Panel A shows protein schematics, illustrating human AQP1 constructs with inserted or substituted yolk platelet targeting domains. Panel B shows immunoblot images, comparing membrane localization of human AQP1 and control aquaporin constructs. Panel C shows immunofluorescence microscopy images, depicting localization of human AQP1 constructs in oocytes and isolated yolk platelets. Panel D shows bar graphs, quantifying water uptake by isolated yolk platelets expressing human AQP1 constructs. Panel E shows protein schematics, illustrating zebrafish Aqp1aa constructs with inserted or substituted yolk platelet targeting domains. Panel F shows immunoblot images, comparing membrane localization of zebrafish Aqp1aa and control aquaporin constructs. Panel G shows immunofluorescence microscopy images, depicting localization of zebrafish Aqp1aa constructs in oocytes and isolated yolk platelets. Panel H shows bar graphs, quantifying water uptake by isolated yolk platelets expressing zebrafish Aqp1aa constructs. Panel I shows protein schematics, illustrating human AQP3 and zebrafish Aqp3b constructs with modified targeting motifs. Panel J shows immunoblot images, comparing membrane localization of human AQP3 and zebrafish Aqp3b constructs. Panel K shows immunofluorescence microscopy images, depicting localization of human AQP3 and zebrafish Aqp3b constructs in oocytes. Panel L shows a bar graph, comparing glycerol permeability of isolated yolk platelets expressing AQP3 constructs.
YPD is sufficient to confer YPM targeting of constitutive plasma membrane AQP1 and AQP3 channels. (A and E) Schematic diagrams of different HA-tagged HsAQP1 (A) and DrAqp1aa (E) constructs in which the species-specific YPD was inserted at the end of the C termini of the proteins (HsAQP1-YPD-HA and DrAqp1aa-YPD-HA), or by substituting the SD (HsAQP1-ΔSD-YPD-HA and DrAqp1aa-ΔSD-YPD-HA). (B and F) Immunoblots of TM, PM, and YPM protein extracts from X. laevis–uninjected (control) oocytes or expressing HsAQP12-WT-HA, HsAQP12-ΔYPD-HA, DrAqp12-WT-HA, or DrAqp12-ΔYPD-HA as controls, or the different HsAQP1 or DrAqp1aa mutant constructs. Blots were probed with α-HA or α-PDI antibodies. Part of the blot from F is shown in Fig. 4 F. Arrows indicate aquaporin monomers, whereas the arrowheads indicate likely posttranslational modifications of the channels. Molecular mass markers (kDa) are on the left. (C and G) Immunolocalization of HsAQP1-WT-HA and the two YPDs carrying HsAQP1 channels (C), or DrAqp1aa-WT-HA and the two YPDs carrying DrAqp1aa constructs (F), in oocytes and isolated YPs, and in controls, as indicated. Scale bars, 10 µm (left panels), 5 µm (right panels). (D and H) Water permeability of YPs isolated from oocytes injected with the different human and zebrafish AQP12 and AQP1 constructs. (I) Diagrams of HA-tagged HsAQP3-WT (A) and DrAqp3b-WT (D), and constructs generated for each channel in which the YPD was inserted at the end of the C terminus, while the YSMs in each amino acid sequence (indicated in yellow) were erased by point mutations (HsAQP3-ΔYSM-YPD-HA and DrAqp3b-ΔYSM-YPD-HA). (J) Immunoblots of TM, PM, and YPM protein extracts from uninjected oocytes or oocytes expressing the different constructs as indicated, using the α-HA antibody. Protein loading was normalized by PDI, while molecular mass markers (kDa) are on the left. Arrows indicate aquaporin monomers, whereas the arrowheads indicate likely posttranslational modifications of the channels. (K) Immunolocalization of the different AQP3 constructs (green) in injected and uninjected oocytes. Scale bars, 20 and 10 µm (insets). (L) Glycerol permeability of YPs isolated from oocytes expressing the different human and zebrafish AQP3 constructs. In D, H, and L, data (mean ± SEM, n = 4 biological replicates from one experiment) were statistically analyzed by unpaired Student’s t test (**P < 0.01; ***P < 0.001; with respect to the uninjected oocytes). Source data are available for this figure: SourceData F5.