Cell Surface Levels of Organellar Na+/H+ Exchanger Isoform 6 Are Regulated by Interaction with RACK1

Abstract

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. 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. Maintenance of the luminal pH of intracellular compartments is fundamental for various cellular events, such as receptor-mediated endocytosis and recycling to the plasma membrane (1Carnell L. Moore H.P. J. Cell Biol. 1994; 127: 693-705Crossref PubMed Scopus (67) Google Scholar, 2Cosson P. de Curtis I. Pouyssegur J. Griffiths G. Davoust J. J. Cell Biol. 1989; 108: 377-387Crossref PubMed Scopus (107) Google Scholar). Tight regulation of the pH is accomplished by the coordinated functioning of a subset of ion transporters. The vacuolar H+-ATPase (V-ATPase) 2The abbreviations used are: V-ATPasevacuolar H+-ATPaseNHENa+/H+ exchangersiRNAsmall interfering RNAHAhemagglutininPBSphosphate-buffered salineTfntransferrinFITCfluorescein isothiocyanateTfRtransferrin receptorPNGase Fpeptide: N-glycosidase FNHSN-hydroxysuccinimide. is a primary mediator of acidification (3Futai M. Oka T. Sun-Wada G. Moriyama Y. Kanazawa H. Wada Y. J. Exp. Biol. 2000; 203: 107-116Crossref PubMed Google Scholar). A number of observations have also indicated the existence of proton leak systems. For example, pharmacological inhibition of V-ATPase results in an immediate dissipation of the proton gradient across the organellar membrane (4Kim J.H. Lingwood C.A. Williams D.B. Furuya W. Manolson M.F. Grinstein S. J. Cell Biol. 1996; 134: 1387-1399Crossref PubMed Scopus (125) Google Scholar). The luminal pH along the secretory pathway becomes gradually acidic from endoplasmic reticulum to Golgi and secretory vesicles. A previous fluorescence imaging study using live endocrine cells estimated that proton pump activity increases and, on the contrary, proton leakage decreases along the secretory pathway (5Wu M.M. Grabe M. Adams S. Tsien R.Y. Moore H.P. Machen T.E. J. Biol. Chem. 2001; 276: 33027-33035Abstract Full Text Full Text PDF PubMed Scopus (222) Google Scholar). Although our understanding of the molecular identities responsible for proton leakage is incomplete, it is assumed that organelle-specific pH is controlled by a balance between two major activities (i.e. proton pumping by V-ATPases and proton leakage from the organellar lumen) (6Paroutis P. Touret N. Grinstein S. Physiol. (Bethesda). 2004; 19: 207-215Crossref PubMed Scopus (356) Google Scholar). vacuolar H+-ATPase Na+/H+ exchanger small interfering RNA hemagglutinin phosphate-buffered saline transferrin fluorescein isothiocyanate transferrin receptor peptide: N-glycosidase F N-hydroxysuccinimide. To date, nine Na+/H+ exchanger (NHE) isoforms have been identified in mammals. NHE1-NHE5 are localized to the plasma membrane in various cell types (7Sardet C. Franchi A. Pouyssegur J. Cell. 1989; 56: 271-280Abstract Full Text PDF PubMed Scopus (700) Google Scholar, 8Orlowski J. Kandasamy R.A. Shull G.E. J. Biol. Chem. 1992; 267: 9331-9339Abstract Full Text PDF PubMed Google Scholar, 9Tse C.M. Levine S.A. Yun C.H. Montrose M.H. Little P.J. Pouyssegur J. Donowitz M. J. Biol. Chem. 1993; 268: 11917-11924Abstract Full Text PDF PubMed Google Scholar, 10Attaphitaya S. Park K. Melvin J.E. J. Biol. Chem. 1999; 274: 4383-4388Abstract Full Text Full Text PDF PubMed Scopus (121) Google Scholar, 11Baird N.R. Orlowski J. Szabo E.Z. Zaun H.C. Schultheis P.J. Menon A.G. Shull G.E. J. Biol. Chem. 1999; 274: 4377-4382Abstract Full Text Full Text PDF PubMed Scopus (144) Google Scholar). NHE6 and NHE9 are predominantly localized to sorting and recycling endosomes in several cultured cell lines (12Nakamura N. Tanaka S. Teko Y. Mitsui K. Kanazawa H. J. Biol. Chem. 2005; 280: 1561-1572Abstract Full Text Full Text PDF PubMed Scopus (283) Google Scholar). NHE7 and NHE8 are localized primarily to the trans-Golgi network and mid-trans-Golgi stacks, respectively (12Nakamura N. Tanaka S. Teko Y. Mitsui K. Kanazawa H. J. Biol. Chem. 2005; 280: 1561-1572Abstract Full Text Full Text PDF PubMed Scopus (283) Google Scholar, 13Brett C.L. Wei Y. Donowitz M. Rao R. Am. J. Physiol. 