Na+/H+ exchangers (NHE) are integral membrane proteins that catalyze the electroneutral exchange of Na+ (or K+) for H+. The nine human isoforms identified to date share the same topology of twelve membrane-spanning domains and a cytoplasmic regulatory tail, and diverge in their tissue expressions and subcellular localizations. This thesis focused on the identification and characterization of proteins interacting with the ubiquitous NHE6 isoform, which resides predominantly in recycling endosomes. Recent yeast two-hybrid (Y2H) screening of a human brain cDNA library using the regulatory tail of NHE6 as a probe resulted in the tentative identification of about 250 partial or full-length NHE6-interacting proteins. Thirty other potential NHE6-interacting partners were identified by isolating NHE6 complexes from embryonic kidney 293 cells by tandem affinity purification (TAP) and subjecting them to mass spectrometry analysis. The interaction between NHE6 and eight of these proteins (four from each Y2H and TAP analyses) was tested by co-immunoprecipitation, co-localization by immunofluorescence microscopy and in vitro GST-fusion protein pull-down assays. We show that apolipoprotein D interacts weakly with NHE6, while myosin light chain kinase and the mitochondrial ATP synthase subunit-alpha are true NHE6-binding partners. The remaining five proteins exhibited poor and/or non-reproducible association with NHE6.
| Identifer | oai:union.ndltd.org:LACETR/oai:collectionscanada.gc.ca:QMM.100793 |
| Date | January 2007 |
| Creators | Davidora, Albena. |
| 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 | Master of Science (Department of Physiology.) |
| Rights | © Albena Davidora, 2007 |
| Relation | alephsysno: 002586679, proquestno: AAIMR32689, Theses scanned by UMI/ProQuest. |
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