Exchanger Nhx1p from Saccharomyces cerevisiae Plays an Important Role in the

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A Novel Implication of the Physiological Importance of Local Protons at the Membrane Surface of Organellar Vesicles in Eukaryote Cells: Organellar Type Na

⁺

/H

⁺

Exchanger Nhx1p from Saccharomyces cerevisiae Plays an Important Role in the

Formation of Multivesicular Bodies

Hiroshi Kanazawa^1, 2) and Keiji Mitsui²⁾

Intracellular pH homeostasis is the essential element for living cells. Optimal pH has to be provided for proteins to function properly within the cells. pH is kept at around 7.3 in most cells from bacterial to mammalian. In contrast, for various endosomes in the cytoplasm their own weak acidic pH is maintained. For the homeostatic pH regulation in the compartments like cytoplasm and endosomal lumen are performed by H⁺ transporting membrane integral proteins. One of these is the Na⁺/H⁺ exchanger (NHE or Nha). Here the roles of Na⁺/H⁺ exchangers in the cells are summarized based on our recent findings. One of the most important findings is that the acidic pH regulated by NHE in the yeast endosomes contributes to recruitment of various factors required for membrane protein trafficking on the membrane surface of the endosomes. This acidic pH leads to multivesicular body (MVB) formation. These observations raise a new important role for NHE, not only in the pH regulation of the endosome lumen but also for pH regulation on the cytoplasmic surface of the endosomes. Based on this new physiological role of organellar NHE from yeast (Nhx1) we propose a new conceptual idea of the importance of local protons regulated by the NHEs in cell physiology.

Key Words : pH regution, Na^＋/H^＋exchangers, ion transporter, multivesicular body, membrane traﬃcking

Homeostasis of pH within the cells Intracellular ionic condition is an essential factor for living cells to survive. In particular, H^＋ is the most important element to be kept at a certain value, the concentration because it provides the best

environment for proteins to perform their roles. It is well established that cytoplasmic pH is kept at around 7.2‑7.4 in the cells from most bacteria to humans [1, 2]. For eukaryotic cells, intracellular vesicular organelles are found in which pH shows an acidic value for the luminal condition (Fig. 1) [1]. For instance, lysosomes, one of these organelles, in which pH is set at around less than 5.5 [1], provide the best pH condition for the lysosomal enzymes to play a role.

The cytoplasmic pH becomes acidic as a result of whole metabolism in the cells. To maintain the neutral pH in the cytoplasm, excess protons should

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http://www.cjc.ac.jp/

Corresponding author.

Hiroshi Kanazawa

Department of Human Nutrition, Faculfy of Contemporary Life Science, Chyugoku Gakuen University, 83, Niwase, Kitaku, Okayama 701ﾝ0197, Japan

Tel & Fax ; + 81 86 293 1100

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be removed from the cytoplasm. For this, various H^＋ transporters and pumps (H^＋ transporting ATPases) have been provided in the plasma membranes of living cells from bacteria to humans [1]. H^＋ is exported from inside the cells to the outside by H^＋ transporting ATPase for bacterial and yeast cells.

H^＋ extrusion is also performed by the respiratory chain for bacterial cells. This extrusion leads to the formation of a proton electrochemical gradient across the plasma membranes [3, 4]. The proton gradient drives various transporters for the nutrients and ions in bacterial and yeast cells. The H^＋ extrusion is also performed by Na^＋/H^＋ exchangers (NHE) for mammalian cells. To drive H^＋ extrusion by NHEs a Na^＋ gradient is used that is established by Na^＋/K^＋ ATPase. Excess H^＋ in the cytoplasm is also removed by uptake of H^＋ into organellar lumen by V-type H^＋ transporting ATPase [1, 2].

An extensively acidic condition outside of the cells eventually leads to acidification of cytoplasm and inhibits the growth of cells [5]. For a NHE1- deﬁcient cultured cell line under an acidic condition in a culture medium cell growth was inhibited, supporting the idea of functional importance of NHE1 in the pH regulation. Thus pH regulation inside cells is accomplished by total functional regulation of various H^＋ transporters such as NHEs together with H^＋ or Na^＋ transporting ATPases. The precise molecular mechanisms of this coordinated control of various H^＋ transporters are still unclear.

Na^＋/H^＋ exchangers (NHE)

One of the most important H^＋ transporters is the Na^＋/H^＋ exchanger (NHE) also named Na^＋/H^＋ antiporter (Nha) found in bacterial to human cells.

NHE and Nha form a family of several isoforms. Na^＋ and H^＋ are transported in the opposite direction.

The transport direction of the ions is determined primarily by the ion gradient formed by the ion transporting ATPases. For bacteria and yeast Na^＋ is exported to the outside of cells to maintain the optimal osmotic pressure of the cells by NHEs [3]. This extrusion of Na^＋ is driven by the H^＋ gradient formed by H^＋-transporting ATPase. Thus Nha plays an important role for bacterial and yeast cells to survive under high saline environments [3].

