Diss. (sammanfattning) Uppsala : Univ., 2003. / Härtill 4 uppsatser.

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Die lichtgetriebene Protononenpumpe Bacteriorhodopsin (BR) aus Halobacterium salinarum stellt den Prototyp lichtgetriebener Protonenpumpen dar und wurde durch strukturelle und spektroskopische Untersuchungen als einfaches Modellsystem für Protonentransferschritte in Proteinen etabliert. Aufgrund der schlechten heterologen Expression von BR in Wirtszellen konnten elektrophysiologische Untersuchungen unter kontrollierter Membranspannung jedoch nur vereinzelt durchgeführt werden und erlaubten kaum Rückschlüsse zum Einfluss einzelner Aminosäuren auf die Pumpkraft des Proteins. Das dem BR nahverwandte Coccomyxa subellipsoidea Rhodopsin (CsR) hingegen zeigte gegenüber BR etwa 8-fach größere Photoströme in elektrophysiologischen Messungen in Xenopus laevis Oozyten und ermöglichte so eine umfangreiche elektrophysiologische Charakterisierung. Dabei konnten Unterschiede zu BR, wie etwa die erhöhte Spannungsabhängigkeit oder die erhöhte Toleranz der Pumpkraft gegenüber dem extrazellulären pH, auf Grundlage des Sequenzvergleiches nicht geklärt werden. Entsprechend wurden, um weitere mechanistische Details zu klären, im Rahmen dieser Arbeit spektroskopische und röntgenkristallographische Untersuchungen an CsR vorgenommen. Dabei konnte eine hochauflösende Kristallstruktur von CsR gelöst werden, die es erlaubt, mechanistische Unterschiede zu anderen lichtgetriebenen Protonenpumpen auf strukturelle Unterschiede zurück zu führen. So konnte die erhöhte Spannungsabhängigkeit mit der besonderen Komposition des zytoplasmatischen Halbkanals und die erhöhte Toleranz gegenüber dem äußeren pH mit der einzigartigen Konfiguration des Protonenfreisetzungskomplexes in CsR assoziiert werden. Letztlich konnte auf Grundlage der Struktur CsR, durch rationales Design, in einen licht-induzierten Protonenkanal transformiert werden, der bereits bei physiologischen Bedingungen quasisymmetrische bidirektionale Ströme zeigt. / The light-driven proton pump bacteriorhodopsin (BR) from Halobacterium salinarum represents the prototype of light-driven proton pumps and was established by structural and spectroscopic investigations as a simple model system for proton transfer steps in proteins. However, due to the poor heterologous expression of BR in host cells, electrophysiological investigations under controlled membrane potential could only be carried out in some cases and hardly allowed conclusions to be drawn about the influence of individual amino acids on the pumping force of the protein. Coccomyxa subellipsoidea rhodopsin (CsR), which is closely related to BR, showed about 8 times larger photocurrents in electrophysiological measurements in Xenopus laevis oocytes than BR and thus enabled an extensive electrophysiological characterization. Observed differences to BR, such as the increased voltage dependence or the increased tolerance of the pumping force to the extracellular pH, could not be clarified solely on the basis of the sequence comparison. Accordingly, in order to clarify further mechanistic details, spectroscopic and X-ray crystallographic investigations on CsR were carried out within the scope of this work. A high-resolution crystal structure of CsR was solved, which allows to link mechanistic differences to other light-driven proton pumps to structural differences. Thus, the increased voltage dependence could be addressed by the special composition of the cytoplasmic half channel and the increased tolerance to the external pH could be associated with the unique configuration of the proton release complex in CsR. Finally, by a structure-based rational design approach, CsR was transformed into a light-induced, which shows almost symmetrical bidirectional currents already under physiological conditions.

