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These results are in agreement with the notion that TGN-derived vesicles are acidic and that the V-ATPase plays an active role in this process. Vesicle acidification induces the recruitment of coat proteins and small GTPases of the ADP-ribosylation factor (ARF) family, a process that is important for the appropriate targeting and recycling of various membrane proteins, including GLUT4 and AQP2. (more…)

Because of its brighter apical staining in clear cells and its high expression in kidney intercalated cells, d2 appears to be the predominant isoform in proton-secreting narrow and clear cells. Principal Cell Localization Interestingly, subunit A was detected in principal cells, where it was localized in intracellular structures closely associated with but not identical to the TGN38-labeled trans-Golgi network (TGN). (more…)

The presence of G2 in the epididymis was not investigated here because this subunit has been previously shown to be specific for the brain and absent from the kidney. In addition to their localization in the apical plasma membrane and subapical vesicles, a faint cytosolic staining was also observed for A, C1, C2, G1, and G3, similarly to the previously reported localization of other subunits of the V1 domain, including subunit E2, in the soluble fraction of clear cells. (more…)

The precise colocalization of subunit A with subunit E2 in apical microvilli and subapical vesicles is consistent with the assembly of these unique subunit isoforms to form the V-ATPase complex. This result indicates that clear cells use the A subunit for net luminal proton secretion in the epididymis. Both the ubiquitous isoform C1 and the more restricted C2 isoform were expressed in clear cells. Two C2 isoforms, C2a and C2b, resulting from alternative mRNA splicing, have been identified. C2a was detected in the lung and C2b in the kidney. (more…)

Immunofluorescence was used to examine the expression and the cellular and subcellular localization of five subunits of the V-ATPase and some of their respective isoforms in rat epididymis and vas deferens: A, C1, C2, G1, G3, a1, a2, a4, d1, and d2. All five subunits were detected in epithelial cells of these organs but showed specific patterns of expression (Fig. 10). (more…)

Localization of the d Subunit of the V-ATPase: d1 and d2 Isoforms The d1 isoform was detected in the apical pole of narrow cells (data not shown) and of clear cells in the epididymis (arrows in Fig. 9A) and vas deferens (data not shown), where it colocalizes with the E2 subunit (arrows in Fig. 9, C and E). The staining for d1 in clear cells was weaker compared to other subunits and did not allow for the exact localization of this subunit in apical microvilli and/or subapical endosomes. Immunogold electron microscopy will be required to determine with precision the subcellular localization of this subunit in clear cells. (more…)

Localization of the a Subunit of the V-ATPase: a1, a2, and a4 Isoforms Isoforms al and a4 were detected in the apical pole of narrow cells (data not shown) and in clear cells of the epididymis (Fig. 7, A and B) and vas deferens (data not shown). Double labeling for the E2 subunit (Fig. 7C) revealed that al colocalized with E2 in subapical vesicles but was absent from the microvilli (arrows in Fig. 7, A, C, and E). Subunit a4 showed a complete colocalization with E2 in subapical vesicles and microvilli of clear cells (Fig. 7, B, D, and F). Expression of the osteoclast-specific a3 isoform was not examined. (more…)

However, the cytosolic staining for Cl seemed stronger compared to that of C2. Double labeling for the E2 subunit (Fig. 5, B, B, E, and E ) revealed colocalization of both C isoforms with E2 in subapical vesicles (Fig. 5, C, C0, F, and F0; yellow staining). In contrast, Cl and C2 were barely detected in apical microvilli (Fig. 5, C, C0, F, and F0; red staining). No significant staining was detected in principal cells. (more…)

Similarly to narrow cells, clear cells were identified by their coexpression of the E2 subunit (Fig. 3B). A weak cytosolic staining was also detected in these cells, as we have previously reported for other subunits of the V1 domain of the pump, including subunit E2. Figure 3C shows a complete colocalization of subunits A and E2 in subapical vesicles and apical microvilli (yellow staining). Immunogold electron microscopy confirmed the localization of subunit A in apical microvilli in addition to its subapical localization (Fig. 3D). Interestingly, this subunit was also detected in intracellular structures of principal cells located in the proximal regions of the epididymis (Fig. 4A). (more…)

Specificity of the V-ATPase Antibodies Each of the V-ATPase subunit antibodies used in this study, except the novel anti-subunit A antibody, has been characterized previously. The specificity of these antibodies in epididymis samples was further confirmed by Western blotting. As shown in Figure 1, all antibodies gave one single band at the appropriate molecular weight, showing their purity. A more complete analysis was performed for the novel anti-A antibody by repeating the Western blot after preincubation of the antibody with the immunizing peptide. (more…)