For mammalian cells, NHE1 to NHE5 are found in the plasma membranes [2, 6]. For organellar membranes, NHE6 to NHE9 are found [7] (Fig.

2). For yeast cells, one organellar type antiporter (Nhx1p) and one plasma membrane type Nha (Nha1p) are known.

Organellar type NHEs

In eukaryotic cells various types of vesicular structures are found as organelles. These organellar vesicular structures include lysosomes, peroxisomes, and other endosomes. The internal acidic pH in these organelles (Fig. 1) is established as described earlier here by V type‑ATPase transporting H^＋ from the cytoplasmic space to the inside of the vesicles (lumen) (Fig. 3). The acidic value of a type of organellar lumen is speciﬁc for each type of organelles as shown in Fig. 3. The organellar type NHEs can export H^＋ to the cytoplasmic space driven by a K^＋ gradient across the membranes instead of Na^＋ because high K^＋ and low Na^＋ are established by Na^＋/K^＋ ATPase [7, 8]. For mammalian cells, NHE6 is localized to the early and recycling endosomes [8, 9]. NHE 9 is found at the late endosomes. NHE 7 is localized at the Trans-Golgi network, while NHE8 is found at the mid-Golgi. Thus the organellar type NHEs are localized to different organelles and play a role to keep the weak acidic pH value in the lumen speciﬁc to each organelle together with V-ATPase [7, 8, 10].

Although acidic pH for various organellar lumen seems to be regulated by both V-ATPase and NHEs,

14 Kanazawa et al. CHUGOKUGAKUEN J. Vol. 12

Fig. 1 Luminal pH values of various organelles are shown at the side of an organelle located on the secretory or endocytic pathway in a mammalian cell [7].

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the physiological significance of this pH regulation is not fully known in detail. NHE6 is localized at the early and recycling endosomes. However, about 20 percent of NHE6 in HeLa cells is also found in the plasma membranes [11]. Knockdown of NHE6 expression in HeLa cells caused reduction of transferrin uptake from the outside of cells,

especially at early stage of endocytosis of transferrin receptor (Fig. 4). NHE6 may affect clathrin recruitment to the endosome surface [12]. NHE6 binds RACK1 that had been found as a cytoplasmic scaffold protein for a protein kinase and other transporters [11]. Knockdown of RACK1 expression leads to a decrease of NHE6 molecules at the plasma membrane and accumulation at the early endosomes.

NHE6 shuttles between the plasma membrane and endosomes which is regulated by RACK1 [11].

NHE6 is also found at endosomes in HepG2 cells, a hepatoma cell line, in which formation of cell-polarity as observed for the formation of distinct apical and basolateral structures in a cell is observed during cell culture [13]. This polarity formation involves pH regulation of endosomes by NHE6 that is supported by knockdown experiments of NHE6.

This knockdown caused a decrease of bile canalicular structure observed for the apical area of HepG2 cells [13].

NHE6 is identiﬁed as a causative gene for human mental retardation syndrome such as Angelman syndrome like syndrome [14]. We have found a

Fig. 2 Schematic illustration of various Na^＋

/H

^＋ exchangers. NhaA, NhaB, and ChaA are family members of bacteria exchangers found in the cytoplasmic membranes. NHE1 to NHE9 are family members of mammalian Na^＋

/H

^＋ exchangers located at distinct organelles in mammalian cells [7].

Fig. 3 Schematic illustration of pH regulation in organellar lumen by V‑ATPase, the Cl⁻ channel and NHEs.

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Japanese family lacking the normal NHE6 gene [15].

Because of the defect in the primary sequence in the NHE6 gene found as a nucleotide deletion at position 441, a termination codon and resultant truncation of the NHE6 protein sequence was taken place. This mutation causes a malfunction of NHE6. Detailed studies to clarify the correlation between the disease and gene defect will be required. Interestingly our preliminary experimental results suggest that NHE6 is required for neurite growth for neuronal cell model PC12 cells [16]. NHE9 is found at the late endosomes and has been found as the causative gene in case of the autism [17]. Mechanistic studies to reveal the physiological role of NHE9 in neuronal cells are needed.

Organellar type Nhx1p from yeast and its physiological role

As found for mammalian NHE6 and NHE9, organellar type NHEs are an important pH regulator in cell physiology, probably through membrane protein trafficking in the cells [7]. However, the mechanisms that involve NHEs including NHE6 are largely unknown. The yeast counterpart of NHE6 is Nhx1p [18] (Fig. 5). Knockout of Nhx1 affects protein traﬃcking of CPY (carboxy peptidase Y) that is localized to the vacuole [19]. For the knockout cells CPY is excreted to the outside of the cells.

