Dataset DOI: 10.5061/dryad.2bvq83c4f

Description of the data and file structure

All the raw files and data used in Figures 1-5 and Supplementary material are organized per figure.

"Source Data 1" files include a pdf file with all uncropped blots used for the corresponding figure. "Source Data" files contain all the raw images for the corresponding figure in the original captured format.

Files and variables

File: FIGURE_1-SOURCE_DATA.zip

Description: This folder contains 3 sub-folders with source data for Figure 1. Figure 1 is a 6-panel figure showing the location of amino acid substitutions reported in this manuscript (panel A), protein abundance (panel B), protein localization by confocal microscopy (panel C), protein stability (panel D), a schematic cartoon explaining kidney anion exchanger 1 (kAE1) activity and expected changes in intracellular pH (pHi) in variant-expressing cells. Panel F shows a graph reporting pHi in various cell lines and panel F shows a graph reporting the rate of intracellular alkalinisation normalised to wild-type (WT) cells incubated with doxycycline (+Dox).

The Folder entitled "A. PYMOL IMAGES" contains the pictures extracted from PYMOL to generate Figure 1 A 3D model showing kAE1 structure with zoomed images highlighting the location of the specific variants studied in the article. Files named "ALL MUTATIONS.pdf" and files in ".png" format can be opened using Adobe Acrobat. File "ALL MUTATIONS" shows in a single structure model the 3D structure of human kidney anion exchanger 1 with the location of the 3 variations described in the manuscript shown in navy blue. Individual files in ".png" format show a zoomed view of the location of amino acid substitution in the 3D structure of the anion exchanger 1.

The Folder entitled "B. Immunoblot on polarized cells" includes the source image of kAE1 for wildtype (WT), R295H, Y413H, S525F and R589H variants and housekeeping protein actin from immunoblot used for Figure 1 B. HA stands for hemagglutinin. This file can be opened in Adobe Acrobat or Preview.

The Folder "C. Immunofluorescence with beta-catenin" contains the original immunostaining images from polarized cells shown in Figure 1 C. “IMCD” indicates inner medullary collecting duct cells, “Cy3” and “Alexa488” correspond to the fluorophores attached to the secondary antibodies, beta-catenin (B-catenin) is a basolateral membrane marker. “Dapi” corresponds to nuclear marker 4′,6-diamidino-2-phenylindole. Each image can be opened with Adobe Acrobat or Preview.

The Folder "D. Cycloheximide Assay" contains the images for kAE1 WT and Y413H variant shown in Figure 1 D, both can be opened with Adobe Acrobat or Preview.

The Folder "F. pHi measurements" contains the Prism file with raw data used to generate Figure 1F. This experiment measured the initial, steady-state 2′,7′-bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein (BCECF) fluorescence from cells expressing WT or kAE1 variants, as an indication of steady-state pHi. “PTI” Stands for Photon Technologies International, the company that built the fluorometer. The file can be opened with Prism 11. Although both ANOVA and unpaired t test were calculated, the manuscript statistics only report the appropriate one-way ANOVA analysis.

The Folder "G. Rate of pHi alkalinisation" contains the Prism file with source data used to generate Figure 1G. The rates of intracellular alkalinisation upon switching the perfusion buffer to a chloride-free buffer data are normalized to WT +Dox in each experiment. The file can be opened with Prism 11.

File: FIGURE_2-SOURCE_DATA.zip

Description: Figure 2 is a 12-panel figure showing LC3B, p62 proteins and corresponding actin in kAE1 WT-, R295H-, S525F-, R589H-expressing cells (A-C), and in kidney homogenates from kAE1 R607H knockin (KI) homozygous, heterozygous or WT mice (panel J). Corresponding quantification graphs are shown in panels D-I for cells, and panels K&L for mouse kidneys.

The FIGURE 2_SOURCE_DATA Folder contains one folder titled “A-L LC3B &p62” corresponding to source data for panels A to L in Figure 2. “LC3” stands for Microtubule-associated protein 1A/1B-light chain 3. This folder is further divided between source data obtained in cells (panels A, B and C) or kidney lysates (panel J).

