Sorting motifs involved in the trafficking and localization of the PIN1 auxin efflux carrier Journal Article

Author(s): Sancho-Andrés, Gloria; Soriano-Ortega, Esther; Gao, Caiji; Bernabé-Orts, Joan M; Narasimhan, Madhumitha; Müller, Anna; Tejos, Ricardo; Jiang, Liwen; Friml, Jiří; Aniento, Fernando; Marcote, Maria J
Article Title: Sorting motifs involved in the trafficking and localization of the PIN1 auxin efflux carrier
Affiliation IST Austria
Abstract: 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.
Journal Title: Plant Physiology
Volume: 171
Issue 3
ISSN: 1532-2548
Publisher: American Society of Plant Biologists  
Date Published: 2016-07-01
Start Page: 1965
End Page: 1982
DOI: 10.1104/pp.16.00373
Notes: 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.
Open access: yes (repository)