Norihiro Nakamura

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.

Regulation and Reversibility of Vacuolar H+-ATPase

Arabidopsis thaliana vacuolar H+-translocating pyrophosphatase (V-PPase) was expressed functionally in yeast vacuoles with endogenous vacuolar H+-ATPase (V-ATPase), and the regulation and reversibility of V-ATPase were studied using these vacuoles. Analysis of electrochemical proton gradient (ΔμH) formation with ATP and pyrophosphate indicated that the proton transport by V-ATPase or V-PPase is not regulated strictly by the proton chemical gradient (ΔpH). On the other hand, vacuolar membranes may have a regulatory mechanism for maintaining a constant membrane potential (ΔΨ). Chimeric vacuolar membranes showed ATP synthesis coupled with ΔμH established by V-PPase. The ATP synthesis was sensitive to bafilomycin A1 and exhibited two apparent K m values for ADP. These results indicate that V-ATPase is a reversible enzyme. The ATP synthesis was not observed in the presence of nigericin, which dissipates ΔpH but not ΔΨ, suggesting that ΔpH is essential for ATP synthesis.

A Novel Kinesin-Like Protein, KIF1Bβ3 Is Involved in the Movement of Lysosomes to the Cell Periphery in Non-Neuronal Cells

The kinesin superfamily protein, KIF1Bβ, a splice variant of KIF1B, is involved in the transport of synaptic vesicles in neuronal cells, and is also expressed in various non‐neuronal tissues. To elucidate the functions of KIF1Bβ in non‐neuronal cells, we analyzed the intracellular localization of KIF1Bβ and characterized its isoform expression profile. In COS‐7 cells, KIF1B colocalized with lysosomal markers and expression of a mutant form of KIF1Bβ, lacking the motor domain, impaired the intracellular distribution of lysosomes. A novel isoform of the kinesin‐like protein, KIF1Bβ3, was identified in rat and simian kidney. It lacks the 5th exon of the KIF1Bβ‐specific tail region. Overexpression of KIF1Bβ3 induced the translocation of lysosomes to the cell periphery. However, overexpression of KIF1Bβ3‐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 KIF1Bβ3 is involved in the translocation of lysosomes from perinuclear regions to the cell periphery.

Identification of the copper chaperone, CUC‐1, in Caenorhabditis elegans: tissue specific co‐expression with the copper transporting ATPase, CUA‐1

A cDNA encoding a putative copper chaperone protein, CUC‐1, was cloned from Caenorhabditis elegans. CUC‐1 had the characteristic motifs of MTCXXC and KKTGK, and showed 49.3 and 39.1% sequence identity with yeast Atx1p and human HAH1, respectively. Expression of CUC‐1 cDNA complemented a null atx1 mutant, the yeast copper chaperone gene, thus demonstrating that CUC‐1 is a functional copper chaperone. Studies with transgenic worms indicated that cuc‐1 and cua‐1, which encodes the copper transporting ATPase, are expressed together in intestinal cells of adult and hypodermal cells in the larvae. cua‐1 was also expressed in pharyngeal muscle but cuc‐1 was not. These results suggest that CUC‐1 and CUA‐1 constitute a copper trafficking pathway similar to the yeast counterparts in intestinal and hypodermal cells, and CUA‐1 may have a different function in pharyngeal muscle.

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.

Detection of Oligomerization and Conformational Changes in the Na+/H+ Antiporter from Helicobacter pylori by Fluorescence Resonance Energy Transfer

Oligomerization and conformational changes in the Na+/H+ antiporter from Helicobacter pylori (HPNhaA) were studied by means of fluorescence resonance energy transfer (FRET) analysis. Na+/H+ antiporter-deficient Escherichia coli cells expressing C-terminal fusions of HPNhaA to green fluorescent protein (GFP) variants exhibited wild-type levels of antiporter activity in their everted membrane vesicles. Vesicles containing both HPNhaA-CFP and HPNhaA-YFP or HPNhaA-Venus exhibited FRET from CFP (donor) to YFP or Venus (acceptor), suggesting that HPNhaA forms an oligomer. Co-precipitation of HPNhaA tagged by Venus and FLAG sequences confirmed oligomerization. FRET decreased extensively after treatment of the vesicles with proteinase K, which released GFP variants from the fusion proteins. FRET was not observed by merely mixing vesicles expressing the donor or acceptor fusion alone. Fluorescence of Venus is less sensitive to anions and stronger than that of anion-sensitive YFP. Using HPNhaA-Venus as the acceptor, Li+ was found to cause a significant decrease in FRET regardless of the presence or absence of ΔpH across the membranes, whereas Na+ caused a much weaker effect. This Li+ effect was minimal in vesicles prepared from cells expressing HPNhaA containing an Asp141 to Asn mutation, which results in defective Li+/H+ antiporter activity, possibly Li+ binding. These results demonstrate that monomer interactions within the HPNhaA oligomer are weakened possibly by Li+ binding. Dynamic interactions between HPNhaA monomers were detectable in membranes by FRET analysis, thus providing a new approach to study dynamic conformational changes in NhaA during antiport activity.

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.