Tierärztliche Hochsch., Diss., 2005--Hannover.

Universiẗat, Diss., 2001--Köln.

München, Techn. Univ., Diss., 2002.

University, Diss., 2003--Düsseldorf. / Parallelt.: The role of annexin A1 in the activation and transport of the EGF receptor. Text engl.

Universiẗat, Diss., 2005--Bielefeld.

In der vorliegenden Arbeit konnte am rekombinanten Tom40 aus Neurospora crassa und aus Saccharomyces cerevisiae gezeigt werden, dass in beiden Spezies ein einziges Tom40-Molekül in der Lage ist ein membrandurchspannendes Beta-Barrel Protein aus 16 transmembranen Beta-Strängen auszubilden. Tom40 aus beiden Spezies bildet einen kationenselektiven Kanal dessen charakteristischer Hauptleitwert eine gute Übereinstimmung zu publizierten Werten zeigt und genau einer porenbildenden Einheit im TOM-Komplex entspricht. Ein generell unterschiedliches Verhalten von Tom40 durch eine Fehlfaltung des zuvor denaturierten Proteins, kann also ausgeschlossen werden. Der Kanal interagiert seitenabhängig mit aminoterminalen mitochondrialen Präsequenzen. Die Spezifität der Wechselwirkung mit Tom40 ist jedoch geringer als die mit dem TOM-Komplex. In elektrophysiologischen Untersuchungen des SAM-Komplexes aus Saccharomyces cerevisiae konnte gezeigt werden, dass Sam50 die charakteristische porenbildende Einheit im Komplex darstellt. Sam50 aus Saccharomyces cerevisiae und Homo sapiens bilden kationenselektive Kanäle, wobei der Hauptleitwert im humanen Protein signifikant größer als im Hefe-Protein ist. Der evolutiv konservierte C-Terminus von Sam50 reicht hierbei aus diese Pore zu bilden, ist aber im Vergleich zum Volllängenprotein stark im Schaltverhalten beeinträchtigt. Die elektrophysiologischen Eigenschaften von Sam50, spiegeln exemplarisch die Werte verwandter Poren der Omp85-Familie wider. Der Sam50-Kanal wird im SAM-Komplex durch die ebenfalls essenzielle Komponente Sam35 reguliert. Es konnte gezeigt werden, dass sich durch eine Interaktion von Sam35-Protein mit einem konservierten Beta-Signalpeptid der Komplex in einen aktiven Zustand größerer Leitfähigkeit überführen lässt, der sich durch ein dynamisches Schaltverhalten und das Auftreten multipler Leitwerte auszeichnet, wobei im Komplex mehrere Porenproteine am Stromfluss beteiligt sind.

The structure and functions of lipid rafts and the mechanisms of intracellular membrane trafficking are major topics in current cell biological research. Rafts have been proposed to act as sorting platforms during biosynthetic transport, especially along pathways that deliver proteins to the apical membrane of polarised cells. Based on this, the aim of this work was to contribute to the understanding of apical sorting in epithelial cells. The study of how lipid rafts are structured has been hampered by the scarcity of techniques for their purification. Rafts are thought to be partially resistant to solubilisation by mild detergents, which has made the isolation of detergent-resistant membranes (DRMs) the primary method to characterise them biochemically. While a growing number of detergents is being used to prepare DRMs, it is not clear what can be inferred about the native structure of cell membranes from the composition of different DRMs. This issue was addressed by an analysis of DRMs prepared with a variety of mild detergents. The protein and lipid content of different DRMs from two cell lines, Madin-Darby canine kidney (MDCK) and Jurkat cells, was compared. It was shown that the detergents differed considerably in their ability to selectively solubilise membrane proteins and lipids. These results make it unlikely that different DRMs reflect the same underlying principle of membrane organisation. Another obstacle for understanding apical sorting is that the evidence implicating certain proteins in this process has come from various disparate approaches. It would be helpful to re-examine the putative components of the apical sorting machinery in a single experimental system. To this end, a retroviral system for RNA interference (RNAi) in MDCK cells was established. Efficient suppression of thirteen genes was achieved by retroviral co-expression of short hairpin RNAs and a selectable marker. In addition, the system was extended to simultaneously target two genes, giving rise to double knockdowns.Retroviral RNAi was applied to deplete proteins implicated in apical sorting. Surprisingly, none of the knockdowns analysed caused defects in surface delivery of influenza virus hemagglutinin, a common marker protein for apical transport. Therefore, none of the proteins examined is absolutely required for transport to the apical membrane of MDCK cells. Cells may adapt to the depletion of proteins involved in membrane trafficking by activating alternative pathways. To avoid such adaptation, a visual transport assay was established. It is based on the adenoviral expression of fluorescent marker proteins whose surface transport can be followed microscopically as soon as RNAi has become effective. With this assay, it should now be possible to screen the knockdowns for defects in surface transport. Taken together, this work has provided a number of experimental tools for the study of membrane trafficking in epithelial cells. First, the biochemical analysis of DRMs highlighted that DRMs obtained with different detergents are unlikely to correspond to distinct types of membrane microdomains in cell membranes. Second, the retroviral RNAi system should be valuable for defining the function of proteins, not only in membrane transport, but also in processes like epithelial polarisation. Third, the visual assay for monitoring the surface transport of adenovirally expressed marker proteins should be suitable to detect defects in polarised sorting.