CoordenaÃÃo de AperfeiÃoamento de Pessoal de NÃvel Superior / O acÃmulo de Na+ no vacÃolo central representa um importante mecanismo de defesa de plantas contra o estresse salino. A regulaÃÃo dos volumes e conteÃdos dos vacÃolos de cÃlulas vegetais depende da atividade de transportadores e canais localizados no tonoplasto (membrana vacuolar). A membrana vacuolar possui duas distintas bombas de prÃtons (V-ATPase e V-PPase), aquoporinas e vÃrios sistemas de transportes ativos e/ou secundÃrios, como os contra-transportadores Na+/H+ vacuolares. As duas bombas de prÃtons transmembranares funcionam como sistemas de transporte primÃrio nas cÃlulas vegetais e ambas as enzimas geram uma diferenÃa de potencial eletroquÃmico de prÃtons atravÃs da membrana vacuolar. Os contra-transportadores vacuolares Na+/H+ utilizam o gradiente eletroquÃmico de prÃtons gerado pelos transportadores primÃrios para transportar Na+ para dentro do vacÃolo. No presente trabalho inicialmente foram determinados os conteÃdos de Ãons Na+ e K+ em raÃzes, hipocÃtilos e folhas e em seguida a anÃlise da expressÃo dos genes das bombas de prÃtons (VHA-A, VHA-E e HVP) e dos contra-transportadores vacuolares (NHX2 e NHX6) em plÃntulas de Vigna unguiculata (L.) Walp cv. Vita 5 submetidas a estresse salino e osmÃtico. As plÃntulas foram crescidas em meio nutritivo na ausÃncia de NaCl e PEG (controle), na presenÃa de 100 mM de NaCl (estresse salino) ou na presenÃa de 200,67 g/L de PEG (estresse osmÃtico). O conteÃdo de Ãons Na+ aumentou em todos os tecidos da planta quando submetidos ao estresse salino (NaCl 100 mM) enquanto que o conteÃdo de Ãons K+ diminuiu na mesma condiÃÃo. A expressÃo dos genes das bombas de prÃtons e dos contra-transportadores vacuolares de folhas e de raÃzes no estresse salino aumentou em todas as condiÃÃes estudadas, porÃm o aumento foi mais expressivo para os genes da V-PPase, NHX2 e NHX6 sugerindo uma regulaÃÃo paralela entre esses genes. JÃ no estresse osmÃtico, os resultados para as folhas mostraram que a expressÃo dos genes VHA-A e VHA-E aumentaram enquanto que os outros genes nÃo sofreram mudanÃas significativas. Nossos resultados sugerem que o estresse salino e o estresse osmÃtico induziram uma regulaÃÃo diferenciada em todos os genes sendo o contra-transportador Na+/H+ importante na homeostase celular quando as plantas foram submetidas ao estresse salino e osmÃtico. / The acummulation of Na+ in the central vacuole represents an important mechanism for plants to cope with salt stress. The vacuolar content and the regulations of their volumes in vegetable cells depend on the activity of transporters and channels located in the tonoplast (vacuolar membrane). The vacuolar membrane possesses two different proton pumps (V-ATPase and V-PPase), aquoporine, and systems of primary and secondary transporters like the vacuolar Na+/H+ antiporter (NHX). The two transmembrane proton pumps work as systems of primary transport in vegetable cells and both enzymes generate a difference of proton electrochemical potential through the vacuolar membrane which can provide energy to antiport system, H+/substrate. The vacuolar Na+/H+ antiporter, uses the electrochemical gradient generated by the primary transporters to pump Na+ ions inward the vacuole. In the present work were first determined the Na+ and K+ content followed by the gene expression of the vacuolar proton pumps (VHA-A, VHA-E and HVP) and the vacuolar antiporters (NHX2 and NHX6) from seedlings of Vigna unguiculata subjected to salt and osmotic stress. The seedlings were grown on nutritive medium in the absence of NaCl and PEG (control condition), presence of NaCl 100 mM (salt stress) or in the presence of PEG 6000 200,67g.L-1 (osmotic stress). The ion Na+ content essay showed an increase in all plant tissues when submitted to salt stress, while the K+ ions decreased in the same condition. The gene expression of the vacuolar proton pumps and the Na+ antiporter from roots and leaves showed an increase in all studied conditions being more expressive to V-PPase, NHX2 and NHX6 suggesting a coordinated regulation of these genes. The results from leaves showed that VHA-A and VHA-E were increased, while the others genes tend to remain constant in the osmotic stress. These results suggest that salt and osmotic stress induced a differential regulation of all studied genes, being the vacuolar Na+ antiporters an important part on keep the cellular homeostasis when the plants were submitted to salt stress

Diss. (sammanfattning) Stockholm : Karol inst., 2004. / Härtill 4 uppsatser.

