The Na+/H+ exchangers (NHE) mediate the electroneutral exchange of sodium for protons and play integral roles in sodium, acid-base and cell volume homeostasis. Presently, eight isoforms of the NHE (NHE1 to NHE8) have been identified that are targeted to distinct membrane compartments. The focus of this study is to characterize in greater detail the biosynthesis and differential sorting of two closely related organellar NHE isoforms, NHE6 and NHE7. Previous studies have established that NHE7 accumulates in the trans-Golgi network and associated endosomes, whereas the localization of NHE6 remains controversial. In one study, HeLa cells transiently expressing low levels of a green fluorescent protein-tagged construct of NHE6 showed close co-localization with mitochondrion-specific dyes. However, when NHE6 was overexpressed in COS7 cells, significant accumulation was observed throughout the cell in membrane vesicles derived from the endoplasmic reticulum. To further address this discrepancy, NHE6 engineered to contain the influenza virus hemagglutinin (HA) epitope at its C-terminus (NHE6HA), was subcloned into the ecdysone-inducible expression vector pIND and stably transfected into Chinese hamster ovary cells that constitutively express the ecdysone receptor. NHE6HA expression was stimulated by ponasterone A, an ecdysone analogue. The localization of NHE6 was determined biochemically by subcellular fractionation of cell lysates and visually by immunofluorescence confocal microscopy of intact cells using antibodies that recognize the HA-epitope and various organellar specific markers. Similar studies were conducted with an NHE7-inducible mammalian expression system. Our findings indicate that NHE6 is differentially processed through distinct Golgi-dependent and -independent pathways, ultimately accumulating in recycling endosomal vesicles. Little evidence was found to support sorting to mitochondria. Furthermore, we show that NHE6 is synthesiz
| Identifer | oai:union.ndltd.org:LACETR/oai:collectionscanada.gc.ca:QMM.81451 |
| Date | January 2004 |
| Creators | Virdee, Inderpreet |
| 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 | All items in eScholarship@McGill are protected by copyright with all rights reserved unless otherwise indicated. |
| Relation | alephsysno: 002166689, proquestno: AAIMR06467, Theses scanned by UMI/ProQuest. |
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