Keiji Mitsui

Four Na+/H+ Exchanger Isoforms Are Distributed to Golgi and Post-Golgi Compartments and Are Involved in Organelle pH Regulation

Four isoforms of the Na+/H+ exchanger (NHE6–NHE9) are distributed to intracellular compartments in human cells. They are localized to Golgi and post-Golgi endocytic compartments as follows: mid- to trans-Golgi, NHE8; trans-Golgi network, NHE7; early recycling endosomes, NHE6; and late recycling endosomes, NHE9. No significant localization of these NHEs was observed in lysosomes. The distribution of these NHEs is not discrete in the cells, and there is partial overlap with other isoforms, suggesting that the intracellular localization of the NHEs is established by the balance of transport in and out of the post-Golgi compartments as the dynamic membrane trafficking. The overexpression of NHE isoforms increased the luminal pH of the compartments in which the protein resided from the mildly acidic pH to the cytosolic pH, suggesting that their in vivo function is to regulate the pH and monovalent cation concentration in these organelles. We propose that the specific NHE isoforms contribute to the maintenance of the unique acidic pH values of the Golgi and post-Golgi compartments in the cell.

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.

Na+/H+ exchanger isoform 6 (NHE6/SLC9A6) is involved in clathrin-dependent endocytosis of transferrin.

In mammalian cells, nine conserved isoforms of the Na(+)/H(+) 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 ∼15% 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 clathrin-dependent endocytosis of transferrin via pH regulation.

Dual functional significance of calcineurin homologous protein 1 binding to Na+/H+ exchanger isoform 1

Calcineurin homologous protein 1 (CHP1) binds to the hydrophilic tail of the Na(+)/H(+) exchanger isoform 1 (NHE1). Previous gene knockout of CHP1 revealed that the loss of CHP1 caused a decrease in the total amount of NHE1, suggesting the destabilization of NHE1 molecules without CHP1 (Matsushita et al., Am J Physiol Cell Physiol 293: C246-C254, 2007). However, Pang et al. (J Biol Chem 276: 17367-17372, 2001) reported that NHE1 without a CHP1 binding site was found in the plasma membrane, suggesting no requirement of CHP1 binding for plasma membrane localization of NHE1. Here, the functional significance of CHP1 binding to NHE1 was examined to resolve these contradictory results. In CV1 cells, which overexpressed wild-type NHE1, overexpression of CHP1 caused an increase in both the total amount of NHE1 and the colocalization of NHE1 and CHP1 at the plasma membrane. This provided new visual evidence of the localization of NHE1 from endoplasmic reticulum to the plasma membrane upon CHP1 binding. An immunoprecipitation assay showed that the expression of CHP1 reduced the ubiquitination of NHE1 and/or its associated proteins. Mutant NHE1s without CHP1 binding site exhibited a modest localization to the plasma membrane. After reaching the plasma membrane, these mutant NHE1s exhibited shorter half-lives than the wild-type NHE1 with CHP1. The results suggest a dual functional significance of CHP1 and its binding region: 1) binding of CHP1 stabilizes NHE1 and increases its plasma membrane localization by masking a NHE1 disposal signal, and 2) CHP1 binding is required for the antiporter activity.

A novel membrane protein capable of binding the Na+/H+ antiporter (Nha1p) enhances the salinity-resistant cell growth of Saccharomyces cerevisiae.

The Na+/H+ antiporter Nha1p of Saccharomyces cerevisiae plays an important role in maintaining intracellular pH and Na+ homeostasis. Nha1p has a two-domain structure composed of integral membrane and hydrophilic tail regions. Overexpression of a peptide of ∼40 residues (C1+C2 domains) that is localized in the juxtamembrane area of its cytoplasmic tail caused cell growth retardation in highly saline conditions, possibly by decreasing Na+/H+ antiporter activity. A multicopy suppressor gene of this growth retardation was identified from a yeast genome library. The clone encodes a novel membrane protein denoted as COS3 in the genome data base. Overexpression or deletion of COS3 increases or decreases salinity-resistant cell growth, respectively. However, in nha1Δ cells, overexpression of COS3 alone did not suppress the growth retardation. Cos3p and a hydrophilic portion of Cos3p interact with the C1+C2 peptide in vitro, and Cos3p is co-precipitated with Nha1p from yeast cell extracts. Cos3p-GFP mainly resides at the vacuole, but overexpression of Nha1p caused a portion of the Cos3p-GFP proteins to shift to the cytoplasmic membrane. These observations suggest that Cos3p is a novel membrane protein that can enhance salinity-resistant cell growth by interacting with the C1+C2 domain of Nha1p and thereby possibly activating the antiporter activity of this protein.

