SummaryProteins are synthesized by large molecular machines, the ribosomes. Ribosomes consist of two subunits of unequal size, the small subunit and the large subunit. The small subunit contains the decoding site, where the sequence information contained in the messenger RNA (mRNA) is translated into protein sequence, while the large subunit contains the peptidyl transferase center, which catalyzes the peptide bond formation between amino acids of the nascent protein. Translation initiation in eukaryotes requires twelve initiation factors in addition to the ribosome.The aim of this thesis was to determine the crystal structure of an initiation complex of the eukaryotic ribosome in order to make high-resolution structural information on eukaryotic ribosomes and their initiation complexes available. The complex of the Tetrahymena thermophila 40S ribosomal subunit with eukaryotic initiation factor 1 (eIF1) was crystallized in three space groups. Phasing of the dataset with tantalum bromide clusters and subsequent non-crystallographic symmetry multicrystal averaging resulted in electron density maps at 3.9Å that were of sufficient quality to build the entire structure. The resulting model comprises the entire 18S rRNA, 33 ribosomal proteins and initiation factor eIF1.The structure gives insights into the evolution of the eukaryotic ribosome, into signaling at the eukaryotic ribosome and into the function of eIF1 during initiation. The eukaryotic 40S ribosomal subunit contains more proteins that the bacterial 30S subunit, which are engaged in stronger protein-protein interactions and have replaced a bacterial rRNA feature at the beak. Expansion segments of the rRNA are clustered at the back of the 40S subunit. The signaling hub protein RACK1 is an integral part of the ribosome that contacts three ribosomal proteins (rpS3e, rpS16e and rpS17e). The phosphorylation site of ribosomal protein rpS6e, which is a downstream target of the mTOR pathway, is located at the end of a long C-terminal helix, which stretches from the bottom to the back of the 40S subunit. In Tetrahymena, rpS4e, which is directly adjacent to rpS6e, is phosphorylated instead.The study yielded insights into the structural basis of the ability of eIF1 to sense the start codon recognition during translation initiation. Initiation factor eIF1 was found bound to the 40S on top of helix 44, directly below the P site. In the structure eIF1 extends a basic loop into the mRNA channel and is therefore in principle able to sense the conformation of mRNA and tRNA by interaction with their phosphate backbone. The structure presented in this thesis is the first structure of an entire eukaryotic ribosomal subunit and will form the foundation for structure-guided studies of eukaryotic translation. 5 ZusammenfassungProteine werden von den Ribosomen synthetisiert. Ribosomen sind grosse molekulare

The subcellular localization of the epidermal growth factor receptor (EGFR) in polarized epithelial cells profoundly affects the activity of the intracellular signaling pathways activated after EGF ligand binding. Therefore, changes in EGFR localization and signaling are implicated in various human diseases, including different types of cancer. We have performed the first in vivo EGFR localization screen in an animal model by observing the expression of the EGFR ortholog LET-23 in the vulval epithelium of live C. elegans larvae. After systematically testing all genes known to produce an aberrant vulval phenotype, we have identified 81 genes regulating various aspects of EGFR localization and expression. In particular, we have found that ERM-1, the sole C. elegans Ezrin/Radixin/Moesin homolog, regulates EGFR localization and signaling in the vulval cells. ERM-1 interacts with the EGFR at the basolateral plasma membrane in a complex distinct from the previously identified LIN-2/LIN-7/LIN-10 receptor localization complex. We propose that ERM-1 binds to and sequesters basolateral LET-23 EGFR in an actin-rich inactive membrane compartment to restrict receptor mobility and signaling. In this manner, ERM-1 prevents the immediate activation of the entire pool of LET-23 EGFR and permits the generation of a long-lasting inductive signal. The regulation of receptor localization thus serves to fine-tune the temporal activation of intracellular signaling pathways.

The intracellular trafficking of growth factor receptors determines the activity of their downstream signaling pathways. Here, we show that the putative HSP-90 co-chaperone CHP-1 acts as a regulator of EGFR trafficking in C. elegans. Loss of chp-1 causes the retention of the EGFR in the ER and decreases MAPK signaling. CHP-1 is specifically required for EGFR trafficking, as the localization of other transmembrane receptors is unaltered in chp-1(lf) mutants, and the inhibition of hsp-90 or other co-chaperones does not affect EGFR localization. The role of the CHP-1 homolog CHORDC1 during EGFR trafficking is conserved in human cells. Analogous to C. elegans, the response of CHORDC1-deficient A431 cells to EGF stimulation is attenuated, the EGFR accumulates in the ER and ERK2 activity decreases. Although CHP-1 has been proposed to act as a co-chaperone for HSP90, our data indicate that CHP-1 plays an HSP90-independent function in controlling EGFR trafficking through the ER.