Sorting Motifs Involved in the Trafficking and Localization of the PIN1 Auxin Efflux Carrier

• Published in: Plant physiology (Bethesda) 2016-07, Vol.171 (3), p.1965-1982
• Main Authors: Sancho-Andrés, Gloria, Soriano-Ortega, Esther, Gao, Caiji, Bernabé-Orts, Joan Miquel, Narasimhan, Madhumitha, Müller, Anna Ophelia, Tejos, Ricardo, Jiang, Liwen, Friml, Jiří, Aniento, Fernando, Marcote, María Jesús
• Format: Article
• Language: English
• Subjects: Adaptor Protein Complex mu Subunits - genetics; Adaptor Protein Complex mu Subunits - metabolism; Arabidopsis - genetics; Arabidopsis - metabolism; Arabidopsis Proteins - genetics; Arabidopsis Proteins - metabolism; CELL BIOLOGY; Clathrin - metabolism; Cytosol - metabolism; Endocytosis - genetics; Endoplasmic Reticulum - genetics; Endoplasmic Reticulum - metabolism; Green Fluorescent Proteins - genetics; Green Fluorescent Proteins - metabolism; Guanine Nucleotide Exchange Factors - genetics; Membrane Transport Proteins - genetics; Membrane Transport Proteins - metabolism; Mutation; Phenylalanine - genetics; Plants, Genetically Modified; Protein Sorting Signals - genetics; Protein Transport
• ISSN: 0032-0889; 1532-2548
• DOI: 10.1104/pp.16.00373

In 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) PINFORMED1 (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.