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Journal of Biochemistry Advance Access originally published online on March 2, 2009
Journal of Biochemistry 2009 145(6):709-720; doi:10.1093/jb/mvp032
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© The Authors 2009. Published by Oxford University Press on behalf of the Japanese Biochemical Society. All rights reserved

Saccharomyces cerevisiae Na+/H+ Antiporter Nha1p Associates with Lipid Rafts and Requires Sphingolipid for Stable Localization to the Plasma Membrane

Keiji Mitsui, Ken Hatakeyama, Masafumi Matsushita and Hiroshi Kanazawa*

Department of Biological Sciences, Graduate School of Science, Osaka University, Toyonaka City, Osaka, Japan

*To whom correspondence should be addressed. Tel: +81-6-6850-5812; Fax: +81-6-6850-5812; E-mail: kanazawa{at}bio.sci.osaka-u.ac.jp

Received February 2, 2009; Accepted February 13, 2009


  Abstract

The plasma membrane-type Na+/H+ antiporter Nha1p from budding yeast plays an important role in intracellular Na+ and pH homeostasis by mediating the exchange of Na+ for H+ across the plasma membrane. However, the mechanism of intracellular targeting of Nha1p to the plasma membrane remains unknown. Here, we found that Nha1p exists predominantly in detergent-resistant membrane fractions (DRMs) following density gradient centrifugation. When ergosterol was extracted from membranes, Nha1p was transferred to a detergent-soluble fraction, suggesting that Nha1p associates with ergosterol-containing DRMs, also known as lipid rafts. Density gradient centrifugation of cell extracts of yeast mutants that were defective in different stages of the secretory pathway revealed that, unlike previously identified raft proteins, the association of Nha1p with DRMs occurs mainly at the plasma membrane. In lcb1-100 cells, which are temperature-sensitive for sphingolipid synthesis, newly synthesized Nha1p failed to localize to the plasma membrane at the non-permissive temperature. Rather, Nha1p was distributed in an intracellular punctate pattern. The addition of phytosphingosine or the inhibition of endocytosis in lcb1-100 cells restored the targeting of Nha1p to the plasma membrane. The results of the current study suggest that sphingolipids are required for the stable localization of Nha1p to the plasma membrane.

Key Words: lipid rafts, membrane traffic, Na+/H+ antiporter, Saccharomyces cerevisiae, sphingolipid

Abbreviations: Nha1p, Na+/H+ antiporter 1 protein; SDS–PAGE, SDS polyacrylamide gel electrophoresis; DRM, detergent-resistant membrane; MβCD, methyl β-cyclodextrin; ER, endoplasmic reticulum; TCA, trichloroacetic acid; PHS, phytosphigosine


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