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Título: Characterization of protein-protein interactions involved in clathrin-mediated budding at the plasma membrane and the trans-Golgi network
Autores: Wasiak, Sylwia
Fecha: 2004
Publicador: McGill University - MCGILL
Fuente:
Tipo: Electronic Thesis or Dissertation
Tema: Biology, Cell.
Descripción: Clathrin-mediated membrane budding mainly occurs at the cell surface, where it drives the formation of endocytic vesicles necessary for the internalization of membrane proteins, and at the trans-Golgi network (TGN), where it mediates the trafficking of cargo proteins from the TGN to the endosomal/lysosomal system. The general objective of my doctoral research was to study adaptor and accessory proteins that play structural and regulatory roles in the formation of clathrin-coated vesicles (CCVs). This was achieved principally through the characterization of two protein complexes: a first, which functions at the cell surface and a second, which regulates vesicle formation at the TGN/endosome.
First, we discovered that the endocytic protein PACSIN 1 is a binding partner for the signaling molecule mSos1. Further analysis of this interaction revealed that both proteins form a complex in vivo, that they co-localize at actin-rich sites, and that their interaction is regulated by phosphorylation. These data strengthen the link between endocytosis, signaling and actin cytoskeleton dynamics and provide the basis for further investigation of the role of this protein complex.
Second, we used subcellular proteomics to identify novel components of brain-derived CCVs. Among these was an ENTH domain-containing protein, which we named enthoprotin. Further work demonstrated that enthoprotin localizes to CCVs, interacts with clathrin adaptors AP-1 and GGA2 at the TGN, and stimulates clathrin assembly in vitro. Altogether, our data suggest a role for enthoprotin in clathrin coat assembly on internal membranes. Analysis of the enthoprotin protein sequence led to the discovery of two peptide motifs responsible for interactions with AP-1 and GGA2. We used alanine-scan mutagenesis and nuclear magnetic resonance to biochemically and structurally characterize the high-affinity site responsible for the interaction between enthoprotin and the TGN adaptors. This experimental approach, coupled to alignments, allowed us to deduce an AP-1/GGA-binding consensus motif that can be used towards the identification of novel TGN adaptor-binding partners. Altogether, my doctoral research contributed to furthering knowledge of the mechanisms that underlie clathrin-mediated membrane budding.
Idioma: en