Further accumulation of prevacuolar compartments was observed [19]. Based on these observations we analyzed Nhx knockout on the traﬃcking of another protease CPS (carboxy peptidase S) localized at the vacuole (Fig. 5) because we thought that the role of Nhx1 in protein trafficking might give a clue to the mechanism involving NHE6 and other organellar type NHEs [20]. Knockout of Nhx1

Fig. 4 Schematic illustration of transferrin uptake and endocytosis. NHE6 is located at the plasma membrane and early endosomes (left panel). Uptake of transferrin by HeLa cells is reduced for the cells with reduced levels of NHE6 expression by the gene knockdown (right panel). Details of the experiments are described in [12].

Fig. 5 Traﬃcking of membrane receptors (Ste3) and vacuolar protease CPS in yeast cells. Nhx1p is located at the late endosomes with multivesicular bodies (MVB). Ste3 is degraded while Cps1p is processed in vacuole and becomes matured. For more details, see the reference [20].

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caused the accumulation of CPS in the prevacuolar compartment which blocks the destination of CPS to the vacuole. It has been described that traﬃcking of CPS from prevacuoles to the vacuole, multivesicular body structures (MVBs) [20] occurs (Fig. 5).

In this process a series of proteins named VPS (vacuolar protein sorting) are involved [19, 21].

The first step involves Vps27. Vps27 knockout also caused accumulation of CPS in the prevacuolar compartments [20]. These results suggested that Nhx1p might affect the process of Vps27p recruitment to the early endosomes by pH regulation of the endosomes. We successfully established an system of MVB formation and found that Nhx1 knockout affects MVB formation [20] (Fig.

6). Vps27p recruitment on the early endosomes was also aﬀected for the Nhx1 knockout cells [20]. The most important ﬁnding was that for the MVB formation, acidic pH is better than neutral pH (Fig.

7). We further analyzed pH on the endosome surface

facing to the cytoplasm by using chimeric protein of Vps27p and pHluorin, a pH indicator GFP variant.

The results indicated that pH on the endosome surface is signiﬁcantly more acidic than the cytoplasm detected by free pHluorin (Fig. 8). Based on these observations we concluded that acidic pH environment on the endosome surface provided by Nhx1p is required for recruitment of Vps27p and acts as the trigger for MVB formation. This acidic pH may be also required for fusion of MBV to the vacuole. It was reported that Nhx1p is required for the fusion of MVB to the vacuole although it is not shown how pH regulation by Nhx1p is involved in the process [22]. It has been shown that Vps27p has an amino acid sequence called FYVE required for binding to phosphatidyl inositol phosphate (PIP) [23]. We found that Vps27p binds to liposomes with PIP more eﬃciently in acidic pH conditions [24].

Conclusion

We found that acidic pH on the early endosome surface formed by Nhx1p is an important factor for the recruitment of Vps27p, the ﬁrst element of MVB formation. This ﬁnding reveals a new physiological role for Nhx1p because pH regulation by Nhx1 is

Fig. 6 Schematic illustration of the assay procedure of MVB formation (top panel). Yeast lysate prepared from yeast cells are incubated at 30℃ with ﬂuorescent dye HPTS and ATP. After the incubation ﬂuorescent dye quencher DPX is added to stop the uptake of HPTS and then the vesicles are separated from the free dyes by centrifugation. MVB formation was indicated by measuring HPTS fluorescence intensity taken up into the vesicles. Time course of the uptake of HPTS is shown for the wild type vesicles and the mutant vesicles forΔ 1 andΔ 27 in the bottom panel

[20].

Fig. 7 MVB formation detected by the uptake of HPTS (Fig. 6) is pH dependent [20].

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thought to be important for the luminal pH regulation.

This observation also raises a new possibility of the importance of H^＋ localized in a certain area of the cells. In this connection two similar observations should be noted. Firstly, it is shown that local pH regulation by NHE5 in neuronal cells is important in the feedback regulation of NMDA (N-methyl-D- aspartate) receptors [25]. NHE5 shuttles between the endosomes and plasma membrane in nerve cells.

NMDA induces accumulation of NHE5 molecules at the plasma membrane of the dendritic spine and causes decrease in the activity of receptors in the synapse.

Thus the pH based negative feedback mechanism of NMDA receptor is proposed. Secondly, it has been described that plasma membrane type NHE1 provides a local microdomain with higher pH on the plasma membrane surface that induces recruitment of carcineurin B and subsequent enhancement of NFκB [26]. These two instances support the importance of local pH regulation in the cell physiology.

The precise molecular mechanisms regulated by NHE6 are still open to the future studies. However, the new physiological role of Nhx1 proposed here will give a clue for future studies to elucidate the roles of pH on the surface of the plasma membrane or endosome membrane by NHE6 in mammalian cells.

Acknowledgement. Our studies described in this review were supported ﬁnancially by Grant-in aid for a Scientiﬁc Research from the

Ministry of Education, Science, Culture, Sports and Technology of Japan. This manuscript was kindly reviewed by Dr. Paul Moritoshi of Chygokugakuen University.

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Accepted March 29, 2013.

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