n contrast with the wealth of recent reports about the function of μ-adaptins and clathrin adaptor protein (AP) complexes, there is very little information about the motifs that determine the sorting of membrane proteins within clathrin-coated vesicles in plants. Here, we investigated putative sorting signals in the large cytosolic loop of the Arabidopsis (Arabidopsis thaliana) PIN-FORMED1 (PIN1) auxin transporter, which are involved in binding μ-adaptins and thus in PIN1 trafficking and localization. We found that Phe-165 and Tyr-280, Tyr-328, and Tyr-394 are involved in the binding of different μ-adaptins in vitro. However, only Phe-165, which binds μA(μ2)- and μD(μ3)-adaptin, was found to be essential for PIN1 trafficking and localization in vivo. The PIN1:GFP-F165A mutant showed reduced endocytosis but also localized to intracellular structures containing several layers of membranes and endoplasmic reticulum (ER) markers, suggesting that they correspond to ER or ER-derived membranes. While PIN1:GFP localized normally in a μA (μ2)-adaptin mutant, it accumulated in big intracellular structures containing LysoTracker in a μD (μ3)-adaptin mutant, consistent with previous results obtained with mutants of other subunits of the AP-3 complex. Our data suggest that Phe-165, through the binding of μA (μ2)- and μD (μ3)-adaptin, is important for PIN1 endocytosis and for PIN1 trafficking along the secretory pathway, respectively.
We thank Dr. R. Offringa (Leiden University) for providing the GST- PIN-CL construct; Sandra Richter and Gerd Jurgens (University of Tübin- gen) for providing the estradiol-inducible PIN1-RFP construct and the gnl1 mutant expressing BFA-sensitive GNL1; F.J. Santonja (University of Valencia) for help with the statistical analysis; Jurgen Kleine-Vehn, Elke Barbez, and Eva Benkova for helpful discussions; the Salk Institute Genomic Analysis Laboratory for providing the sequence-indexed Arabidopsis T-DNA in- sertion mutants; and the greenhouse section and the microscopy section of SCSIE (University of Valencia) and Pilar Selvi for excellent technical assistance.
1965 - 1982
Sancho Andrés G, Soriano Ortega E, Gao C, et al. Sorting motifs involved in the trafficking and localization of the PIN1 auxin efflux carrier. Plant Physiology. 2016;171(3):1965-1982. doi:10.1104/pp.16.00373
Sancho Andrés, G., Soriano Ortega, E., Gao, C., Bernabé Orts, J., Narasimhan, M., Müller, A., … Marcote, M. (2016). Sorting motifs involved in the trafficking and localization of the PIN1 auxin efflux carrier. Plant Physiology. American Society of Plant Biologists. https://doi.org/10.1104/pp.16.00373
Sancho Andrés, Gloria, Esther Soriano Ortega, Caiji Gao, Joan Bernabé Orts, Madhumitha Narasimhan, Anna Müller, Ricardo Tejos, et al. “Sorting Motifs Involved in the Trafficking and Localization of the PIN1 Auxin Efflux Carrier.” Plant Physiology. American Society of Plant Biologists, 2016. https://doi.org/10.1104/pp.16.00373.
G. Sancho Andrés et al., “Sorting motifs involved in the trafficking and localization of the PIN1 auxin efflux carrier,” Plant Physiology, vol. 171, no. 3. American Society of Plant Biologists, pp. 1965–1982, 2016.
Sancho Andrés G, Soriano Ortega E, Gao C, Bernabé Orts J, Narasimhan M, Müller A, Tejos R, Jiang L, Friml J, Aniento F, Marcote M. 2016. Sorting motifs involved in the trafficking and localization of the PIN1 auxin efflux carrier. Plant Physiology. 171(3), 1965–1982.
Sancho Andrés, Gloria, et al. “Sorting Motifs Involved in the Trafficking and Localization of the PIN1 Auxin Efflux Carrier.” Plant Physiology, vol. 171, no. 3, American Society of Plant Biologists, 2016, pp. 1965–82, doi:10.1104/pp.16.00373.
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