SOBREIRA, Alana Cecília de Menezes. Estudo da expressão dos genes das bombas de prótons (V-ATPase e V-PPase) e dos contra-transportadores vacuolares (NHX) de Vigna unguiculata (L.) Walp submetidos a estresses abióticos.2009 f. Dissertação (Mestrado em Bioquímica) - Centro de Ciências, Departamento de Bioquímica e Biologia Molecular, Universidade Federal do Ceará, Fortaleza, 2009. / Submitted by JOANA BEZERRA (joanabib@yahoo.com.br) on 2012-11-09T21:07:31Z No. of bitstreams: 1 2009_tese_acmsobreira.pdf: 1938746 bytes, checksum: 064fea08dd8578e2f6b67de140140d86 (MD5) / Approved for entry into archive by Érica Barros(ericabarros@ufc.br) on 2012-11-12T22:49:16Z (GMT) No. of bitstreams: 1 2009_tese_acmsobreira.pdf: 1938746 bytes, checksum: 064fea08dd8578e2f6b67de140140d86 (MD5) / Made available in DSpace on 2012-11-12T22:49:16Z (GMT). No. of bitstreams: 1 2009_tese_acmsobreira.pdf: 1938746 bytes, checksum: 064fea08dd8578e2f6b67de140140d86 (MD5) / The acummulation of Na+ in the central vacuole represents an important mechanism for plants to cope with salt stress. The vacuolar content and the regulations of their volumes in vegetable cells depend on the activity of transporters and channels located in the tonoplast (vacuolar membrane). The vacuolar membrane possesses two different proton pumps (V-ATPase and V-PPase), aquoporine, and systems of primary and secondary transporters like the vacuolar Na+/H+ antiporter (NHX). The two transmembrane proton pumps work as systems of primary transport in vegetable cells and both enzymes generate a difference of proton electrochemical potential through the vacuolar membrane which can provide energy to antiport system, H+/substrate. The vacuolar Na+/H+ antiporter, uses the electrochemical gradient generated by the primary transporters to pump Na+ ions inward the vacuole. In the present work were first determined the Na+ and K+ content followed by the gene expression of the vacuolar proton pumps (VHA-A, VHA-E and HVP) and the vacuolar antiporters (NHX2 and NHX6) from seedlings of Vigna unguiculata subjected to salt and osmotic stress. The seedlings were grown on nutritive medium in the absence of NaCl and PEG (control condition), presence of NaCl 100 mM (salt stress) or in the presence of PEG 6000 200,67g.L-1 (osmotic stress). The ion Na+ content essay showed an increase in all plant tissues when submitted to salt stress, while the K+ ions decreased in the same condition. The gene expression of the vacuolar proton pumps and the Na+ antiporter from roots and leaves showed an increase in all studied conditions being more expressive to V-PPase, NHX2 and NHX6 suggesting a coordinated regulation of these genes. The results from leaves showed that VHA-A and VHA-E were increased, while the others genes tend to remain constant in the osmotic stress. These results suggest that salt and osmotic stress induced a differential regulation of all studied genes, being the vacuolar Na+ antiporters an important part on keep the cellular homeostasis when the plants were submitted to salt stress. / O acúmulo de Na+ no vacúolo central representa um importante mecanismo de defesa de plantas contra o estresse salino. A regulação dos volumes e conteúdos dos vacúolos de células vegetais depende da atividade de transportadores e canais localizados no tonoplasto (membrana vacuolar). A membrana vacuolar possui duas distintas bombas de prótons (V-ATPase e V-PPase), aquoporinas e vários sistemas de transportes ativos e/ou secundários, como os contra-transportadores Na+/H+ vacuolares. As duas bombas de prótons transmembranares funcionam como sistemas de transporte primário nas células vegetais e ambas as enzimas geram uma diferença de potencial eletroquímico de prótons através da membrana vacuolar. Os contra-transportadores vacuolares Na+/H+ utilizam o gradiente eletroquímico de prótons gerado pelos transportadores primários para transportar Na+ para dentro do vacúolo. No presente trabalho inicialmente foram determinados os conteúdos de íons Na+ e K+ em raízes, hipocótilos e folhas e em seguida a análise da expressão dos genes das bombas de prótons (VHA-A, VHA-E e HVP) e dos contra-transportadores vacuolares (NHX2 e NHX6) em plântulas de Vigna unguiculata (L.) Walp cv. Vita 5 submetidas a estresse salino e osmótico. As plântulas foram crescidas em meio nutritivo na ausência de NaCl e PEG (controle), na presença de 100 mM de NaCl (estresse salino) ou na presença de 200,67 g/L de PEG (estresse osmótico). O conteúdo de íons Na+ aumentou em todos os tecidos da planta quando submetidos ao estresse salino (NaCl 100 mM) enquanto que o conteúdo de íons K+ diminuiu na mesma condição. A expressão dos genes das bombas de prótons e dos contra-transportadores vacuolares de folhas e de raízes no estresse salino aumentou em todas as condições estudadas, porém o aumento foi mais expressivo para os genes da V-PPase, NHX2 e NHX6 sugerindo uma regulação paralela entre esses genes. Já no estresse osmótico, os resultados para as folhas mostraram que a expressão dos genes VHA-A e VHA-E aumentaram enquanto que os outros genes não sofreram mudanças significativas. Nossos resultados sugerem que o estresse salino e o estresse osmótico induziram uma regulação diferenciada em todos os genes sendo o contra-transportador Na+/H+ importante na homeostase celular quando as plantas foram submetidas ao estresse salino e osmótico.