The Na<sup>+</sup>/H<sup>+</sup>Exchanger NHE6 in the Endosomal Recycling System Is Involved in the Development of Apical Bile Canalicular Surface Domains in HepG2 Cells

Ohgaki, Ryuichi; Matsushita, Masafumi; Kanazawa, Hiroshi; Ogihara, Satoshi; Hoekstra, Dick; IJzendoorn, Sven C. D. van

doi:10.1091/mbc.e09-09-0767

Cited by 67 publications

(70 citation statements)

References 40 publications

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“…9D). These results indicate that Nhx1p contributes to the regulation of endosome luminal pH by means of proton leakage from the endosome, as do organellar NHEs in mammalian cells (24,25). Lowering the pH of the endosome lumen would not account for the impaired MVB formation in nhx1⌬ cells because MVB formation observed in vitro was enhanced by acidic pH, as shown in Fig.…”

Section: Endosomal Na ϩ /H ϩ Exchange Activity Is Required For Efficimentioning

confidence: 74%

“…Although NHE1-5 are mainly found in plasma membranes, NHE6 -9 are mainly distributed to organellar membranes. We have shown previously that in mammalian cells Na ϩ /H ϩ exchangers contribute to endosomal pH homeostasis by allowing proton leakage from the endosomal lumen in cooperation with V-ATPase activity (23)(24)(25)(26). The budding yeast S. cerevisiae also expresses Na ϩ /H ϩ exchangers, one of which, Nhx1p, localizes to the late endosomes (prevacuolar compartment) and is highly homologous to the NHEs found in mammalian cells (27,28).…”

mentioning

confidence: 99%

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

Mitsui

Koshimura

Yoshikawa

et al. 2011

Journal of Biological Chemistry

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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 endosomelocalized 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.Multivesicular bodies (MVBs) 3 are a subset of late endosomes that contain internal vesicles, permitting precursor and plasma membrane proteins to reach the lysosome or vacuole lumen for degradation or maturation, respectively. In eukaryotic cells, the MVB pathway controls various important processes such as receptor down-regulation, antigen presentation, cytokinesis, retroviral budding, and autophagy (1-6). In the budding yeast Saccharomyces cerevisiae, previous studies identified a number of genes involved in vacuolar protein sorting (VPS) (7,8). These Vps proteins function at distinct steps of protein transport between the Golgi complex and the vacuole (7,8). A subset of Vps proteins, class E proteins, is involved in MVB formation. Most class E proteins are components of five distinct complexes; Vps27p-Hse1p (also called endosomal sorting complexes required for transport-0 (ESCRT-0), -I, -II, and -III and Vps4p. These complexes are required for the formation of internal vesicles and sorting of cargo proteins to the vesicles from the endosomal membrane (3, 9). Vps27p (ESCRT-0) recruits ESCRT-I, which in turn recruits and/or activates ESCRT-II and ESCRT-III at the endosome (3, 10). Recent in vitro studies have provided new insights into the precise role of each ESCRT complex in MVB formation (11-13). ESCRT-0 initiates the MVB sorting process by recruiting ESCRT-I to the endosomal membrane and concentrating ubiquitylated MVB cargos to the vesicle budding site (12). ESCRT-I and ESCRT-II are directly involved in membrane budding by stabilizing the bud necks, whereas ESCRT-III is responsible for the cleavage of bud necks (11-13). Finally, the AAA-type ATPase Vps4p dissociates the ESCRT-III complex from the endosome (14). The sequential activity of these complexes on the cytoplasmic surface of endosomes directs the budding and fission of vesicles into the lumen of the endosome and the sequestration of cargo proteins in the vesicle. Defects in ESCRT proteins prevent internal vesicle formation and result in exagg...

