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.
The kinesin superfamily protein, KIF1Bb, a splice variant of KIF1B, is involved in the transport of synaptic vesicles in neuronal cells, and is also expressed in various nonneuronal tissues. To elucidate the functions of KIF1Bb in non-neuronal cells, we analyzed the intracellular localization of KIF1Bb and characterized its isoform expression profile. In COS-7 cells, KIF1B colocalized with lysosomal markers and expression of a mutant form of KIF1Bb, lacking the motor domain, impaired the intracellular distribution of lysosomes. A novel isoform of the kinesin-like protein, KIF1Bb3, was identified in rat and simian kidney. It lacks the 5th exon of the KIF1Bb-specific tail region. Overexpression of KIF1Bb3 induced the translocation of lysosomes to the cell periphery. However, overexpression of KIF1Bb3-Q98L, which harbors a pathogenic mutation associated with a familial neuropathy, Charcot-Marie-Tooth disease type 2 A, resulted in the abnormal perinuclear clustering of lysosomes. These results indicate that KIF1Bb3 is involved in the translocation of lysosomes from perinuclear regions to the cell periphery.
Mammalian Na+/H+ exchangers (NHEs) play a fundamental role in cellular ion homeostasis. NHEs exhibit an appreciable variation in expression, regulation, and physiological function, dictated by their dynamics in subcellular localization and/or interaction with regulatory proteins. In recent years, a subgroup of NHEs consisting of four isoforms has been identified, and its members predominantly localize to the membranes of the Golgi apparatus and endosomes. These organellar NHEs constitute a family of transporters with an emerging function in the regulation of luminal pH and in intracellular membrane trafficking as expressed, for example, in cell polarity development. Moreover, specific roles of a variety of cofactors, regulating the intracellular dynamics of these transporters, are also becoming apparent, thereby providing further insight into their mechanism of action and overall functioning. Interestingly, organellar NHEs have been related to mental disorders, implying a potential role in the brain, thus expanding the physiological significance of these transporters.
This study underscores the emerging role of NHE6 as a novel regulatory protein in the apical surface development of human hepatoma HepG2 cells. A limited range of endosomal pH facilitated by NHE6.1 is suggested to be important for securing the polarized distribution of membrane lipids and proteins and maintenance of apical bile canaliculi.
In mammalian cells, nine conserved isoforms of the Na ϩ /H ϩ exchanger (NHE) are known to be important for pH regulation of the cytoplasm and organellar lumens. NHE1-5 are localized to the plasma membrane, whereas NHE6 -9 are localized to distinct organelles. NHE6 is localized predominantly in endosomal compartments but is also found in the plasma membrane. To investigate the role of NHE6 in endocytosis, we established NHE6-knockdown HeLa cells and analyzed the effect of this knockdown on endocytotic events. The expression level of NHE6 in knockdown cells was decreased to ϳ15% of the level seen in control cells. Uptake of transferrin was also decreased. No effect was found on the endocytosis of epidermal growth factor or on the cholera toxin B subunit. Moreover, in the NHE6-knockdown cells, transferrin uptake was found to be affected in the early stages of endocytosis. Microscopic analysis revealed that, at 2 min after the onset of endocytosis, colocalization of NHE6, clathrin, and transferrin was observed, which suggests that NHE6 was localized to endocytotic, clathrin-coated vesicles. In addition, in knockdown cells, transferrin-positive endosomes were acidified, but no effect was found on cytoplasmic pH. In cells overexpressing wild-type NHE6, increased transferrin uptake was observed, but no such increase was seen in cells overexpressing mutant NHE6 deficient in ion transport. The luminal pH in transferrin-positive endosomes was alkalized in cells overexpressing wild-type NHE6 but normal in cells overexpressing mutant NHE6. These observations suggest that NHE6 regulates clathrindependent endocytosis of transferrin via pH regulation. pH regulation; endosome; exocytosis IN MAMMALIAN CELLS, endocytosis and exocytosis are important for various intracellular events such as receptor-mediated ligand uptake and signal transduction via the receptors. The ligands and receptors are internalized through vesicles that form from the plasma membrane, migrate to endosomes, and are sorted in recycling pathways for relocation to the plasma membrane or to late endosomes/lysosomes for degradation. The lumina of endocytotic membrane compartments are gradually acidified as they are trafficked through the pathways, and the regulation of this acidification has been suggested to be important for these trafficking events (21). This acidification is primarily achieved by proton pumping into lumens by vacuolar H ϩ -ATPase (V-ATPase) (9). Recent studies suggest that luminal pH is regulated by a counterbalance between the influx of protons mediated by V-ATPase and proton leakage from the lumen through ion transport proteins such as anion channels, ClϪ /H ϩ exchangers, and Na ϩ /H ϩ exchangers (NHE) (5,15,26,37).For mammals, nine conserved NHEs (NHE1-9, also referred to as SLC9A1-9) have been identified (31). NHA1-2 (SLC9B1-2) and SLC9C1-2 have also been identified as SLC9 members, but their primary structures are distantly related to SLC9A1-9 (3, 18) (www.bioparadigms.org/slc/pdf/SLC09_2010 -09-06.pdf). In NHE1-9, five of those isoforms ...
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