Dokument: The Schizosaccharomyces pombe bifunctional enzyme Asp1 regulates cellular levels of 1,5-bisdiphosphoinositol tetrakisphosphate
| Titel: | The Schizosaccharomyces pombe bifunctional enzyme Asp1 regulates cellular levels of 1,5-bisdiphosphoinositol tetrakisphosphate | |||||||
| URL für Lesezeichen: | https://docserv.uni-duesseldorf.de/servlets/DocumentServlet?id=44727 | |||||||
| URN (NBN): | urn:nbn:de:hbz:061-20180130-095737-8 | |||||||
| Kollektion: | Dissertationen | |||||||
| Sprache: | Englisch | |||||||
| Dokumententyp: | Wissenschaftliche Abschlussarbeiten » Dissertation | |||||||
| Medientyp: | Text | |||||||
| Autor: | Pascual Ortiz, Marina [Autor] | |||||||
| Dateien: |
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| Beitragende: | PD Dr. Fleig, Ursula [Gutachter] Prof. Dr. Feldbrügge, Michael [Gutachter] | |||||||
| Dewey Dezimal-Klassifikation: | 500 Naturwissenschaften und Mathematik » 570 Biowissenschaften; Biologie | |||||||
| Beschreibungen: | Inositol pyrophosphates are signalling molecules present in all eukaryotic cells. They are phosphate-rich molecules consisting of an inositol ring with at least one di-phosphate group (pyrophosphate). Although these molecules were discovered only 25 years ago, an extremely wide range of biological processes have already been defined to be controlled by inositol pyrophosphates. These include the phosphate starvation response in Saccharomyces cerevisiae, chromosome transmission fidelity in Schizosaccharomyces pombe, cell morphogenesis in a number of fungi, insulin secretion in mammals, and immune defense mechanisms in mammals and plants. Understanding how these molecules are synthesized, hydrolysed and their cellular concentration regulated is essential to understand how they modulate biological processes. In this context, the functional analysis of the S. pombe Asp1 protein is of a great interest as this protein controls intracellular pyrophosphate levels.
Asp1 belongs to the highly conserved Vip1 protein family. All members of this family present a dual domain structure: the N-terminal domain synthesizes inositol pyrophosphates and a C-terminal domain has homology to histidine acid phosphatases. Prior to this study, the function of the C-terminal domain of Vip1 proteins was unclear. Using different methods such as direct quantification of cellular inositol pyrophosphates with a HPLC-based approach and analysis of microtubule stability as an in vivo read-out of cellular inositol pyrophosphate production, I have demonstrated that the C-terminal domain of Asp1 is enzymatically active in vivo and identified the amino acids essential for this activity. I determined the in vivo substrate specificity of both domains of Asp1; the N-terminal domain synthesizes IP8 (1,5-bisdiphosphoinositol tetrakisphosphate) using 5-IP7 (5-diphosphoinositol pentakisphosphate) as a substrate and the C-terminal domain dephosphorylates IP8 to 5-IP7 (5-diphosphoinositol pentakisphosphate). Interestingly, the pyrophosphatase activity of Asp1 reduced 2-fold cellular IP8 levels. Thus the C-terminal Asp1 pyrophosphatase domain controls intracellular IP8 levels. How the two opposing activities of Asp1 (synthesis and hydrolysis of IP8) are regulated was another goal of my study. An interaction partner of Asp1, the mitochondrial-associated protein Met10, inhibited Asp1 pyrophosphatase activity in vitro. Interestingly, microscopical analysis showed that some Asp1 mutant variants can also associate with mitochondria, suggesting a spatial subcellular regulation of Asp1 bifunctional activity. Previous studies showed that Asp1-generated IP8 modulate polarized growth, a microtubule-regulated process in S. pombe. Microtubules regulate mitochondria distribution via the protein Mmb1. This work presents new data to understand how Asp1-generated IP8 regulates the microtubule cytoskeleton possibly through association with the mitochondrial network. asp1+ and mmb1+ genetically interact and strikingly Asp1-generated IP8 are required for proper mitochondrial distribution. Finally, I