Properties of the Na+/H+ exchanger protein: Detergent‐resistant aggregation and membrane microdistribution
2002
Bullis, Bonnie L. | Li, Xiuju | Rieder, Carmen V. | Singh, Dyal N. | Berthiaume, Luc G. | Fliegel, Larry
The Na+/H+ exchanger is a ubiquitous membrane protein of bacteria, plants and mammals. The first isoform discovered (NHE1) is present on the mammalian plasma membrane and transports one H+ out of cells in exchange for one extracellular Na+. With solubilization in standard SDS/PAGE buffer, this protein had a high tendency to aggregate when subjected to elevated temperature. The aggregates were stable and did not dissociate in high concentrations of SDS or 2‐mercaptoethanol. We examined the distribution of the Na+/H+ exchanger within membrane subfractions. The Na+/H+ exchanger was found both in caveolin‐containing fractions and, in lesser amounts, in higher density membrane fractions where the bulk of proteins were contained. Treatment with cytochalasin D caused only a minor reduction of the amount of Na+/H+ exchanger present in caveolin‐enriched fractions suggesting an intact cytoskeleton was not important for NHE1 localization to these microdomains. Treatment of cells with methyl β‐cyclodextrin had a small stimulatory effect on Na+/H+ exchanger activity and reduced the amount of Na+/H+ exchanger in low density membrane fractions. Our study demonstrates that SDS cannot maintain the protein in a monomeric state suggesting that strong hydrophobic interactions are responsible for this temperature dependent aggregation behavior. In addition a large proportion of the Na+/H+ exchanger protein is found to be enriched in low density caveolin‐containing fractions.
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