In the “CELLS” folder, there are 2 sub-folders with source data identifying LC3B and P62. Each contain 3 sub-folders (SET1, SET2, SET3) corresponding to the three biological replicate experiments used for quantification. In these folders, images are in TIF format and can be opened with Preview or Adobe Acrobat. The “CELLS” folder also contains two Prism files with corresponding quantifications used to generate panels D-I. Prism files can be opened using GraphPad Prism 11.

In “KIDNEY LYSATES” folder, there are two sub-folders named “P62” and “LC3” with source data for quantification of p62 and LC3 in kidney lysates (panel J). Files can either be opened with Adobe Acrobat or Preview. This folder also contains two Prism files with corresponding P62 and LC3 quantifications (panels K & L) from kidney lysates. These files can be opened using GraphPad Prism 11.

File: FIGURE_3-SOURCE_DATA.zip

Description: Figure 3 is a 11 panel figure showing cell surface biotinylation images for kAE1 WT and mutants, positive control sodium/potassium ATPase (Na⁺/K⁺ ATPase) and housekeeping protein actin (panel A), corresponding quantification (panel B), confocal images of cells expressing kAE1 WT, S525F or R589H mutants and transfected with a vector expressing LC3 coupled to fluorophores Red Fluorescent protein (RFP) and green fluorescence protein (GFP) (panel C) and corresponding quantification (panels D-F). Panels G-I show LC3B I and II, lysosomal associated membrane protein 1 (LAMP1) and housekeeping protein actin in WT and mutant cells exposed to control or conditions reducing pHi, with corresponding quantification in panels J&K.

This Folder contains 3 sub-folders. The Folder named "A&B. BIOTINYLATION BLOTS" contains the original blots in sub-folders named after each cell line and protein detected, and the Prism file named “Biotinylation” used to generate Figure 3A & B. Images can be opened with Preview or Adobe Acrobat. Folder named "C-F. LC3 GFP RFP” contains 3 sub-folders named “R589H”, “S525F” and “WT” which each contain the captures of live cell images from WT, R589H and S525F mutant cells used to generate Figure 3C. These files can be opened with Adobe Acrobat or Preview. For panels D, E and F, the file “eGFP-RFP-LC3 data” contains the source data and graphs used for these panels and can be opened with GraphPad Prism 11.

Folder named "G-K CELLS INCUBATED IN LOW pH MEDIA" contains original images, two corresponding Prism files named “LC3B” for LC3B protein quantification (panel J) and “LAMP1” for Lamp1 quantification (panel K) used to generate Figure 3 G-K. “Ctrl” stands for control. Images in TIF format can be opened using Adobe Acrobat or Preview. Prism files in “pzfx” or “prism” format can be opened using GraphPad Prism 11.

File: FIGURE_4-SOURCE_DATA.zip

Description: Figure 4 is a 6-panel figure that shows confocal images of WT, R589H or S525F-expressing mIMCD cells stained for Magic Red or kAE1 (panel A), with corresponding quantification of number of active lysosomes per cell and active lysosomal size (panel B). Panel C shows LAMP1 and kAE1 immunostaining in cortical kidney sections from WT or R607H KI mice (KI/KI) with corresponding lysosomal number and volume quantification in panel D. Panel E and F are the same for medullary kidney sections.

 This Folder contains two sub-folders named “A&B. MAGIC RED” and “C-F. LAMP1 IN TISSUES. The “A&B. MAGIC RED” folder contains two Prism files with source data named “MagicRed -GRAPHS LYSOSOME NUMBER” and “MagicRed Lysosome size “from Magic Red staining, as well as subfolders with source images for R589H, S525F mutants and WT. In each of these subfolders are the source images used to generate Figure 4, panel A. Images can be opened with Preview or Adobe Acrobat, files in “prism” or “pzfx” format can be opened with GraphPad Prism 11.

