A growing repertoire of proteins is involved in the molecular events surrounding clathrin-coated vesicle (CCV) formation, which is seminal to the regeneration of synaptic vesicles in nerve terminals, as well as to more universal membrane trafficking mechanisms. Amphiphysin-I and endophilin-I are two nerve terminal-enriched proteins thought to function in CCV formation. In the first study of this thesis, we identified a novel amphiphysin-I isoform (termed amphiphysin-II), through a homology search of the expressed sequence tag (EST) database. We demonstrated that like amphiphysin-I, amphiphysin-II is highly enriched in nerve terminals, where it interacts with dynamin-I and synaptojanin-I through its SH3 domain. Further, both amphiphysins were found to bind directly to the vesicle coat protein, clathrin, in an SH3 domain-independent manner. / In the next two studies, we cloned a number of amphiphysin-II splice variants that were found to display differential clathrin binding. The use of these splice variants, together with amphiphysin-II truncation and deletion mutants, allowed us to identify two distinct clathrin-binding domains that are also conserved in amphiphysin-I. Using these amphiphysin constructs we also determined that the amphiphysins are targeted to the plasma membrane through their N-termini, which also appears to be important in facilitating amphiphysin oligomerization. / In the final study of this thesis, we turned our attention to a related endocytic SH3 domain-containing protein, endophilin-I. In an attempt to further understand its complete role within the cell, we screened a brain expression library with its SH3 domain, and identified the rat germinal center kinase-like kinase (rGLK), a member of the germinal center kinase (GCK) family of c-jun N-terminal kinase (JNK) activating enzymes, as a novel endophilin-I-binding partner. We characterized the interaction both in vitro and in vivo and determined the sequence on rGLK that mediates the interaction. Importantly, overexpression of full-length endophilin-I was found to enhance rGLK-mediated JNK activation, whereas fragments of endophilin-I blocked JNK activation, suggesting a novel role for endophilin-I in JNK intracellular signaling. / Taken together, the data in this thesis not only adds to the growing list of endocytic accessory proteins, but also contributes to our understanding of their individual molecular interactions and functions within the cell.
| Identifer | oai:union.ndltd.org:LACETR/oai:collectionscanada.gc.ca:QMM.38088 |
| Date | January 2001 |
| Creators | Ramjaun, Antoine. |
| Contributors | McPherson, Peter (advisor) |
| Publisher | McGill University |
| Source Sets | Library and Archives Canada ETDs Repository / Centre d'archives des thèses électroniques de Bibliothèque et Archives Canada |
| Language | English |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Format | application/pdf |
| Coverage | Doctor of Philosophy (Division of Neuroscience.) |
| Rights | All items in eScholarship@McGill are protected by copyright with all rights reserved unless otherwise indicated. |
| Relation | alephsysno: 001837494, proquestno: NQ75671, Theses scanned by UMI/ProQuest. |
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