Archazolid is a novel and highly efficient inhibitor of the V-ATPase, a ubiquitous membrane energizing protein complex consisting of a cytosolic ATP-hydrolyzing V1 domain and a VO domain which mediates proton translocation via a membrane embedded ring of c subunits. The intention of the present thesis was to characterize the archazolid binding site within the V-ATPase on the molecular level. Initial labeling experiments with 14C-derivatives of archazolid and the Manduca sexta V-ATPase clearly identified the c subunit as binding partner for the inhibitor. Concurrently performed site-directed mutagenesis studies in Saccharomyces cerevisiae as well as continuous wave electron paramagnetic resonance spectroscopy, revealed that the amino acids tyrosine 142 and leucine 144, located within the fourth transmembrane helix of subunit c, contribute to archazolid binding. Strikingly, mutation of these amino acids to either asparagine or isoleucine resulted in a V-ATPase approximately 10-fold more sensitive to the inhibitor, indicating increased inhibitor-target interaction in both cases. In addition, inhibition assays with different derivatives of archazolid suggested close proximity of the C-15 of archazolid and tyrosine 142 of subunit c. Competition assays with NCD-4, a covalently binding inhibitor which specifically targets a highly conserved glutamate within subunit c that is essential for proton translocation, revealed that the archazolid binding site also comprises this amino acid. Since the three amino acids tyrosine 142, leucine 144 and glutamate 137 (positions according to the S. cerevisiae c subunit) form a triangle within the central part of subunit c, the archazolid binding site most likely resides within a single c subunit and the inhibitor probably directly prevents proton translocation by the c ring. A spin labeled derivative of archazolid was used to enlighten the stoichiometry of c subunits within the VO ring. The measurements, performed via double electron electron resonance spectroscopy on spin labeled archazolid bound to the M. sexta V-ATPase, revealed a clear distance distribution that suggested, based on the supposed binding site of archazolid, a number between 9 and 11 subunits in the ring. This number is in line with a previously suggested decameric arrangement of the M. sexta ring and excludes a hexameric structure which is frequently assumed to be valid for V-ATPases.

V-ATPasen sind eukaryotische Protonenpumpen, die viele sekundär aktive Transporte energetisieren und eine Reihe von intrazellulären Kompartimenten ansäuern. In der vorliegenden Doktorarbeit wurde die Interaktion der V-ATPase aus Manduca sexta mit dem Aktin-Zytoskelett untersucht. Nachdem die Co-Lokalisation der V-ATPase mit F-Aktin in den apikalen Membranen der Gobletzellen immunzytochemisch festgestellt worden war, wurde auch die direkte Wechselwirkung zwischen der V-ATPase und den Aktinfilamenten durch Co-Pelletierungsstudien nachgewiesen. Mit Hilfe von Overlayblots wurde gezeigt, dass sowohl die Untereinheit B als auch Untereinheit C die Bindung an Aktin vermitteln. Co-Pelletierung der Aktinfilamente mit rekombinanter Untereinheit C wiesen eine direkte Interaktion zwischen beiden Proteinen nach. Mit rekombinanter Untereinheit C rekonstituierter V1-Komplex hatte eine deutlich höhere Affinität zu Aktinfilamenten als C-freier V1-Komplex. In Overlayblots wurde nachgewiesen, dass die Untereinheit C beide Formen (F- und G-) des Aktins bindet. Die Interaktion mit G-Aktin wurde mit NBD-markiertem Aktin quantifiziert, wobei sich Dissoziationskonstanten von 50-60 nM ergaben; eine Bevorzugung von ATP-Aktin gegenüber ADP-Aktin oder umgekehrt konnte nicht festgestellt werden. Die Untereinheit C beeinflusste die Dynamik des Aktin-Zytoskeletts dadurch, dass sie Aktinfilamente unabhängig vom pH stabilisierte und einen positiven Einfluss auf die Vernetzung von Aktinfilamenten hatte. Die auch im Fluoreszenzmikroskop nachgewiesene Fähigkeit zur Bündelung kann sowohl durch die zwei Aktinbindungsstellen in der Untereinheit C als auch durch ihre Di- und Oligomerisierung unter oxidierenden Bedingungen ermöglicht werden. Mit Hilfe von radioaktivem ATP wurde gezeigt, dass die Untereinheit C durch die Proteinkinase A phosphoryliert werden kann. Diesem Phänomen könnte eine große Bedeutung für die Regulation der Eigenschaften der Untereinheit C zukommen.

