Cloning and Expression of Vacuolar Proton-Pumping ATPase Subunits in the Follicular Epithelium of the Bullfrog Endolymphatic Sac

Abstract

In an investigation aimed at clarifying the mechanism of crystal dissolution of the calcium carbonate lattice in otoconia (the mineral particles embedded in the otolithic membrane) of the endolymphatic sac (ELS) of the bullfrog, cDNAs encoding the A-and E-subunits of bullfrog vacuolar protonpumping ATPase (V-ATPase) were cloned and sequenced. The cDNA of the A-subunit consisted of an 11-bp 5'-untranslated region (UTR), a 1,854-bp open reading frame (ORF) encoding a protein comprising 617 amino acids with a calculated molecular mass of 68,168 Da, and a 248-bp 3'-UTR followed by a poly(A) tail. The cDNA of the E-subunit consisted of a 72-bp 5'-UTR, a 681-bp ORF encoding a protein of 226 amino acids with a calculated molecular mass of 26,020 Da, and a 799-bp 3'-UTR followed by a poly(A) tail. Western blot and immunofluorescence analyses using specific anti-peptide antisera against the V-ATPase A-and E-subunits revealed that these subunits were present in the ELS, urinary bladder, skin, testes, and kidneys. In the ELS, positive cells were scattered in the follicular epithelium which, as revealed by electron microscopy, corresponds to the location of mitochondria-rich cells. These findings suggest that V-ATPase, including the A-and Esubunits, exists in mitochondria-rich cells of the ELS, which might be involved in dissolution of the calcium carbonate crystals in the lumen of the ELS.

Detection and quantification of the vacuolar H+-ATPase using the Legionella effector protein SidK

“…The high signal-to-noise ratio, along with specific organellar markers allowed us to clearly discern V-ATPase-positive organelles, primarily endo/lysosomes and the trans -Golgi. This labeling was in sharp contrast to the staining we obtained using a commercially available antibody to the A subunit and the conditions for its use described in the literature (Ramirez et al, 2019), which yielded results similar to those reported by others using other available antibodies (Yajima et al, 2007; McGuire et al, 2019; Michel et al, 2013). V-ATPase complexes have been shown to interact with several accessory proteins involved in endocytic sorting and signaling (Merkulova et al, 2015; Maxson and Grinstein, 2014; Marshansky et al, 2014).…”

“…When tested in HeLa cells (Figs. 4G and H), this antibody yielded a diffuse punctate pattern reminiscent of that reported by Ramirez et al (2019) for tumour cells and elsewhere for a variety of other cells stained with other A subunit antibodies (Yajima et al, 2007;McGuire et al, 2019;Michel et al, 2013). However, its co-localization with acidic compartments was poor: the Manders' coefficient of the antibody with LAMP1 was M = 0.08 (compared to an M = 0.79 with SidK-AL568), while that with anti-TGN46, the trans-Golgi marker used, was M = 0.05 (compared to an M = 0.81 with SidK-AL568).…”

Gene expression and localization of two types of AQP5 inXenopus tropicalisunder hydration and dehydration

“…Antibodies were raised in a rabbit or guinea pig immunized with the ST-200 peptide coupled to keyhole limpet hemocyanin, as described previously (43). Rabbit or guinea pig anti-peptide antibodies were previously generated for X. laevis AQP5 homologue, AQP-x5(b) (26), and X. laevis AQP3, AQP-x3BL (30), bullfrog vacuolar H ϩ -ATPase (V-ATPase) E-subunit (46).…”

“…Double-immunofluorescence staining was carried out to elucidate the detailed distribution of AQPs in the acinar cells of the small granular gland. Staining against AQP-xt5a and V-ATPase E-subunit, a marker molecule for mitochondrion-rich cells (46), depicted AQP-xt5a in secretory cells of the small granular gland of dehydrated frogs, but not in the mitochondrion-rich cells (Fig. 9A).…”

Immunolocalization of a mammalian aquaporin 3 homolog in water-transporting epithelial cells in several organs of the clawed toad Xenopus laevis

“…The antibody was raised in a rabbit immunized with the ST-203 peptide coupled to keyhole limpet hemocyanin (Pierce, Rockford, Ill., USA), as described by Tanaka and Kurosumi (1992). Antibodies against Xenopus AQP-x5 (ST-156; Kubota et al 2006) homologous to mammalian AQP5, against the bullfrog vacuolar H + -ATPase (V-ATPase) E-subunit (ST-173; Yajima et al 2007) for identifying mitochondria-rich (MR) cells, and against Hyla AQP-h2 (ST-140; were generated in guinea pigs and characterized as described previously.…”