title: 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.
authors.affiliation: Harvard Medical School, Boston, MA, USA.
authors.forename: Nicolas
authors.initials: N
authors.lastname: Da Silva
authors.forename: Trairak
authors.initials: T
authors.lastname: Pisitkun
authors.forename: Clémence
authors.initials: C
authors.lastname: Belleannée
authors.forename: Lance R
authors.initials: LR
authors.lastname: Miller
authors.forename: Raoul
authors.initials: R
authors.lastname: Nelson
authors.forename: Mark A
authors.initials: MA
authors.lastname: Knepper
authors.forename: Dennis
authors.initials: D
authors.lastname: Brown
authors.forename: Sylvie
authors.initials: S
authors.lastname: Breton
mesh_terms: D000818:Animals; D002469:Cell Separation; D002853:Chromatography, Liquid; D030562:Databases, Protein; D004822:Epididymis / Q000166:cytology / Q000201:enzymology*; D005434:Flow Cytometry; D005786:Gene Expression Regulation; D005838:Genotype; D049452:Green Fluorescent Proteins / Q000096:biosynthesis / Q000235:genetics; D007668:Kidney / Q000166:cytology / Q000201:enzymology*; D008297:Male; D051379:Mice; D008822:Mice, Transgenic; D010641:Phenotype; D011401:Promoter Regions, Genetic; D040901:Proteomics / Q000379:methods; D012333:RNA, Messenger / Q000378:metabolism; D011980:Receptors, Progesterone / Q000235:genetics / Q000378:metabolism; D053719:Tandem Mass Spectrometry; D025262:Vacuolar Proton-Translocating ATPases / Q000235:genetics / Q000378:metabolism*
chemical_list: D012333:RNA, Messenger; D011980:Receptors, Progesterone; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins; C480628:Atp6v1b1 protein, mouse; D025262:Vacuolar Proton-Translocating ATPases