GGA1 interacts with the endosomal Na+/H+ exchanger NHE6 governing localization to the endosome compartment

Mutations in the endosomal Na+/H+ exchanger 6 (NHE6) cause Christianson syndrome, an X-linked neurological disorder. NHE6 functions in regulation of endosome acidification and maturation in neurons. Using yeast two-hybrid screening with the NHE6 carboxyl terminus as bait, we identify Golgi-associated, gamma adaptin ear-containing, ADP-ribosylation factor (ARF) binding protein 1 (GGA1) as an interacting partner for NHE6. We corroborated the NHE6-GGA1 interaction using: coimmunoprecipitation; overexpressed constructs in mammalian cells; and coimmunoprecipitation of endogenously expressed GGA1 and NHE6 from neuroblastoma cells, as well as from the mouse brain. We demonstrate that GGA1 interacts with organellar NHEs (NHE6, NHE7, and NHE9) and that there is significantly less interaction with cell-surface localized NHEs (NHE1 and NHE5). By constructing hybrid NHE1/NHE6 exchangers, we demonstrate the cytoplasmic tail of NHE6 interacts most strongly with GGA1. We demonstrate the colocalization of NHE6 and GGA1 in cultured, primary hippocampal neurons, using super-resolution microscopy. We test the hypothesis that the interaction of NHE6 and GGA1 functions in the localization of NHE6 to the endosome compartment. Using subcellular fractionation experiments, we show that NHE6 is mislocalized in GGA1 KO cells, wherein we find less NHE6 in endosomes, but more NHE6 transport to lysosomes, and more Golgi retention of NHE6, with increased exocytosis to the surface plasma membrane. Consistent with NHE6 mislocalization, and Golgi retention, we find the intraluminal pH in Golgi to be alkalinized in GGA1-null cells. Our study demonstrates a new interaction between NHE6 and GGA1 which functions in the localization of this intracellular NHE to the endosome compartment.


Supplementary figures SUPPLEMENTAL
(A) Cell lysates from HEK293T cells expressing HA-tagged NHE5, NHE7, and NHE9 were subjected to western blot analysis with anti-HA to detect expression of NHE5, NHE7, and NHE9 constructs.
NHE6-HA was used as a control.Red asterisks denote the monomer and dimer of NHE5, NHE7, NHE9 and NHE6.
(C) Cell lysates from HEK293T cells expressing c-Myc-GGA1 with HA-tagged NHE5, NHE7, NHE9, and NHE6 were immunoprecipitated with an anti-c-Myc antibody.The precipitates were probed with HA and c-Myc antibodies.Western blot analysis was performed with anti-HA to detect the expression of NHEs.Red asterisks denote the monomer and dimer of NHE5, NHE7, NHE9 and NHE6.(B) Representative image of rabbit anti-NHE6 antibody specificity test (1:2500 dilution) using NHE6 wild type (WT) and NHE6 null mouse primary hippocampal neurons at 14 days in vitro (14 DIV).

Figure S4. Specificity of NHE6 primary antibody in 5x expanded rat neuronal cell cultures.
Hippocampal neuronal rat cell cultures of DIV 14 were imaged after 5x hydrogel expansion of cells nuclear stained with sytox green, shown in green and the cells were immuno-stained with (A) secondary only in wild type cells with anti-rabbit Alexa 647 (blue), (B) shows stained for rabbit NHE6 primary antibody with anti-rabbit Alexa 647 (blue) in wild type rat hippocampal neurons, and (C) stained for rabbit NHE6 primary antibody with anti-rabbit Alexa 647 (blue) in NHE6 KO rat hippocampal neurons.
The left panels show a single frame, and right panels show the average intensity projections of z-stacks imaged on confocal microscope.Intensities were adjusted to similar thresholds for better visualization.
Scale bar: 10 μm.(A) Western blot analysis of cell lysates harvested from HAP1 knockout (KO) and wildtype (WT) cell lines using GGA1 antibody.Tubulin was used as a loading control and GGA3 was used for the specificity of GGA1 KO.

Figure
Figure S2.GGA3 interaction with NHE6 and NHE9.(A)Cell lysates from HEK293T cells expressing Flag-hGGA3 with GFP-tagged hNHE6, mNHE9, and mGGA1 were immunoprecipitated with anti-Flag antibody.The precipitates were probed with GFP

Figure S5 .
Figure S5.Shortest distance of NHE6 and GGA1 in rat primary hippocampal neurons.(A) Representative max projection z-stack images of unexpanded (a) or expanded (b) primary rat hippocampal neurons at 14 days in vitro (14 DIV) stained for nucleus using Sytox TM Green Nucleic acid