The sodium/proton exchanger NHE8 regulates late endosomal morphology and function.
The pH and lumenal environment of intracellular organelles is considered essential for protein sorting and trafficking through the cell. We provide the first evidence that a mammalian NHE sodium (potassium)/proton exchanger, NHE8, plays a key role in the control of protein trafficking and endosome morphology. At steady state, the majority of epitope-tagged NHE8 was found in the trans-Golgi network of HeLa M-cells, but a proportion was also localized to multivesicular bodies (MVBs). Depletion of NHE8 in HeLa M-cells with siRNA resulted in the perturbation of MVB protein sorting, as shown by an increase in epidermal growth factor degradation. Additionally, NHE8-depleted cells displayed striking perinuclear clustering of endosomes and lysosomes, and there was a ninefold increase in the cellular volume taken up by LAMP1/LBPA-positive, dense MVBs. Our data points to a role for the ion exchange activity of NHE8 being required to maintain endosome morphology, as overexpression of a nonfunctional point mutant protein (NHE8 E225Q) resulted in phenotypes similar to those seen after siRNA depletion of endogenous NHE8. Interestingly, we found that depletion of NHE8, despite its function as a sodium (potassium)/proton antiporter, did not affect the overall pH inside dense MVBs.
Lawrence
Scott P
SP
Institute for Structural and Molecular Biology, Division of Biosciences, University College London, London, United Kingdom.
Bright
Nicholas A
NA
Luzio
J Paul
JP
Bowers
Katherine
K
D002451:Cell Compartmentation; D015536:Down-Regulation; D011992:Endosomes / Q000378:metabolism* / Q000648:ultrastructure; D004815:Epidermal Growth Factor / Q000378:metabolism; D000939:Epitopes / Q000276:immunology; D006367:HeLa Cells; D006801:Humans; D006863:Hydrogen-Ion Concentration; D008247:Lysosomes / Q000378:metabolism; D057146:Multivesicular Bodies / Q000378:metabolism / Q000648:ultrastructure; D050505:Mutant Proteins / Q000378:metabolism; D053140:Organelle Shape / Q000502:physiology*; D021381:Protein Transport; D034741:RNA, Small Interfering / Q000378:metabolism; D017923:Sodium-Hydrogen Exchangers / Q000378:metabolism*; D021601:trans-Golgi Network / Q000378:metabolism / Q000648:ultrastructure
D000939:Epitopes; D050505:Mutant Proteins; D034741:RNA, Small Interfering; C486775:SLC9A8 protein, human; D017923:Sodium-Hydrogen Exchangers; D004815:Epidermal Growth Factor