Sekretorische Proteine und Proteine der Kompartimente des sekretorischen Transportweges müssen die Membran des Endoplasmatischen Retikulums überqueren, um an ihren Wirkungsort zu gelangen. In der vorliegenden Arbeit wurden frühe Schritte des kotranslationalen Transports von Proteinen durch die ER-Membran untersucht. Signalsequenzen leiten diese Proteine als ribosomengebundene Intermediate an die ER-Membran. Die Ribosomen binden dort an den Sec61p-Komplex, der als Ribosomenrezeptor wirkt und gleichzeitig den proteinleitenden Kanal in der Membran bildet. Die Assoziation von Ribosomen mit dem Sec61p-Komplex verläuft in zwei Phasen. Die initiale Bindung ist sensitiv gegenüber hohen Salzkonzentrationen. Die Ribosomenbindung wird salzresistent, wenn die naszierende Kette in den Kanal inseriert und der Sec61p-Komplex die Signalsequenz erkennt. Sowohl Ribosomen ohne naszierende Kette als auch Ribosomen, die Proteine ohne Signalsequenzen synthetisieren, sind nur zur initialen salz-sensitiven Bindung an den Sec61p-Komplex fähig. Signalsequenzen interagieren im Cytosol mit SRP (engl.: Signal Recognition Particle). In dieser Arbeit wurde gezeigt, daß Signalsequenzen außerdem von Calmodulin gebunden werden. SRP und Calmodulin scheinen für die Interaktion mit Signalsequenzen einen ähnlichen Mechanismus zu benutzen, der wiederum mit der Signalsequenzerkennung durch den Sec61p-Komplex verwandt ist. Alle Ribosomen, unabhängig davon ob und welches Protein sie translatieren, können mit dem Sec61p-Komplex interagieren und daher um Bindungsplätze an der ER-Membran kompetitieren. Wenn SRP an die Signalsequenz einer naszierenden Kette gebunden ist, erhalten diese Ribosomen jedoch einen Vorteil in der Kompetition. Nur sie können Ribosomen ohne naszierende Kette oder Ribosomen, die ein cytosolisches Protein translatieren, vom Sec61p-Komplex verdrängen und sich selbst dann einen Translokationsort sichern, wenn alle Bindingsplätze an der Membran besetzt sind. In der vorliegenden Arbeit wurden dreidimensionale Strukturen von Komplexen aus Ribosom und proteinleitendem Translokationskanal vorgestellt, die der ersten und zweiten Phase der Ribosomenbindung entsprechen. Überraschenderweise unterscheiden sich diese beiden Stadien strukturell nicht. In beiden Fällen existieren definierte Verbindungen zwischen Ribosom und Kanal, die eine Lücke von etwa 20 Angström zwischen dem Ribosom und der Membranoberfläche überbrücken. Die Lücke stellt eine Verbindung zum Cytosol her, die eventuell dazu dient, naszierende Ketten ins Cytosol zu entlassen, wenn diese nicht ins Lumen des ER transportiert werden sollen. Weiterhin zeigen wir, daß der Kanal in nativen Membranen größer ist als der Kanal, der nur aus gereinigtem Sec61p-Komplex besteht. Dieser größere Kanal besitzt eine zusätzliche lumenale Domäne, die von der Oligosaccharyltransferase oder vom TRAP-Komplex gebildet wird. / The first step in the secretory pathway is the translocation of proteins across the membrane of the endoplasmic reticulum (ER). In this thesis project, early stages of cotranslational protein translocation in mammalian cells were studied. Proteins following the secretory pathway are targeted to the ER as ribosome-nascent chain complexes by their N-terminal hydrophobic signal sequences. The nascent chain is translocated across the ER membrane through a hydrophilic channel formed by the Sec61p complex, which also functions as the ribosome receptor. The initial binding of ribosomes to the ER membrane is salt-sensitive. After insertion of the nascent chain into the translocation channel and signal sequence recognition by the Sec61p complex, the ribosome is bound in a salt-resistant manner. The membrane binding of ribosomes lacking nascent chains and of ribosomes carrying nascent chains without signal sequences is always salt-sensitive. It is known that in the cytosol, the signal sequence binds to the signal recognition particle (SRP). Here we show that another cytosolic factor, the small regulatory protein calmodulin, can interact with signal sequences. Our data suggest that both SRP and calmodulin use a similar mechanism for substrate binding and recognition. In fact, this mechanism may be related to signal sequence recognition by the Sec61p complex. Previously the question has been raised of how efficient targeting of ribosome-nascent chain complexes (RNCs) carrying a signal sequence is possible when all ribosomes, regardless of the presence or nature of a nascent chain, can bind to the Sec61p complex. We demonstrate that all ribosomes compete for common binding sites at the ER membrane and that SRP functions as a positive effector to give RNCs carrying a signal sequence an advantage over other ribosomes. RNCs with a signal sequence and bound SRP can displace ribosomes without a nascent chain and ribosomes synthesizing cytosolic proteins from the membrane and can therefore secure a translocation site even when all ribosome binding sites at the ER membrane are occupied. A structural analysis by single particle cryo electron microscopy revealed that ribosome-translocation channel complexes do not differ in the salt-sensitive or the salt-resistant stage of ribosome binding to the ER membrane. Furthermore our data show that the ribosome is linked to the translocation channel by a discrete number of connections. Even in the presence of a translocating nascent chain the ribosome-membrane junction is not completely sealed towards the cytosol. Instead, a sizable gap exists between the ribosome and the surface of the membrane that may allow nascent polypeptide chains to enter the cytosol when their translocation across the ER membrane is prevented. We also show that translocation channels derived from native microsomes are larger than channels derived from purified Sec61p complex. These larger channels contain a wider central pore and an additional lumenal domain, which is formed by the oligosaccharyl transferase or by the TRAP complex.

