The lysosomal-associated protein transmembrane 5 (LAPTM5) is a protein preferentially expressed in the immune cells. LAPTM5 was isolated in our laboratory as an interacting partner of the ubiquitin ligase, Nedd4. The intracellular domains of LAPTM5 contain three PY (L/PPxY) motifs, which bind the WW domains of Nedd4, as well as a ubiquitin-interacting motif (UIM). Here, I show that sorting of LAPTM5 from the Golgi to the lysosomes requires its association with Nedd4 and the clathrin adaptor GGA3. Although the Nedd4-LAPTM5 interaction leads to the ubiquitination of LAPTM5, this event is not necessary for LAPTM5 sorting. Rather, the Nedd4-LAPTM5 complex recruits ubiquitinated GGA3, which binds the UIM of LAPTM5. Hence, I propose a novel mechanism by which the ubiquitin ligase Nedd4, via interactions with GGA3 and cargo (LAPTM5), regulates cargo trafficking to the lysosome without requiring cargo ubiquitination. Because nothing was known about the biological function of LAPTM5, at the beginning of my Ph.D. training, I set out to determine the role of LAPTM5 in the innate immune cells. I demonstrate that LAPTM5 interacts with kinesin, a motor protein previously implicated in the anterograde movement of the late endosomal/lysosomal compartments. In dendritic cells, I show that upon maturation LAPTM5 is present within endolysosomal tubules formed by class II MHC molecules. Although I find that LAPTM5 is dispensable for the translocation of peptide-loaded MHC II molecules to the cell surface, this study extends our knowledge of the repertoire of proteins present within tubules formed by the MHC II compartments in activated dendritic cells. In macrophages, I demonstrate that LAPTM5 acts as a positive regulator of NFκB and MAPK signaling cascades, and promotes efficient proinflammatory cytokine production in response to several inducers of macrophage activation. During TNFα stimulation, LAPTM5 is required for proper initiation of NFκB signaling by acting at the receptor-proximate level. Thus, my findings indicate that LAPTM5 is an important component of inflammatory signaling cascades in macrophages and highlight a role for the endosomal/lysosomal system in regulating these cascades. Collectively, the work presented in this thesis broadens our understanding of lysosomal membrane protein sorting and function.

The lysosomal-associated protein transmembrane 5 (LAPTM5) is a protein preferentially expressed in the immune cells. LAPTM5 was isolated in our laboratory as an interacting partner of the ubiquitin ligase, Nedd4. The intracellular domains of LAPTM5 contain three PY (L/PPxY) motifs, which bind the WW domains of Nedd4, as well as a ubiquitin-interacting motif (UIM). Here, I show that sorting of LAPTM5 from the Golgi to the lysosomes requires its association with Nedd4 and the clathrin adaptor GGA3. Although the Nedd4-LAPTM5 interaction leads to the ubiquitination of LAPTM5, this event is not necessary for LAPTM5 sorting. Rather, the Nedd4-LAPTM5 complex recruits ubiquitinated GGA3, which binds the UIM of LAPTM5. Hence, I propose a novel mechanism by which the ubiquitin ligase Nedd4, via interactions with GGA3 and cargo (LAPTM5), regulates cargo trafficking to the lysosome without requiring cargo ubiquitination. Because nothing was known about the biological function of LAPTM5, at the beginning of my Ph.D. training, I set out to determine the role of LAPTM5 in the innate immune cells. I demonstrate that LAPTM5 interacts with kinesin, a motor protein previously implicated in the anterograde movement of the late endosomal/lysosomal compartments. In dendritic cells, I show that upon maturation LAPTM5 is present within endolysosomal tubules formed by class II MHC molecules. Although I find that LAPTM5 is dispensable for the translocation of peptide-loaded MHC II molecules to the cell surface, this study extends our knowledge of the repertoire of proteins present within tubules formed by the MHC II compartments in activated dendritic cells. In macrophages, I demonstrate that LAPTM5 acts as a positive regulator of NFκB and MAPK signaling cascades, and promotes efficient proinflammatory cytokine production in response to several inducers of macrophage activation. During TNFα stimulation, LAPTM5 is required for proper initiation of NFκB signaling by acting at the receptor-proximate level. Thus, my findings indicate that LAPTM5 is an important component of inflammatory signaling cascades in macrophages and highlight a role for the endosomal/lysosomal system in regulating these cascades. Collectively, the work presented in this thesis broadens our understanding of lysosomal membrane protein sorting and function.

