Clathrin-mediated endocytosis (CME) plays a central role in the regulation of multiple cellular processes such as uptake of nutrients, recycling of housekeeping receptors and transporters, as well as for cell surface removal and downregulation of signaling receptors. Once endocytosed, cargo passes through early endosomes where sorting mechanisms traffic the cargo to the recycling pathway or to degradation in the lysosome. The general objectives of this doctoral research were to identify and characterize new players of the clathrin-mediated trafficking pathway to reveal differences between the abundant components of the trafficking machinery in two tissues, and to examine the mechanisms of endosomal sorting. / We used subcellular proteomics to reveal the differences in components of clathrin-coated vesicles (CCVs) isolated from brain and liver and to identify new molecules participating in clathrin trafficking. We demonstrated that the ratio between the clathrin adaptor proteins AP-1 and AP-2 is different in brain and liver, which indicates differential functions between the two tissues. We also discovered that clathrin-light chains, which have been proposed for many years to be regulatory proteins in the assembly of CCVs, were less abundant relative to clathrin-heavy chain in liver and in non-brain tissues compared to brain. / We identified a new DnaJ domain-containing protein, receptor-mediated endocytosis protein 8 (RME-8) that was detected in liver CCVs specifically. Further characterization revealed that the RME-8 DnaJ domain binds to the chaperone heat-shock cognate 70 (Hsc70) in an ATP-dependent manner. RME-8 is a ubiquitously expressed protein that tightly associates with endosomes, and its depletion causes intracellular trafficking defects. Moreover, we demonstrated that RME-8 depletion also leads to a decrease in levels of epidermal growth factor receptor (EGFR), as a result of an increase in EGFR degradation. RME-8 knock-down causes decreased EGFR levels even in cancer cells lines where EGFR is generally protected from degradation. / Globally this doctoral project revealed new insights on specialized functions for c1athrin-mediated trafficking in different tissues and allowed the identification and characterization of a novel protein implicated in sorting decisions occurring on endosomes.

No description available.

Utilization of iron by hemoglobin-producing cells is highly efficient. The acquisition of iron from plasma requires the binding of diferric transferrin (Tf) to its cognate receptor (Tf-R) on the erythroid cell membrane, followed by internalization of the Tf - Tf-R complexes via receptor-mediated endocytosis. Through a poorly understood mechanism, iron is targeted to mitochondria, the site of heme biosynthesis. We believe that a direct interaction between iron-containing endosomes and mitochondria is essential for iron transfer to mitochondria and its efficient incorporation into heme. / In order to illustrate the interaction between endosomes and mitochondria, we have employed flow cytometry. Flow cytometry analysis of reticulocytes (erythrocyte precursors which still synthesize hemoglobin) stained with fluorescent dyes specific to mitochondria and endosomes revealed three distinct populations: mitochondria, endosomes and a population labeled with both dyes. This double-labeled population suggests a population composed of endosomes associated with mitochondria. Using non-fluorescent diferric-Tf, we were able to remove the double population, leaving only the endosomal and the mitochondrial population. This finding has confirmed that the double population is the result of the interaction between the two organelles. / Additionally, we established a cell-free assay consisting of fluorescent mitochondria and endosomes isolated from erythroid cells. Using confocal microscopy, we demonstrated a colocalization between the two organelles. We repeated the assay using fluorescent mitochondria and endosomes isolated from HeLa spinner cells. Using the mitochondrial uncoupler CCCP, we were able to significantly reduce the colocalization between the two organelles, indicating that the interaction between the organelles is specific and that the mitochondrial potential is a requirement for organellar interaction. / Based on our results from flow cytometry and confocal microscopy, we conclude that a specific and direct interaction exists between the two organelles.

No description available.