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Section: Endosomal Na ϩ /H ϩ Exchange Activity Is Required For Efficimentioning

confidence: 74%

mentioning

confidence: 99%

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

Mitsui

Koshimura

Yoshikawa

et al. 2011

Journal of Biological Chemistry

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“…Knockdown of RACK1 increases NHE6 levels in endosomes, elevates endosomal pH, and disturbs transferrin uptake (29). Previously, we reported that knockdown or overexpression of NHE6 in hepatoma HepG2 cells disturbs endosomal pH and polarized membrane traffic, and that consequently it causes a reduction in the number of apical bile canalicular surface domains (30). These studies suggest that NHE6 plays important roles in the maintenance of membrane traffic in endosomal compartments via pH regulation.…”

Section: CLmentioning

confidence: 86%

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

Xinhan

Matsushita

Numaza

et al. 2011

American Journal of Physiology-Cell Physiology

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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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“…Another important function of NHE6 was demonstrated in hepatoma HepG2 cells, where NHE6 localizes to recycling endosomes and colocalizes with transcytosing bulk membrane lipids [122]. Knock-down of NHE6 lowered recycling endosomal pH and surprisingly, disrupted the apical canalicular plasma membrane with failure to traffick or maintain apical proteins; these effects were associated with reduced abundance of apical lipids.…”

Section: Slc9a6 -Nhe6mentioning

confidence: 99%

“…Knock-down of NHE6 lowered recycling endosomal pH and surprisingly, disrupted the apical canalicular plasma membrane with failure to traffick or maintain apical proteins; these effects were associated with reduced abundance of apical lipids. Thus, in HepG2 cells endosomal NHE6 is important for maintaining the polarized distribution of membrane lipids at the apical surface, the maintenance of apical bile canaliculi and consequently cell polarity [122]. In addition to cell polarity, NHE6 participates in clathrin-dependent endocytosis by alkalinizing clathrin containing early endocytic vesicles [179].…”

Section: Slc9a6 -Nhe6mentioning

confidence: 99%

Traditional and emerging roles for the SLC9 Na+/H+ exchangers

Fuster

Alexander

2013

Pflugers Arch - Eur J Physiol

141

122

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The SLC9 gene family encodes Na + /H + exchangers (NHEs). These transmembrane proteins transport ions across lipid bilayers in a diverse array of species from prokaryotes to eukaryotes, including plants, fungi and animals. They utilize the electrochemical gradient of one ion to transport another ion against its electrochemical gradient. Currently, 13 evolutionarily conserved NHE isoforms are known in mammals [46,22,128]. The SLC9 gene family (solute carrier classification of transporters:www.bioparadigms.org) is divided into three subgroups [46]. The SLC9A subgroup encompasses plasmalemmal isoforms NHE1-5 (SLC9A1-5) and the predominantly intracellular isoforms NHE6-9 (SLC9A6-9). The SLC9B subgroup consists of two recently cloned isoforms, NHA1 and NHA2 (SLC9B1 and SLC9B2, respectively). The SLC9C subgroup consist of a sperm specific plasmalemmal NHE (SLC9C1) and a putative NHE, SLC9C2, for which there is currently no functional data [46].NHEs participate in the regulation of cytosolic and organellar pH as well as cell volume. In the intestine and kidney, NHEs are critical for transepithelial movement of Na + and HCO 3 -and thus for whole body volume and acid-base homeostasis [46]. Mutations in the NHE6 or NHE9 genes cause neurological disease in humans and are currently the only NHEs directly linked to human disease.However, it is becoming increasingly apparent that members of this gene family contribute to the pathophysiology of multiple human diseases.

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The Na⁺/H⁺Exchanger NHE6 in the Endosomal Recycling System Is Involved in the Development of Apical Bile Canalicular Surface Domains in HepG2 Cells

Cited by 67 publications

References 40 publications

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

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

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

Traditional and emerging roles for the SLC9 Na+/H+ exchangers

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The Na+/H+Exchanger NHE6 in the Endosomal Recycling System Is Involved in the Development of Apical Bile Canalicular Surface Domains in HepG2 Cells

Cited by 67 publications

References 40 publications

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

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

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

Traditional and emerging roles for the SLC9 Na+/H+ exchangers

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The Na⁺/H⁺Exchanger NHE6 in the Endosomal Recycling System Is Involved in the Development of Apical Bile Canalicular Surface Domains in HepG2 Cells

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