2002; 282: C1031-C1041Crossref PubMed Scopus (147) Google Scholar, 14Numata M. Orlowski J. J. Biol. Chem. 2001; 276: 17387-17394Abstract Full Text Full Text PDF PubMed Scopus (218) Google Scholar). Four organellar NHEs may mediate K+/H+ exchange rather than Na+/H+ exchange under physiological conditions and could be responsible for proton leakage from organelles, contributing to the establishment of a unique organellar luminal pH. The boundaries of such intracellular compartments are not static and not definite, since the organelles are connected by continuous membrane trafficking. Therefore, the steady state localization of NHE isoforms is under a dynamic equilibrium. However, little is known about the mechanisms that regulate the localization and activity of organellar NHEs. NHEs are composed of a relatively conserved N-terminal transmembrane domain (10-12 transmembrane helices) and a more variable C-terminal hydrophilic domain. In NHE1, the most extensively studied isoform, the C-terminal domain is cytoplasmic (15Wakabayashi S. Pang T. Su X. Shigekawa M. J. Biol. Chem. 2000; 275: 7942-7949Abstract Full Text Full Text PDF PubMed Scopus (199) Google Scholar) and serves as a regulatory domain. The interaction of NHE1 and various and plays an role in the activity and localization of NHE1 Zaun H.C. T. Orlowski J. Grinstein S. J. Cell Biol. 2000; PubMed Scopus Google Scholar, S. J. J. Biol. Chem. 2001; 276: Full Text Full Text PDF PubMed Scopus Google Scholar, W. K. J. J. Biol. Chem. 2001; 276: Full Text Full Text PDF PubMed Scopus Google Scholar, T. Su X. S. Shigekawa M. J. Biol. Chem. 2001; 276: Full Text Full Text PDF PubMed Scopus Google Scholar). in the C-terminal of NHEs the existence of NHE and regulatory We a yeast two-hybrid using the C terminus of NHE9 as to that may regulate intracellular NHEs. a scaffold identified from a as an for kinase C C.H. J. L. S. A. 1994; PubMed Scopus Google was that NHE isoforms and The of RACK1 in the distribution of NHE6 between endosomes and the cell and in the of pH was Cell and and HeLa cells and The cells cultured in For of and used to the of RACK1 and a from for RACK1 of was for in and the was by the and The was the For of the for and by from NHE (12Nakamura N. Tanaka S. Teko Y. Mitsui K. Kanazawa H. J. Biol. Chem. 2005; 280: 1561-1572Abstract Full Text Full Text PDF PubMed Scopus (283) Google Scholar). In the used as and and and and and and and and and For the of in was of the and and and The two and by the and for in and as (12Nakamura N. Tanaka S. Teko Y. Mitsui K. Kanazawa H. J. Biol. Chem. 2005; 280: 1561-1572Abstract Full Text Full Text PDF PubMed Scopus (283) Google Scholar). The of was from to the of an a was by the and The was for the of a for to and To an for membrane a N-terminal was from a by the and The is in the was The was further used as a to the A and was the and The N-terminal was from the and in the The was To an for the RACK1 binding region of NHE6 the from the for was and from was from primary NHE1 and transferrin receptor from and was from from and from and in the C terminus. of NHE6 and NHE9 and of was using the yeast two-hybrid screening was using from the C terminus of NHE9 as bait. was used to a the The was and the on yeast and for and and The by using the for the region of the and The to was in cells cultured on in was the using a and for cells and to In was of and