Proteomic analysis of V-ATPase-rich cells harvested from the kidney and epididymis by fluorescence-activated cell sorting

Abstract

Proton-transporting cells are located in several tissues where they acidify the extracellular environment. These cells express the vacuolar H(+)-ATPase (V-ATPase) B1 subunit (ATP6V1B1) in their plasma membrane. We provide here a comprehensive catalog of the proteins that are expressed in these cells, after their isolation by enzymatic digestion and fluorescence-activated cell sorting (FACS) from transgenic B1-enhanced green fluorescent protein (EGFP) mice. In these mice, type A and B intercalated cells and connecting segment cells of the kidney, and narrow and clear cells of the epididymis, which all express ATP6V1B1, also express EGFP, while all other cell types are negative. The proteome of renal and epididymal EGFP-positive (EGFP(+)) cells was identified by liquid chromatography-tandem mass spectrometry (LC-MS/MS) and compared with their respective EGFP-negative (EGFP(-)) cell populations. A total of 2,297 and 1,564 proteins were detected in EGFP(+) cells from the kidney and epididymis, respectively. Out of these proteins, 202 and 178 were enriched by a factor greater than 1.5 in EGFP(+) cells compared with EGFP(-) cells, in the kidney and epididymis respectively, and included subunits of the V-ATPase (B1, a4, and A). In addition, several proteins involved in intracellular trafficking, signaling, and cytoskeletal dynamics were identified. A novel common protein that was enriched in renal and epididymal EGFP(+) cells is the progesterone receptor, which might be a potential candidate for the regulation of V-ATPase-dependent proton transport. These proteomic databases provide a framework for comprehensive future analysis of the common and distinct functions of V-ATPase-B1-expressing cells in the kidney and epididymis.

Fig. 1.

Flow cytometry sorting of B1-enhanced green fluorescent protein (EGFP) cells. Left: dot plot of a representative epididymal cell sorting sample [EGFP vs. phycoerythrin channel autofluorescence (PE-A)]. Events in the top left quadrant are EGFP-positive (EGFP⁺) cells, representing ∼6% of the live cell population. Right: fluorescence microscopy of Cytospin smears of cells isolated from B1-EGFP mouse kidney (A and B) and epididymis (C and D) before (A and C) and after (B and D) sorting. A few vacuolar H⁺-ATPase (V-ATPase)-B1-expressing cells (green) are seen in the cell suspension before fluorescence-activated cell sorting (FACS). After FACS, all cells show positive EGFP fluorescence. Nuclei are visualized with DAPI (blue). Bars = 20 μm (A and C), 30 μm (B), and 15 μm (D).

Fig. 2.

Three examples of epididymal EGFP⁺ cells (green) labeled for the V-ATPase B1 subunit (red) visualized after FACS. Cells retained their polarized expression of the V-ATPase. Nuclei are labeled with DAPI (blue). Bars = 5 μm.

Fig. 3.

Electron microcopy showing several renal EGFP⁺ cells isolated by FACS. All cells retained their characteristic morphology with visible microvilli (arrows) and numerous vesicles. Bar = 3 μm.

Fig. 4.

RT-PCR analysis showing depletion of aquaporin 9 (AQP9) mRNA (top; Aqp9) and high enrichment of the V-ATPase B1 subunit (middle; Atp6v1b1) in epididymal EGFP⁺ cells compared with EGFP-negative (EGFP⁻) cells. Controls using GAPDH-specific primers (Gapdh) are shown (bottom).

Fig. 5.

Pie charts of the top 15 gene ontology terms for the proteins that were significantly enriched in the renal (top) and epididymal (bottom) EGFP⁺ cell populations compared with their respective EGFP⁻ cell samples.

Fig. 6.

RT-PCR analysis of selected genes that were shown to be enriched in EGFP⁺ cells from the kidney (top; GFP⁺) and epididymis (bottom; GFP⁺) compared with their respective EGFP⁻ samples (GFP⁻). Of note, the B1 subunit of the V-ATPase (Atp6v1b1) was significantly enriched in both kidney and epididymis EGFP⁺ cells compared with EGFP⁻ cells. Atp6v0a4, V-ATPase a4 subunit; Prom1 and 2, Prominin 1 and 2, respectively; Lrba, LPS-responsive beige-like anchor; Pgrmc1, progesterone receptor; Anxa6, annexin A6; Dbnl, drebrin-like; Krt18, keratin 18; Myo6, myosin 6; Slco4a1, solute carrier organic anion transporter member 4a1; Hsd17b12, hydroxysteroid (17-β) dehydrogenase 12; Gapdh, GAPDH.