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Fig. 7. A Leu-Ile motif is required for the basolateral targeting of PMCA2xb. (A,B) Alignment of the basolateral targeting motifs of (A) E-cadherin and (B) CD147 with the crucial aa region for the basolateral targeting of PMCA2. Aa in red indicate the targeting motif of E-cadherin Leu587-[Leu/Ile588] and its homolog in PMCA2 Leu1153-Ile1154 (A), and the crucial residue Leu252 of CD147 and its homolog in PMCA2, Leu1165 (B). Aa in green indicate acidic aa residues. (C) Alignment of the distal C-terminal aa sequences of the b-tail of PMCA1, PMCA2, PMCA3, PMCA 4 from rat and from human PMCA4. Aa in red indicate the Leu[Leu/Ile] motif, aa in green indicate acidic residues, green shading indicates the conserved basolateral targeting motif found in all these PMCAs. Note the conservation of Leu1153-[Leu/Ile1154] and the downstream acidic residues in all four PMCAs. However, Leu1165, which is homologous to the residue crucial for targeting of CD147, is not conserved in all PMCAs. (D-I) Apical surface-scan of the hair bundle (plane 2 in J, actin in red) and an orthogonal view of the basolateral plasma membrane (plane 1 in J) of a transfected hair cell expressing a transgene encoding the EGFP-tagged PMCA2xb isoform (green) (wt-2xb; see Fig. 6A) and derived mutated isoforms, presented in separated green and red, and merged channels. High-resolution basolateral cross-sectional scans (plane 3 in J) are also shown in merged channels. Mutations are shown in red on the aa sequence of the b-tail. Substitutions Leu1153Ala and Ile1154Ala alter the basolateral targeting of PMCA2xb, whereas Phe1152Ala does not. The acidic cluster Asp-Asp-Thr-Asp at position 1155-1158 is not essential for the basolateral targeting of PMCA2xb. (K) Summary of our data. aa, amino acid; b, b-tail; AP, apical PM localization; BL, basolateral PM localization. Bars, 5 µm.
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