of in for cells in and a and for for the was for was and bound the of organellar cells in a a of the was to of NHEs. F to the of the was by and The in and the indicated primary and in the using an and by of cells and in and a on for and for and the for of to by and bound RACK1 was by HeLa cells and in and a on for in a a and for The and of of for four and by NHE6 and RACK1 by and cells and in and the primary for by the for In a was to the primary To the localization of transferrin cells for in and The using a a an and Cell cells and in for in and cells in and a Cell in a a and for of of and for by and The by and by The of the was using J. of imaging was to the pH to the K. J. N. J. Cell Biol. 1992; PubMed Scopus Google Scholar) and from HeLa cells for in and and cells using an a using an and The and for and for a pH was and a of of the pH pH of the recycling endosome was and the of fluorescence was The fluorescence was in the of pH In a the pH values of a of cells and are as the of was used for between RACK1 and cells in the of pH and the of cell transferrin receptor and NHE6 A of was to be of RACK1 as an NHE9 of a yeast two-hybrid A was using the C-terminal of NHE9 as bait. the screening of and and of RACK1 and These two on and RACK1 of the C-terminal of RACK1 and We confirmed the interaction using a The C-terminal bound RACK1 in a To further the region responsible for from of the NHE9 C terminus RACK1 the and but not These results that the central region of the NHE9 C-terminal is responsible for binding of RACK1 binding domain the NHE9 C terminus. and of various NHE9 and in and by bound to and cell by and RACK1 was by an The C of NHE9 to the the C terminus of mammalian organellar NHEs and and yeast the organellar the is P.J. Williams J. M. Cell. 19: PubMed Scopus Google Scholar, P.J. Williams Y. Orlowski J. M. J. Cell 2005; PubMed Scopus Google Scholar, Rao R. J. Biol. Chem. 2001; 276: Full Text Full Text PDF PubMed Scopus Google Scholar, T. H. S. A. PubMed Scopus Google Scholar, Y. M. J. 2005; PubMed Scopus Google Scholar, R. C.L. S. Rao R. J. Biol. Chem. 2004; Full Text Full Text PDF PubMed Scopus Google Scholar). The is is a cytoplasmic protein, could the C terminus of NHE9 that region of the the To the of the NHE9 C an in was of We used a transmembrane of known as a to the is a membrane predominantly in the recycling endosomes L. T. H. R. 1993; PubMed Scopus Google Scholar, L. Moore H.P. Grinstein S. J. Cell Biol. 2000; PubMed Scopus Google Scholar). The terminus of the cytoplasm, the C terminus is in the endosome A was to the C terminus of an of and the was in cells. was the of cells, and The localization of was confirmed by the fluorescence the C and the of the was by an the and in cells In in cells lumen) intracellular was by the the was as a the cytoplasm, although the fluorescence localized to the recycling endosome is the to the in the These results that the of by the in the but not in the organellar In cells NHE9 the C by the in the region and the The of by that the the the C terminus of was cells, the was the cytoplasm, as in The used in is of NHE9 and not in cells. These results indicate that the NHE9 C terminus is interaction RACK1 is topologically RACK1 NHEs in and in of the RACK1 binding region of NHE9 is to of the in NHE6 and NHE7 but not NHE8 to that RACK1 also NHE6 and To using of the C terminus of organellar NHEs of RACK1 to and as as was by However, binding to to was not the of the These results indicate that RACK1 may be a binding of NHE7, and To an association between RACK1 and organellar NHEs in cells, co-immunoprecipitation For to and We a the and but of of NHE7 and NHE9 not The of the was by NHE7, and NHE9 in cells, and the was confirmed an NHE6 was the membrane was the are two