In contrast with the wealth of recent reports about the function of m-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 m-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 m-adaptins in vitro. However, only Phe-165, which binds mu A(mu 2)- and mu D(mu 3)-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 mu A (mu 2)-adaptin mutant, it accumulated in big intracellular structures containing LysoTracker in a mu D (mu 3)-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 mu A (mu 2)- and mu D (mu 3)-adaptin, is important for PIN1 endocytosis and for PIN1 trafficking along the secretory pathway, respectively.
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