Programmierter Zelltod (PCD) oder Apoptose ist ein universeller biologischer Prozess, der in multizellulären Organismen essentiell ist für die Differenzierung und Homöostase von Geweben. Bei der Entwicklung eines funktionsfähigen zellulären Immunsystems können autoreaktive Zellen entstehen. Die negative Selektion von unreifen, autoreaktiven B-Zellen erfolgt durch IgM-vermittelte Apoptose und ist von de novo Transkription abhängig. Die genauen Mechanismen der IgM-vermittelten Apoptose und die involvierten Genprodukte sind nur unzureichend charakterisiert. Zur Identifikation Apoptose-assoziierter Gene in B-Zellen wurden die Differential Display-Analyse durchgeführt und die Expressionsmuster apoptotischer und nicht-apoptotischer BL60-Zellen untersucht. Es wurden 38 differentielle Fragmente identifiziert und kloniert. Sequenzanalysen ergaben Homologien eines Fragments mit LAPTM5, einem lysosomal-assoziierten Transmembran-Protein mit vorwiegender Expression in hämatopoetischem Gewebe. Northern Blot-Analysen zeigten 2 Stunden nach Inkubation von BL-60-Zellen mit anti-IgM einen Anstieg der Genexpression von LAPTM5. Das LAPTM5-Gen liegt auf Chromosom 1 (1p34), ist evolutionär konserviert und besitzt keine Homologien zu bekannten Genen. Die Untersuchung der gewebespezifischen Expression von LAPTM5 ergab neben der hohen Expressionsrate in hämatopoetischem Gewebe eine sehr starke Expression in Skelett- und Herzmuskelgewebe. Elektronenmikroskopisch zeigte sich eine Lokalisation des Proteins in späten Endosomen und Lysosomen. Daneben konnte LAPTM5 auch auf der Oberfläche von BL60-Zellen detektiert werden. Während des apoptotischen Prozesses bleibt die Menge an LAPTM5 auf der Zelloberfläche konstant. Zur weiteren Charakterisierung von LAPTM5 und anderen Kandidaten-Genen wurde ein episomales Expressions- und Selektionssystem entwickelt. Cotransfektionsanalysen unter Verwendung eines GFP (green fluorescent protein)- Konstrukts zeigten, dass die aufgereinigten Zellen zu 80% das interessierende Gen exprimieren und die Expression länger als vier Wochen anhält. / Programmed cell death (PCD) or apoptosis is a key feature of normal development and tissue homeostasis. In the development of a functional immunesystem the occurence of autoreactive cells is tightly controlled and prevented by apoptosis. The negative selection of autoreactive immature B cells after encountering self antigen occures via surface IgM (sIgM) mediated apoptosis and depends on de novo gene transcription. The precise mechanism of this process and the possible involvement of different genes in the regulation of sIgM-mediated cell death is not understood so far. In order to identify genes associated with B cell apoptosis Differential Display RT-PCR (DD) was performed to analyze sIgM-mediated apoptosis in the human Burkitt lymphoma line BL60. The expression patterns of apoptotic and non-apoptotic cells were investigated and 38 differentially expressed gene fragments were found. Subsequent northern blot analysis showed that LAPTM5, a lysosomal associated membrane protein preferential ly expressed in adult hematopoietic tissue, is up-regulated 2 hours after induction of apoptosis. LAPTM5 is a protein with five transmembrane domains, it is conserved during evolution and the gene, mapping to chromosome 1p34, has no homology to known genes. In contrast to earlier data a very high expression of the protein was detected not only in hematopoietic tissue but also in skeletal muscle and heart muscle. Electronmicroscopy was performed to investigate the subcellular localization in detail and showed that LAPTM5 is mainly present in late endosomes and lysosomes. FACS analysis revealed a surface expression of LAPTM5 and a constant amount of LAPTM5 at the surface during IgM mediated apoptosis. To further investigate the functional role of candidate genes during apoptosis an episomal expression and selection system was established. Cotransfection analysis with GFP (green fluorescent protein) showed that 80% of the separeted cells express the gene of interest for at least four weeks.