a complex heteroglycan prior to their conversion to hexose phosphate via a cytosolic phosphorylase. The significance of this complex pathway is unclear; conversion of maltose to hexose phosphate in bacteria proceeds via a more typical 4-glucanotransferase that does not require a heteroglycan acceptor. It has recently been suggested that DPE2 generates a heterogeneous series of terminal glucan chains on the heteroglycan that acts as a glucosyl buffer to ensure a constant rate of sucrose synthesis in the leaf at night. Alternatively, DPE2 and/or the heteroglycan may have specific properties important for their function in the plant. To distinguish between these ideas, we compared the properties of DPE2 with those of the Escherichia coli glucanotransferase MalQ. We found that MalQ cannot use the plant heteroglycan as an acceptor for glucosyl transfer. However, experimental and modeling approaches suggested that it can potentially generate a glucosyl buffer between maltose and hexose phosphate because, unlike DPE2, it can generate polydisperse malto-oligosaccharides from maltose. Consistent with this suggestion, MalQ is capable of restoring an essentially wild-type phenotype when expressed in mutant Arabidopsis plants lacking DPE2. In light of these findings, we discuss the possible evolutionary origins of the complex DPE2-heteroglycan pathway. KW - Carbohydrate Metabolism KW - Computer Modeling KW - Metabolic Regulation KW - Oligosaccharide KW - Plant Biochemistry KW - Glucanotransferase KW - Leaf Cell KW - Maltose Metabolism KW - Starch Degradation Y1 - 2013 U6 - https://doi.org/10.1074/jbc.M113.497867 SN - 0021-9258 SN - 1083-351X VL - 288 IS - 40 SP - 28581 EP - 28598 PB - American Society for Biochemistry and Molecular Biology CY - Bethesda ER -