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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. Interestingly, a strong apical d1 staining was also observed in the apical membrane of principal cells (arrowheads in Fig. 9A). This is the first description of one of the subunits of the V-ATPase being highly expressed in the plasma membrane of principal cells. In contrast, the d2 isoform was exclusively found in narrow (not shown) and clear cells (arrows in Fig. 9B), identified by their E2 expression (arrows in Fig. 9, D and F). Whereas d2 was located in subapical endosomes, it was absent from E2-labeled microvilli (middle arrow in Fig. 9F; red staining). FIG. 9. Localization of the V-ATPase dl and d2 subunit isoforms. Rat cauda epididymidis was stained for d1 (A; green) and d2 (B; green). Double labeling for E2 was performed (C and D; red) to identify clear cells. In clear cells (arrows), a strong apical staining for d2 (B) and a weaker apical staining for d1 (A) were detected. The merged picture shown in E indicates colocalization of d1 with E2 in the subapical pole of clear cells (yellow-orange staining). F) Colocalization of subunit d2 with E2 in subapical vesicles (yellow staining) but no colocalization in E2-labeled microvilli (red staining in cell located in the middle of the panel). In addition, a strong d1 staining is seen in the apical membrane of principal cells (A and E; green;arrowheads), whereas principal cells are negative for d2 (B and F). Bar = 20 im. FIG. 10. Diagram illustrating the subunit composition of the V-ATPase and their expression in the epididymis. Subunits that form the membrane-bound V0 domain are labeled in lowercase letters, and subunits of the cytosolic V1 domain are labeled in uppercase letters. Based on Kawasaki-Nishi et al..