NHE6 and an of between and of the M. S. Shigekawa M. K. J. Biol. Chem. 2001; 276: Full Text Full Text PDF PubMed Scopus Google on The molecular of NHE6 was of and on the A previous study that NHE6 is an M. S. Shigekawa M. K. J. Biol. Chem. 2001; 276: Full Text Full Text PDF PubMed Scopus Google Scholar). Therefore, also the of F to a of In the of the F NHE6 was as two (i.e. a major of and a of The molecular to to the of and the to to the M. S. Shigekawa M. K. J. Biol. Chem. 2001; 276: Full Text Full Text PDF PubMed Scopus Google Scholar). The two of NHE6 in to on the that the is in cells. results HeLa cells not We also an of was not NHE6 was and was in the of NHE6 that it was it could be a of is that of NHE6 was accompanied by a in an of NHE6 under was an in the of than NHE6 was These are to be of as (15Wakabayashi S. Pang T. Su X. Shigekawa M. J. Biol. Chem. 2000; 275: 7942-7949Abstract Full Text Full Text PDF PubMed Scopus (199) Google Scholar). F the major of and was accompanied by an in the of the and NHE6 to to and to the of NHE6 to the as that NHE6 an of that is for the in the molecular between and NHE6 not but a in the of the In co-immunoprecipitation NHE6 from a HeLa cell RACK1 was that RACK1 and NHE6 in that than of total RACK1 was Therefore, a of total RACK1 in the cells to be Although identified RACK1 as a binding of RACK1 NHE6 in HeLa the localization of NHE6 in HeLa cells using The of NHE6 was confirmed by between NHE6 and NHE6 the transferrin receptor is localization the sorting and recycling endosomes, as C.L. Wei Y. Donowitz M. Rao R. Am. J. Physiol. 2002; 282: C1031-C1041Crossref PubMed Scopus (147) Google Scholar) In localized predominantly the sorting endosomes in the cell (12Nakamura N. Tanaka S. Teko Y. Mitsui K. Kanazawa H. J. Biol. Chem. 2005; 280: 1561-1572Abstract Full Text Full Text PDF PubMed Scopus (283) Google NHE6 primarily in the recycling endosomes, although NHE6 was in the sorting of RACK1 and NHE6 was further by The localization of RACK1 on the cell I. P.J. Williams M. Cell. 19: PubMed Scopus Google Scholar, C.M. Yun C.H. N. J. Biol. Chem. 2002; Full Text Full Text PDF PubMed Scopus Google Scholar, de M. L. Y. P.J. J. 2000; PubMed Scopus Google Scholar, A. G. 2001; PubMed Scopus Google Scholar, M. R. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, L. A. I. A. J. Biol. Chem. 1999; 274: Full Text Full Text PDF PubMed Scopus Google Scholar, T. J. A. J. Biol. Chem. 2001; 276: Full Text Full Text PDF PubMed Scopus Google Scholar). in RACK1 was the and co-localization of NHE6 and RACK1 was in the cell RACK1 not in the co-localization was not of RACK1 the the of the interaction between NHE6 and an RNA knockdown of The of RACK1 was of In of was not the of the organellar NHEs are to be responsible for proton the of RACK1 RNA on the pH of recycling endosomes, the of NHE6 is We a fluorescence imaging using two and In cells a the luminal pH of recycling endosomes was pH in the RACK1 knockdown cells, pH was elevated to pH The was the was than have that by a vacuolar V-ATPase results in accompanied by the of cell and a of recycling of Y. J. T.E. T. M. 2004; PubMed Scopus Google Scholar, S. T.E. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). such significantly in RACK1 knockdown cells, although the and distribution of the compartments not significantly of by to of the in RACK1 knockdown cells, although the total amount of was of These results are also RACK1 of the recycling We the NHE6 in binding RACK1 the results of RNA A on was in cells the RACK1 binding region of NHE6 In cells the fluorescence of was than in cells. a decrease in the was not in cells These results indicate that the in RACK1 knockdown cells is to of the association between RACK1 and of RACK1 binding region