Panel A shows schematic protein diagrams, illustrating wild type and chimeric aquaporin constructs with exchanged terminal regions. Panel B shows immunofluorescence microscopy images, depicting localization of aquaporin constructs in oocyte membranes and yolk platelets. Panel C shows immunoblot images, comparing expression of human aquaporin constructs in total, plasma membrane, and yolk platelet membrane fractions. Panel D shows immunoblot images, comparing expression of zebrafish aquaporin constructs in membrane fractions. Panel E shows a bar graph, quantifying osmotic water permeability of oocytes expressing human aquaporin constructs. Panel F shows a bar graph, comparing osmotic water permeability of oocytes expressing zebrafish aquaporin constructs. Panel G shows immunofluorescence microscopy images, illustrating localization of zebrafish Aqp7 and chimeric Aqp7-12CT proteins. Panel H shows immunoblot images, comparing membrane localization of zebrafish Aqp7 and chimeric Aqp7-12CT proteins.
AQP12 C terminus is involved in channel targeting to the YPM of X. laevis oocytes. (A) Schematic illustrating the structure of wild-type HsAQP1 (black), HsAQP12 (red), DrAqp1aa (black), and DrAqp12 (red), or the chimeric constructs in which the complete N or C termini of AQP1 were exchanged with those of AQP12, and vice versa. (B) Immunostaining of uninjected oocytes and oocytes expressing the different HA-tagged constructs on oocyte sections and corresponding isolated YPs. The YPs were counterstained with α-Lv antibodies and WGA. In the upper panels, the arrowheads indicate the oocyte plasma membrane, whereas the arrows indicate the YPM. Scale bars, 50 µm (oocyte sections), 10 µm (insets), and 5 µm (YPs). (C and D) Immunoblots of TM, plasma membrane (PM), and YPM extracts from uninjected oocytes and oocytes expressing the different human and zebrafish constructs using α-HA antibody and an α-PDI as a loading control. The arrows indicate aquaporin monomers, and molecular mass markers (kDa) are on the left. (E and F) Osmotic water permeability (Pf) of oocytes (mean ± SEM; n = 4–12 oocytes per group) expressing the constructs indicated in Fig. 2, C and D. The asterisks denote statistically significant differences with respect to uninjected oocytes (unpaired Student t test; ***P < 0.001). (G) Immunostaining of whole oocytes and isolated YPs from oocytes expressing HA-tagged wild-type Aqp7 (DrAqp7-HA) or the chimeric construct carrying the complete Aqp12 C terminus (DrAqp7-12CT-HA). The YPs were counterstained as above. In the upper panels, the arrowheads indicate the oocyte plasma membrane, whereas the arrows indicate the YPM. Scale bars, 50 µm (oocyte sections) and 5 µm (YPs). (H) Immunoblots of TM, PM, and YPM extracts from oocytes expressing the two constructs using the α-HA and α-PDI antibodies. The arrows indicate aquaporin monomers, and molecular mass markers (kDa) are on the left. Source data are available for this figure: SourceData F2.