Folder “C-F. LAMP1 IN TISSUES” contains two Prism files named “LAMP1 CORTEX” and “LAMP1 MEDULLA” corresponding to image quantification from images of LAMP1 staining in cortical (CORTEX) or medullary (MED) kidney sections from WT and homozygous knockin (KI/KI) mice used to generate Figure 4 panels C&E. The files in format “ome.tiff” can be opened with Adobe Acrobat or Preview and include Z-stack pictures from the blue channel (nuclear stain DAPI) and LAMP1 across the sections. The corresponding file in “tiff” format only shows the merged image and can also be opened with Adobe Acrobat or Preview. The Prism files can be opened with GraphPad Prism 11.

File: FIGURE_5-SOURCE_DATA.zip

Description: Figure 5 is a 11-panel figure that reports ATP production rate (A-E) and mitochondrial content (F-K) in cells expressing WT, R589H or S525F mutants, or WT or R607H KI (KI/KI) mouse kidney sections.

This Folder contains 3 sub-folders: The Folder named "A-E ATP production" contains 3 Prism files used to generate the Seahorse XFe96 data shown on Figure 5 A-E. “ECAR” stands for extracellular acidification rate, “OCR” stands for oxygen consumption rate.

The Folder named "F&G. CELLS-TOM20" contains three sub-folders with the images used to quantify TOM20 abundance in cells expressing WT or each of the variant as well as the Prism file used to generate panel G. Images can be opened with Preview, Adobe Acrobat and Prism files with GraphPad Prism 11.

The Folder "H-K. TOM 20 IN VIVO" contains the original pictures from cortical or medullary WT or R607H KI mice (KI/KI) kidney sections. The files in format “ome.tiff” can be opened with Adobe Acrobat or Preview and include Z-stack pictures from each channel across the sections. Files in TIFF format can be opened with Adobe Acrobat or Preview. This folder also includes a Prism file used to generate the graphs shown in panels I & K. Prism files can be opened with GraphPad Prism 11.

File: SUPLEMENTARY.zip

Description: This folder contains two sub-folders named “Figure 3-figure supplement 1 source data” and “Figure 4-figure supplement 1 source data” with original immunoblots and corresponding Prism files from primary cell preparation and quantification presented in Figure 3-Figure supplement 1 and Figure 4-Figure supplement 1.

Figure 3-figure supplement 1 Source data includes a Prism file named “A. 2 hour incubation graph” with measurement of intracellular pH (pHi) after 2 hours incubation with Nigericin, shown in panel A. It also contains a Prism file named “C. P53” that contains the data from images shown in subfolder named “B. P53”, corresponding to panels B-D. Finally, it also contains a folder named “D. LC3 ratio in mice” corresponding to the graph shown in panel D, quantifying LC3B protein abundance in whole kidney lysates. The images can be opened using Adobe Acrobat or Preview. Prism file can be opened in GraphPad Prism 11.

Figure 4-figure supplement 1 Source data includes 3 sub-folders named “B1 ATPASE & ACTIN”, “CATHEPSIN” and “PRDX6” which each contain images in TIFF format used to generate panel A, and a Prism file named “Primary cells” which includes the calculations used to generate the graphs shown in panels B-F.

File: Figure_1_-_Source_Data_1.pdf

Description: A pdf file showing all the uncropped gels used in Figure 1 with corresponding bands indicated.

File: Figure_2-Source_Data_1.pdf

Description: A pdf file showing all the uncropped gels used in Figure 2 with corresponding bands indicated.

File: Figure_3_-_Source_Data_1.pdf

Description: A pdf file showing all the uncropped gels used in Figure 3 with corresponding bands indicated.

Code/software

Cell Profiler (version 4.2.8) was used for image analysis on Figure 3C and 5F; and FiJi (version 2.16.0/1.54p) was used to analyze images on figure 4A. Volocity was used to acquire and analyze images on Figure 1C, 4C and 4E and Figure 5H and 5J.

Access information

Other publicly accessible locations of the data:

• N/A

Data was derived from the following sources:

• N/A