Oligomerization of the Saccharomyces cerevisiae Na+/H+ antiporter Nha1p: implications for its antiporter activity.
The Na(+)/H(+) antiporter (Nha1p) from the budding yeast Saccharomyces cerevisiae plays an important role in intracellular pH and Na(+) homeostasis. Here, we show by co-precipitation of differently tagged Nha1p proteins expressed in the same cell that the yeast Nha1p l forms an oligomer. In vitro cross-linking experiments then revealed that Nha1p-FLAG is present in the membranes as a dimer. Differently tagged Nha1p proteins were also co-precipitated from sec18-1 mutant cells in which ER-to-Golgi traffic is blocked under non-permissive temperatures, suggesting that Nha1p may already dimerize in the ER membrane. When we over-expressed a mutant Nha1p with defective antiporter activity in cells that also express the wild-type Nha1p-EGFP fusion protein, we found impaired cell growth in highly saline conditions, even though the wild-type protein was appropriately expressed and localized correctly. Co-immunoprecipitation assays then showed the inactive Nha1p-FLAG mutant interacted with the wild-type Nha1p-EGFP protein. These results support the notion that Nha1p exists in membranes as a dimer and that the interaction of its monomers is important for its antiporter activity.
Mitsui
Keiji
K
Department of Biological Sciences, Graduate School of Science, Osaka University, Toyonaka City, Osaka 560-0043, Japan.
Yasui
Hidetomo
H
Nakamura
Norihiro
N
Kanazawa
Hiroshi
H
D017920:Antiporters / Q000378:metabolism; D027682:Cation Transport Proteins / Q000737:chemistry / Q000172:deficiency / Q000502:physiology*; D049452:Green Fluorescent Proteins / Q000502:physiology; D008565:Membrane Proteins / Q000737:chemistry / Q000172:deficiency / Q000502:physiology*; D020836:Protein Structure, Quaternary; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000254:growth & development / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry / Q000502:physiology*; D017923:Sodium-Hydrogen Exchangers / Q000737:chemistry / Q000502:physiology*
D017920:Antiporters; D027682:Cation Transport Proteins; D008565:Membrane Proteins; C000631624:NHA1 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins; D017923:Sodium-Hydrogen Exchangers; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins