In Leishmania glycosomes compartmentalize a variety of essential biochemical and metabolic pathways required for parasite viability. Biogenesis and maintenance of glycosomes involves a family of proteins called peroxins, however the molecular mechanisms governing the early events of this pathway have not been fully established. / A structural-functional analysis of the receptor LdPEX5 revealed the formation of a tetrameric structure stabilized by coiled-coil motifs. A biophysical approach showed that the tetrameric structure of LdPEX5 dissociates to dimers upon binding to the PTS1 ligand. However, the tetrameric LdPEX5 is more thermodynamically favorable to bind. Lastly, LdPEX14 modulates the LdPEX5-PTS1 interaction as the presence of LdPEX14 decreases the Kd of LdPEX5-PTS1 by ∼10 folds. / PTS1-loaded LdPEX5 docks onto the glycosomal membrane via the membrane-associated LdPEX14, an interaction that was characterized by molecular mapping and biophysical approaches. In higher eukaryotes this PEX5-PEX14 interaction involves conserved WXXXF/Y pentapeptide motifs found on PEX5 and a signature sequence found on PEX14. These three repeats in LdPEX5 do not appear to be crucial for interaction with LdPEX14 thus suggesting a unique molecular mechanism mediating the docking event. On the other hand, the conserved signature motif is crucial for interaction with LdPEX5. / The topology and nature of the interaction of LdPEX14 with the glycosomal membrane is not clear. In vivo expression of FLAG-LdPEX14-HA together with proteinase digestion confirmed that both N- and C-termini are cytosolic facing. Mapping analysis revealed that the first 63 N-terminal residues of LdPEX14 are critical for anchoring LdPEX14 to the glycosomal membrane. Interestingly, expression of the first 74 amino acids of LdPEX14 is toxic to the parasites. / Finally, the structure of LdPEX14 on the glycosomal membrane was addressed by molecular mapping and biophysical techniques. Partial trypsin digestion of recombinant LdPEX14 and molecular mapping suggested that the first 250 residues of LdPEX14 were involved in the formation of the complex. Biophysical approaches and cross-linking assays suggest that this complex may consist of ∼12-100 LdPEX14 subunits. Interestingly, the structure of LdPEX14 appears to be modulated by LdPEX5. / Considering all the results, these findings have established important molecular information concerning early events in the targeting and import of PTS1 proteins into the glycosome.
| Identifer | oai:union.ndltd.org:LACETR/oai:collectionscanada.gc.ca:QMM.100649 |
| Date | January 2005 |
| Creators | Madrid, Kleber Patricio. |
| 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 (Institute of Parasitology.) |
| Rights | © Kleber Patricio Madrid, 2005 |
| Relation | alephsysno: 002495323, proquestno: AAINR25201, Theses scanned by UMI/ProQuest. |
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