on the RACK1 binding region of NHE6 was in HeLa cells. cultured for and and by fluorescence was confirmed by fluorescence cells are indicated an of NHE6 the Cell in RACK1 an of ion activity an in the amount of NHE6 in the recycling endosomes could for the in RACK1 knockdown cells. To the molecular of the in RACK1 knockdown cells, the intracellular localization of NHE6 was by in RACK1 knockdown cells, the of NHE6 is localized the recycling endosomes and a of co-localization Although organellar NHEs primarily in intracellular in compartments and to the plasma as NHE6 and NHE7 C.L. Wei Y. Donowitz M. Rao R. Am. J. Physiol. 2002; 282: C1031-C1041Crossref PubMed Scopus (147) Google Scholar, P.J. Williams J. M. Cell. 19: PubMed Scopus Google Scholar). Therefore, the amount of NHE6 on the cell A that the amount of NHE6 on the plasma membrane was RACK1 was of The was was not the of of RACK1 the of NHE1 and are also plasma membrane was not and of The total amount of NHE6 in cells was not significantly by RNA and the of NHE6 on was not and The total of NHE1 and also largely results to in the RACK1 results are to the of RACK1 and not not These results that RACK1 the distribution of NHE6 between intracellular compartments and the plasma of a NHE9 identified an interaction between the C terminus of NHE9 and RACK1 is a scaffold identified as an for activated kinase C C.H. J. L. S. A. 1994; PubMed Scopus Google Scholar). RACK1 is to the of in and of are interaction on the of J. A. 1996; PubMed Scopus Google Scholar) that the are to a the to binding of the A. J. 2002; PubMed Scopus Google Scholar). by two-hybrid screening in study the C-terminal of that are for identified to C.H. J. L. S. A. 1994; PubMed Scopus Google cell J. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google that ion C.M. Yun C.H. N. J. Biol. Chem. 2002; Full Text Full Text PDF PubMed Scopus Google and ion exchangers I. P.J. Williams M. Cell. 19: PubMed Scopus Google that RACK1 plays in in intracellular is the an interaction of RACK1 an ion that primarily in intracellular compartments. is that RACK1 binding a plasma membrane Na+/H+ I. P.J. Williams M. Cell. 19: PubMed Scopus Google Scholar). However, the on RACK1 that to and organellar NHEs are to be as are the of two of NHE9 RACK1 is localized to the I. P.J. Williams M. Cell. 19: PubMed Scopus Google Scholar, C.M. Yun C.H. N. J. Biol. Chem. 2002; Full Text Full Text PDF PubMed Scopus Google Scholar, de M. L. Y. P.J. J. 2000; PubMed Scopus Google Scholar, A. G. 2001; PubMed Scopus Google Scholar, M. R. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, L. A. I. A. J. Biol. Chem. 1999; 274: Full Text Full Text PDF PubMed Scopus Google Scholar, T. J. A. J. Biol. Chem. 2001; 276: Full Text Full Text PDF PubMed Scopus Google Scholar). Therefore, interaction NHE9 be the C terminus of NHE9 is exposed to the The of the C terminus of mammalian organellar as as and yeast P.J. Williams J. M. Cell. 19: PubMed Scopus Google Scholar, P.J. Williams Y. Orlowski J. M. J. Cell 2005; PubMed Scopus Google Scholar, Rao R. J. Biol. Chem. 2001; 276: Full Text Full Text PDF PubMed Scopus Google Scholar, T. H. S. A. PubMed Scopus Google Scholar, Y. M. J. 2005; PubMed Scopus Google Scholar, I. P.J. Williams M. Cell. 19: PubMed Scopus Google Scholar). that the C terminus of NHE9 is in the For mammalian organellar NHE several the cytoplasmic of the C the cytoplasmic the C terminus P.J. Williams J. M. Cell. 19: PubMed Scopus Google Scholar, P.J. Williams Y. Orlowski J. M. J. Cell 2005; PubMed Scopus Google Scholar, I. P.J. Williams M. Cell. 19: PubMed Scopus Google Scholar). RACK1 and a in the C interaction RACK1 to be by the central region of the hydrophilic NHE9 C terminus The of region in NHE9 is conserved in NHE6 