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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). Double labeling for TGN38, a protein located in the trans-Golgi network, revealed that subunit A is present in structures that are closely associated with, but are at least partially distinct from, the TGN38-stained trans-Golgi (Fig. 4, B and C). Localization of the C Subunit of the V-ATPase: Isoforms C1 and C2 Both Cl (Fig. 5, A and A0) and C2 (Fig. 5, D and D0) were detected in clear cells of the epididymis. Epididymal narrow cells and clear cells from the vas deferens were also stained (data not shown). Strong apical and weaker cytosolic immunofluorescence staining was seen for these two V1 domain isoforms. FIG. 4. Localization of the A subunit in principal cells in the proximal epididymis. A 5-im section of rat caput epididymidis stained for subunit A (A; green) and TGN-38 (B; red). Subunit A is located in intracellular structures in principal cells. Double labeling showed that these structures are closely associated but do not completely overlap with the trans-Golgi network, labeled with TGN-38 (C). Inset shows that intracellular organelles positive for subunit A (green) run in parallel with TGN-38-positive structures (red). Bar = 5 im.