No description available.

The structure and functions of lipid rafts and the mechanisms of intracellular membrane trafficking are major topics in current cell biological research. Rafts have been proposed to act as sorting platforms during biosynthetic transport, especially along pathways that deliver proteins to the apical membrane of polarised cells. Based on this, the aim of this work was to contribute to the understanding of apical sorting in epithelial cells. The study of how lipid rafts are structured has been hampered by the scarcity of techniques for their purification. Rafts are thought to be partially resistant to solubilisation by mild detergents, which has made the isolation of detergent-resistant membranes (DRMs) the primary method to characterise them biochemically. While a growing number of detergents is being used to prepare DRMs, it is not clear what can be inferred about the native structure of cell membranes from the composition of different DRMs. This issue was addressed by an analysis of DRMs prepared with a variety of mild detergents. The protein and lipid content of different DRMs from two cell lines, Madin-Darby canine kidney (MDCK) and Jurkat cells, was compared. It was shown that the detergents differed considerably in their ability to selectively solubilise membrane proteins and lipids. These results make it unlikely that different DRMs reflect the same underlying principle of membrane organisation. Another obstacle for understanding apical sorting is that the evidence implicating certain proteins in this process has come from various disparate approaches. It would be helpful to re-examine the putative components of the apical sorting machinery in a single experimental system. To this end, a retroviral system for RNA interference (RNAi) in MDCK cells was established. Efficient suppression of thirteen genes was achieved by retroviral co-expression of short hairpin RNAs and a selectable marker. In addition, the system was extended to simultaneously target two genes, giving rise to double knockdowns.Retroviral RNAi was applied to deplete proteins implicated in apical sorting. Surprisingly, none of the knockdowns analysed caused defects in surface delivery of influenza virus hemagglutinin, a common marker protein for apical transport. Therefore, none of the proteins examined is absolutely required for transport to the apical membrane of MDCK cells. Cells may adapt to the depletion of proteins involved in membrane trafficking by activating alternative pathways. To avoid such adaptation, a visual transport assay was established. It is based on the adenoviral expression of fluorescent marker proteins whose surface transport can be followed microscopically as soon as RNAi has become effective. With this assay, it should now be possible to screen the knockdowns for defects in surface transport. Taken together, this work has provided a number of experimental tools for the study of membrane trafficking in epithelial cells. First, the biochemical analysis of DRMs highlighted that DRMs obtained with different detergents are unlikely to correspond to distinct types of membrane microdomains in cell membranes. Second, the retroviral RNAi system should be valuable for defining the function of proteins, not only in membrane transport, but also in processes like epithelial polarisation. Third, the visual assay for monitoring the surface transport of adenovirally expressed marker proteins should be suitable to detect defects in polarised sorting.