Programmed cell death (PCD) is a major regulatory mechanism employed during development and homeostasis. The term PCD was coined to describe the death of the intersegmental muscles (ISMs) of moths at the end of metamorphosis. The timing of ISM death in the Tobacco Hawkmoth, Manduca sexta, is regulated by a fall in the titer of the steroid molting hormone 20-hydroxyecdysone (20E) late on day 17of pupal-adult development. This triggers the release of the peptide hormone, Eclosion Hormone (EH), which mediates its effects via the secondary messenger cGMP. It has been previously demonstrated that ISM death requires de novo gene expression. One induced gene in the ISMs encodes the novel protein Acheron. However, Acheron’s role in PCD is unknown. Acheron is a novel member of the Lupus-Antigen family of RNA binding proteins. In humans, Acheron is expressed in many tissues including the myoepithelial cells in mammary ducts. Analysis of the mammary gland revealed that Acheron mRNA levels were elevated in some basal-like breast cancers in women. Ectopic expression of Acheron in human MDA-MB-231 breast cancer cells results in dramatic elevations in proliferation, angiogenesis and metastasis. Moreover, Acheron expressing MDA-MB-231 cells in mouse xenographs resulted in tumors that were five times larger than control cell tumors. These data suggests that Acheron enhances the growth of some human breast cancers. This thesis describes two primary studies. The first tested the hypothesis that Acheron functions as a survival protein for cells in vitro. MDA-MB-231 cells engineered to express Acheron were challenged with various death-inducing treatments, which act via different signaling pathways, to determine if Acheron expression confers survival. Acheron protects cells from apoptosis induced by nutrient withdrawal, proteosome inhibition, heat stress, mitochondrial toxins, inhibiting cellular respiration, DNA damage, and oxidative stress. The second study tested the hypothesis that Acheron is phosphorylated by a cGMP-dependent kinase in the ISMs when the cells initiate death following adult eclosion. Using a non-radioactive in-vitro kinase assay I observed that Acheron is phosphorylated via a cGMP-dependent kinase, presumed via kinase binding motif predictions to be Protein Kinase G. Furthermore I show that phosphorylation is coupled to Acheron degradation.

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The ecology and evolution of a species is often considered only within the context of pairwise interactions even though a species' distribution and abundance may be determined by interactions with many species within and between trophic levels. Multiple herbivores often share the same host and may interact indirectly by altering the relationships between herbivores, their host plants and their parasitoids. However, the relationships between parasitoids and herbivore hosts have typically been studied in isolation of other herbivore species. I examined how the outcomes of species interactions change when multiple relationships are considered. Chapter 1 examined the potentially conflicting selection pressures Manduca sexta exerts on Nicotiana tabacum (tobacco), since M. sexta has pollinating adults but herbivorous larvae. I demonstrated that high nectar amino acids do not affect floral visitation, but increased oviposition of herbivores on leaves. Thus, the relative costs and benefits of nectar rewards may depend on the community of pollinators and their life histories. In the remaining chapters I examined how feeding on tobacco by the aphid Myzus persicae altered the interactions between a parasitoid (Cotesia congregata) and its hornworm host (M. sexta). Chapter 2 demonstrated that aphids reduced hornworm abundance and parasitism. Changes in hornworm abundance were not due to density-dependent changes in moth oviposition, but the proportion of caterpillars attacked by parasitoids was inversely density dependent with hornworm density. Chapter 3 examined whether changes in hornworm abundance and parasitism reflected aphid-induced changes in host plant quality or volatile emissions. Aphids increased hornworm mortality, did not affect parasitoid performance, and increased parasitoid search time. In combination with Chapter 2, results suggest that aphids can mediate parasitoid-caterpillar interactions through changes in host plants that reduce hornworm survival and alter parasitoid behavior. Chapter 4 addressed how the outcome of interactions that are altered by aphids changed with spatial scale, and found no effect spatial scale on hornworm abundance and parasitism. In this system, aphids alter parasitoid-caterpillar interactions through changes in plant quality that reduce hornworm performance and abundance, and in turn, influence parasitoid attack. This work demonstrates that the outcome of multispecies interactions may not be predictable from pairwise interactions. / Ph. D.

Thèse numérisée par la Division de la gestion de documents et des archives de l'Université de Montréal.

Mémoire numérisé par la Direction des bibliothèques de l'Université de Montréal.