Cell surface levels of organellar Na+/H+ exchanger isoform 6 are regulated by interaction with RACK1.
In mammalian cells, four Na(+)/H(+) exchangers (NHE6 - NHE9) are localized to intracellular compartments. NHE6 and NHE9 are predominantly localized to sorting and recycling endosomes, NHE7 to the trans-Golgi network, and NHE8 to the mid-trans-Golgi stacks. The unique localization of NHEs may contribute to establishing organelle-specific pH values and ion homeostasis in cells. Mechanisms underlying the regulation and targeting of organellar NHEs are largely unknown. We identified an interaction between NHE9 and RACK1 (receptor for activated C kinase 1), a cytoplasmic scaffold protein, by yeast two-hybrid screening using the NHE9 C terminus as bait. The NHE9 C terminus is exposed to the cytoplasm, verifying that the interaction is topologically possible. The binding region was further delineated to the central region of the NHE9 C terminus. RACK1 also bound NHE6 and NHE7, but not NHE8, in vitro. Endogenous association between NHE6 and RACK1 was confirmed by co-immunoprecipitation and co-localization in HeLa cells. The luminal pH of the recycling endosome was elevated in RACK1 knockdown cells, accompanied by a decrease in the amount of NHE6 on the cell surface, although the total level of NHE6 was not significantly altered. These results indicate that RACK1 plays a role in regulating the distribution of NHE6 between endosomes and the plasma membrane and contributes to maintaining luminal pH of the endocytic recycling compartments.
Ohgaki
Ryuichi
R
Department of Biological Science, Graduate School of Science, Osaka University, Machikaneyama, Toyonaka, Osaka 560-0043, Japan.
Fukura
Naomi
N
Matsushita
Masafumi
M
Mitsui
Keiji
K
Kanazawa
Hiroshi
H
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D019556:COS Cells; D002522:Chlorocebus aethiops; D017931:DNA Primers; D019204:GTP-Binding Proteins / Q000737:chemistry / Q000378:metabolism / Q000502:physiology*; D006367:HeLa Cells; D006801:Humans; D047468:Immunoprecipitation; D008565:Membrane Proteins / Q000378:metabolism*; D008969:Molecular Sequence Data; D009363:Neoplasm Proteins / Q000737:chemistry / Q000378:metabolism / Q000502:physiology*; D000075370:Receptors for Activated C Kinase; D011956:Receptors, Cell Surface / Q000737:chemistry / Q000378:metabolism / Q000502:physiology*; D017386:Sequence Homology, Amino Acid; D017923:Sodium-Hydrogen Exchangers / Q000378:metabolism*; D020798:Two-Hybrid System Techniques
D017931:DNA Primers; D008565:Membrane Proteins; D009363:Neoplasm Proteins; C419368:RACK1 protein, human; D000075370:Receptors for Activated C Kinase; D011956:Receptors, Cell Surface; C445316:SLC9A6 protein, human; D017923:Sodium-Hydrogen Exchangers; D019204:GTP-Binding Proteins