Panel A shows a sequence logo and protein schematic, illustrating the conserved yolk platelet targeting domain in vertebrate AQP12. Panel B shows a protein schematic, depicting the human AQP12 construct with the yolk platelet targeting domain and hemagglutinin tag. Panel C shows immunoblot images, comparing membrane localization of human AQP12 wild type and targeting domain mutants. Panel D shows immunofluorescence microscopy images, illustrating localization of human AQP12 wild type and targeting domain deletion proteins in oocytes and isolated yolk platelets. Panel E shows a protein schematic, depicting the zebrafish Aqp12 construct containing the yolk platelet targeting domain. Panel F shows immunoblot images, comparing membrane localization of zebrafish Aqp12 wild type and targeting domain deletion proteins. Panel G shows immunofluorescence microscopy images, illustrating localization of zebrafish Aqp12 wild type and targeting domain deletion proteins. Panel H shows a workflow diagram, illustrating zebrafish embryo overexpression and isolation of yolk platelets. Panel I shows immunofluorescence microscopy images, depicting localization of zebrafish Aqp12 proteins in isolated embryonic yolk platelets. Panel J shows immunoblot images, comparing expression of zebrafish Aqp12 proteins in total and yolk platelet membrane fractions.
Identification of a YP-targeting signal in vertebrate AQP12. (A) Calculated consensus amino acid sequence of the YPD in the AQP12 C terminus after the amino acid sequence alignment of 473 gnathostome AQP12 orthologs. AA, amino acid. (B) Diagram of HA-tagged WT HsAQP12 (HsAQP12-WT-HA) showing the YPD amino acid sequence. (C) Western blot of TM and YPM protein extracts from X. laevis–uninjected oocytes or oocytes expressing HsAQP12-WT-HA or HsAQP12-HA mutants in which different residues comprising the YPD were substituted by Ala, or the complete YPD replaced by Ala residues (ΔYPD). The immunoblots were probed with α-HA or α-PDI antibodies. (D) Immunolocalization of HsAQP12-WT-HA and HsAQP12-ΔYPD-HA (green) in oocytes and isolated YPs. Uninjected control oocytes are shown in the top panels. (E–G) YPD amino acid sequence of DrAqp12-HA (E), immunoblotting of TM, PM, and YPM of oocytes expressing the DrAqp12-WT-HA or the DrAqp12-ΔYPD-HA mutant channel (F), and immunolocalization of the constructs in oocytes and YPs, and control oocytes. Part of the blot from F is shown in Fig. 5 F. (H–J) Overexpression of DrAqp12-WT-HA or DrAqp12-ΔYPD-HA in zebrafish embryos (H), and further analysis of the localization of the channels in isolated YPs from 24-hpf embryos by immunostaining (I; green) and immunoblotting (J). In D, G, and I, the YPM and yolk proteins were respectively labeled with WGA (red) and the α-Lv antibody (blue). Scales bars, 10 µm (D and G, left panels) and 5 µm (D and G, right panels, and I). In C, F, and J, molecular mass markers (kDa) are on the left. hpf, hours postfertilization; WT, wild type. Source data are available for this figure: SourceData F4.