have uncovered a new function relationship between inositol pyrophosphates and phosphate homeostasis in S. pombe. Asp1-generated inositol pyrophosphates levels correlated directly with the accumulation of polyP (inorganic polyphosphate), suggesting a connection between Asp1 function and phosphate homeostasis.Inositol pyrophosphates are signalling molecules present in all eukaryotic cells. They are phosphate-rich molecules consisting of an inositol ring with at least one di-phosphate group (pyrophosphate). Although these molecules were discovered only 25 years ago, an extremely wide range of biological processes have already been defined to be controlled by inositol pyrophosphates. These include the phosphate starvation response in Saccharomyces cerevisiae, chromosome transmission fidelity in Schizosaccharomyces pombe, cell morphogenesis in a number of fungi, insulin secretion in mammals, and immune defense mechanisms in mammals and plants. Understanding how these molecules are synthesized, hydrolysed and their cellular concentration regulated is essential to understand how they modulate biological processes. In this context, the functional analysis of the S. pombe Asp1 protein is of a great interest as this protein controls intracellular pyrophosphate levels. Asp1 belongs to the highly conserved Vip1 protein family. All members of this family present a dual domain structure: the N-terminal domain synthesizes inositol pyrophosphates and a C-terminal domain has homology to histidine acid phosphatases. Prior to this study, the function of the C-terminal domain of Vip1 proteins was unclear. Using different methods such as direct quantification of cellular inositol pyrophosphates with a HPLC-based approach and analysis of microtubule stability as an in vivo read-out of cellular inositol pyrophosphate production, I have demonstrated that the C-terminal domain of Asp1 is enzymatically active in vivo and identified the amino acids essential for this activity. I determined the in vivo substrate specificity of both domains of Asp1; the N-terminal domain synthesizes IP8 (1,5-bisdiphosphoinositol tetrakisphosphate) using 5-IP7 (5-diphosphoinositol pentakisphosphate) as a substrate and the C-terminal domain dephosphorylates IP8 to 5-IP7 (5-diphosphoinositol pentakisphosphate). Interestingly, the pyrophosphatase activity of Asp1 reduced 2-fold cellular IP8 levels. Thus the C-terminal Asp1 pyrophosphatase domain controls intracellular IP8 levels. How the two opposing activities of Asp1 (synthesis and hydrolysis of IP8) are regulated was another goal of my study. An interaction partner of Asp1, the mitochondrial-associated protein Met10, inhibited Asp1 pyrophosphatase activity in vitro. Interestingly, microscopical analysis showed that some Asp1 mutant variants can also associate with mitochondria, suggesting a spatial subcellular regulation of Asp1 bifunctional activity. Previous studies showed that Asp1-generated IP8 modulate polarized growth, a microtubule-regulated process in S. pombe. Microtubules regulate mitochondria distribution via the protein Mmb1. This work presents new data to understand how Asp1-generated IP8 regulates the microtubule cytoskeleton possibly through association with the mitochondrial network. asp1+ and mmb1+ genetically interact and strikingly Asp1-generated IP8 are required for proper mitochondrial distribution. Finally, I have uncovered a new function relationship between inositol pyrophosphates and phosphate homeostasis in S. pombe. Asp1-generated inositol pyrophosphates levels correlated directly with the accumulation of polyP (inorganic polyphosphate), suggesting a connection between Asp1 function and phosphate homeostasis. | |||||||
| Lizenz: |  Urheberrechtsschutz | |||||||
| Fachbereich / Einrichtung: | Mathematisch- Naturwissenschaftliche Fakultät » WE Biologie » Mikrobiologie | |||||||
| Dokument erstellt am: | 30.01.2018 | |||||||
| Dateien geändert am: | 30.01.2018 | |||||||
| Promotionsantrag am: | 06.11.2017 | |||||||
| Datum der Promotion: | 06.11.2017 |