and In indicate that RACK1 also two organellar but not the more NHE8 The interaction in was confirmed by The of the RACK1 binding region of organellar NHEs not to RACK1 binding However, the RACK1 binding in known binding in in A. J. 2002; PubMed Scopus Google Scholar). We is a region the RACK1 binding region of NHE9 that is for binding by of However, was of RACK1 in not the interaction may be by in the NHE9 C terminus rather than by a a of the RACK1 binding region and co-immunoprecipitation NHE the binding region be to about binding than the C terminus contribute to by in the NHE6 in HeLa and cells to be a an of although have not the of been of of NHE6 is and is the that is in cell in cells, localized to the endosomes not Therefore, the of not to the localization of and the of the is not of between NHE6 and a yeast of plays an role in the regulation of pH. of the results in acidification of intracellular compartments and of a vacuolar and of an from endosomes to the K. Biol. Cell. 2000; PubMed Scopus Google Scholar, C.L. S. Rao R. Biol. Cell. 2005; PubMed Scopus Google Scholar). our previous study that of organellar NHEs results in the of intracellular compartments in mammalian cell lines (12Nakamura N. Tanaka S. Teko Y. Mitsui K. Kanazawa H. J. Biol. Chem. 2005; 280: 1561-1572Abstract Full Text Full Text PDF PubMed Scopus (283) Google Scholar). These are intracellular NHEs of the proton leak in compartments. In the knockdown of RACK1 in the of the The of results in an in intracellular may contribute to the pH by the proton from We that the total of NHE6 was not in RACK1 knockdown cells, that RACK1 is not in of acidification of endocytic compartments is for receptor-mediated endocytosis and recycling Y. J. T.E. T. M. 2004; PubMed Scopus Google Scholar, S. T.E. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, A. A. S. A. PubMed Scopus Google Scholar). These results that association RACK1 is in localization of NHE6 the plasma membrane and in receptor-mediated endocytosis of by regulating the pH of the recycling are two mechanisms by RACK1 could organellar pH by interaction interaction RACK1 endocytosis of NHE6 and NHE6 the cell surface, RACK1 association of These two are not and further are to the molecular by RACK1 the distribution of Although a co-localization of NHE6 and RACK1 was by the the interaction to be was small in the cell rather than the recycling Therefore, that the interaction sorting endosomes in the plasma membrane RACK1 was to in A. G. 2001; PubMed Scopus Google Scholar). also the of the intracellular distribution of NHE6 could for the The results in study not the that RACK1 also the of such as V-ATPase and the ion homeostasis in the endosomes of have not been about such an However, it be to the of RACK1 knockdown on the and proton pumping activity of V-ATPase in a We are that the of RACK1 binding could the ion activity of NHE6 as as intracellular the activity be to such as by RACK1 not have than and known is to scaffold such as C. it to the of RACK1 binding on the localization and ion Na+/H+ exchange activity the and the pH the intracellular membrane and the activity of NHEs. of in the and of ion further the of RACK1 on the ion activity of In also to the of the interaction are the in the organellar NHE of Na+/H+ exchangers is by various C terminus. We have identified RACK1 as a binding of and in interaction between RACK1 and NHE6 was confirmed by co-immunoprecipitation and by RACK1 the distribution of and it is that the physiological of ion are localized predominantly could be by in the distribution between intracellular compartments and the plasma a scaffold protein, RACK1 could regulate NHEs in to a of is to that the and localization of NHE6 is kinase in the of organellar NHEs in cell