The Endosomal Na+/H+ Exchanger Contributes to Multivesicular Body Formation by Regulating the Recruitment of ESCRT-0 Vps27p to the Endosomal Membrane

Multivesicular bodies (MVBs) are late endosomal compartments containing luminal vesicles (MVB vesicles) that are formed by inward budding of the endosomal membrane. In budding yeast, MVBs are an important cellular mechanism for the transport of membrane proteins to the vacuolar lumen. This process requires a class E subset of vacuolar protein sorting (VPS) genes. VPS44 (allelic to NHX1) encodes an endosome-localized Na+/H+ exchanger. The function of the VPS44 exchanger in the context of vacuolar protein transport is largely unknown. Using a cell-free MVB formation assay system, we demonstrated that Nhx1p is required for the efficient formation of MVB vesicles in the late endosome. The recruitment of Vps27p, a class E Vps protein, to the endosomal membrane was dependent on Nhx1p activity and was enhanced by an acidic pH at the endosomal surface. Taken together, we propose that Nhx1p contributes to MVB formation by the recruitment of Vps27p to the endosomal membrane, possibly through Nhx1p antiporter activity.

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

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.

A Membrane-Proximal Region in the C-Terminal Tail of NHE7 Is Required for Its Distribution in the Trans-Golgi Network, Distinct from NHE6 Localization at Endosomes

Mammalian Na+/H+ exchanger (NHE) isoform NHE6 is localized in sorting/recycling endosomes, whereas NHE7 is localized in the trans-Golgi network (TGN) and mid-trans-Golgi stacks. The mechanism targeting each NHE to a specific organelle is largely unknown, although the targeting is thought to be important for pH control in the lumen of various organelles. NHE6 and NHE7 exhibit distinct localization despite conserved amino acid sequences. To specify the intramolecular region involved in the specific localization, we examined the intracellular localization of chimeric NHE6 and NHE7 constructs. NHEs are composed of an N-terminal transmembrane domain (TM) and a C-terminal hydrophilic tail domain (Ct). Exchange of the Ct between the isoforms suggested that the Ct is required for the specific localization. We further split the Ct into three regions, and chimeras with various combinations of these small regions indicated that the most membrane-proximal region among the three contributes to the specific localization. Mutant forms of NHE7 with sequential alanine substitutions in the most membrane-proximal region, between residues 530 and 589, showed that two regions (residues 553–559 and 563–568) are required for NHE7-like localization. However, NHE6 with alanine substitutions in the membrane-proximal region exhibited no apparent change in localization. These results suggest that two membrane proximal regions (residues 533–559 and 563–568) play an important role in targeting NHE7 to the TGN.

Altered Motor Activity of Alternative Splice Variants of the Mammalian Kinesin‐3 Protein KIF1B

Several mammalian kinesin motor proteins exist as multiple isoforms that arise from alternative splicing of a single gene. However, the roles of many motor protein splice variants remain unclear. The kinesin‐3 motor protein KIF1B has alternatively spliced isoforms distinguished by the presence or absence of insertion sequences in the conserved amino‐terminal region of the protein. The insertions are located in the loop region containing the lysine‐rich cluster, also known as the K‐loop, and in the hinge region adjacent to the motor domain. To clarify the functions of these alternative splice variants of KIF1B, we examined the biochemical properties of recombinant KIF1B with and without insertion sequences. In a microtubule‐dependent ATPase assay, KIF1B variants that contained both insertions had higher activity and affinity for microtubules than KIF1B variants that contained no insertions. Mutational analysis of the K‐loop insertion revealed that variants with a longer insertion sequence at this site had higher activity. However, the velocity of movement in motility assays was similar between KIF1B with and without insertion sequences. Our results indicate that splicing isoforms of KIF1B that vary in their insertion sequences have different motor activities.

Intermolecular cross-linking of monomers in Helicobacter pylori Na+/H+ antiporter NhaA at the dimer interface inhibits antiporter activity.

We have previously shown that HPNhaA (Helicobacter pylori Na+/H+ antiporter) forms an oligomer in a native membrane of Escherichia coli, and conformational changes of oligomer occur between monomers of the oligomer during ion transport. In the present study, we use Blue-native PAGE to show that HPNhaA forms a dimer. Cysteine-scanning mutagenesis of residues 55-61 in a putative beta-sheet region of loop1 and subsequent functional analyses revealed that the Q58C mutation resulted in an intermolecular disulfide bond. G56C, I59C and G60C were found to be cross-linked by bifunctional cross-linkers. Furthermore, the Q58E mutant did not form a dimer, possibly due to electrostatic repulsion between monomers. These results imply that Gln-58 and the flanking sequence in the putative beta-sheet of the monomer are located close to the identical residues in the dimer. The Q58C mutant of NhaA was almost inactive under non-reducing conditions, and activity was restored under reducing conditions. This result showed that cross-linking at the dimer interface reduces transporter activity by interfering with the flexible association between the monomers. A mutant HPNhaA protein with three amino acid substitutions at residues 57-59 did not form a dimer, and yet was active, indicating that the monomer is functional.