| dc.description.abstracteng | Although the basic principles of the intracellular trafficking of proteins and lipids within the secretory apparatus have been established, the precise regulation of molecular membrane transport events is still not well understood. The evolutionarily conserved Golgi membrane protein UNC-50 has been shown to be essential for cell surface expression of levamisole-sensitive acetylcholine receptors (Lev-nAChRs) in C. elegans. The yeast UNC-50 ortholog Gmh1p was demonstrated to interact with the guanine-nucleotide exchange factors for ADP-ribosylation factor GTPases (ARF-GEFs) Gea1p and Gea2p, which are required for ARF GTPase dependent transport vesicle budding and sorting. These data suggested that UNC-50/Gmh1p might be the membrane receptor that recruits Gea1/2p ARF-GEFs to the Golgi. However, the very mild phenotypes of unc-50/gmh1 mutants yeast and C. elegans argue against a general transport function for UNC-50/Gmh1p. In Chapter I, in order to find the evolutionarily conserved function of UNC-50/Gmh1p, we systematically characterized the intracellular transport unc-50/gmh1 mutants in the two model organisms the yeast S. cerevisiae and C. elegans. We determined that the loss of UNC-50/Gmh1p function results in an enhanced traffic-flow to lysosomes/vacuoles. Yeast gmh1 mutants display an enhanced delivery of the membrane dye FM 4-64 to the vacuole as well as recycling defects of v-SNAREs Snc2p back to the plasma membrane. In support of these observations, we also detected an enhanced recruitment of endo-lysosomal markers as well as lysosomal enlargements in unc-50/gmh1 mutants in yeast as well as in C. elegans. This is in agreement with a function of UNC-50/Gmh1p in endosome-to-Golgi retrograde trafficking, which has previously been shown in synthetic lethal screens. An electron microscopic analysis revealed the accumulation of large lipid filled vesicles and mitochondrial alterations in C. elegans unc-50 mutants. This further suggested that UNC-50/Gmh1p has a much broader role for cellular metabolism. In Chapter II, we analyze UNC-50/Gmh1p’s general function for cellular physiology by unbiased whole proteome as well as whole metabolome quantitative analysis. By metabolic SILAC labeling and quantitative proteomics, we identified proteins that have been up or down-regulated in unc-50 mutants. This analysis revealed that anterograde transport promoting proteins such as VPS-32.1, UNC-18 and GPB-1 as well as actin-myosin motility components ACT-2 and NMY-2 were up-regulated, whereas retrograde transport proteins such as dynein-RAB-6-mediated BICD-1 and lipid binding precursors VIT-3 and VIT-5 were down-regulated. In order to identify UNC-50 interacting proteins by SILAC labeling and mass spectrometry we established the BioID method in C. elegans. This method allows to in-vivo biotinylate proteins in close proximity of target protein tagged with a mutated form of the bacterial biotin-ligase BirA. The BioID method allows the in-vivo labeling of transient or weak interactions that occur during intracellular trafficking events, which otherwise would be difficult to isolate by classical CoIP purifications. By expressing BirA-tagged anti-GFP-nanobodies (αGFPNB) we also show that GFP-fusion protein can be tagged in-vivo, extending the usability of the BioID method. This approach using αGFPNB enabled us to determine the topology of GFP-tagged integral membrane proteins in-vivo. Thus, the proteomic tools established in this work will allow us to further define the UNC-50 dependent trafficking pathway at the molecular as well as cellular level. | de |