Sorting signals for PIN1 trafficking and localization

Abstract

PIN-FORMED (PIN) family proteins direct polar auxin transport based on their asymmetric (polar) localization at the plasma membrane. In the case of PIN1, it mainly localizes to the basal (rootward) plasma membrane domain of stele cells in root meristems. Vesicular trafficking events, such as clathrin-dependent PIN1 endocytosis and polar recycling, are probably the main determinants for PIN1 polar localization. However, very little is known about the signals which may be involved in binding the μ-adaptin subunit of clathrin adaptor complexes (APs) for sorting of PIN1 within clathrin-coated vesicles, which can determine its trafficking and localization. We have performed a systematic mutagenesis analysis to investigate putative sorting motifs in the hydrophilic loop of PIN1. We have found that a non-canonical motif, based in a phenylalanine residue, through the binding of μA(μ2)- and μD(μ3)-adaptin, is important for PIN1 endocytosis and for PIN1 traffcking along the secretory pathway, respectively. In addition, tyrosine-based motifs, which also bind different μ-adaptins, could also contribute to PIN1 trafficking and localization.

Keywords: Clathrin; PIN1; clathrin-adaptor complex; mu-adaptins; sorting signals.

Figure 1.

Sequence of the hydrophilic loop of the PIN1 auxin efflux carrier. Residues analyzed for μ-adaptin binding are highlighted in red and their position in the sequence with an asterisk. The motifs involved in μ-adaptin binding containing these residues are shown in a grey box. Putative ER export signals (containing acidic residues, in blue) are shown in a yellow box.

Figure 2.

Characterization of a PIN1 mutant in tyrosine 480. (A) Binding of the receptor binding domain of Arabidopsis μ-adaptins, with a N-terminal (His)₆-tag, to the C-terminal part of the cytosolic loop of PIN1 (residues 403–482) or to a mutant version in tyrosine 480 (Y480A), both fused with GST, using pull-down experiments and Western blotting with His antibodies (as described in 19). (B) CLSM of primary roots of 4-days-old seedlings expressing a PIN1:GFP-Y480A mutant in pin1 background. Note the accumulation of this mutant in intracellular structures at stele cells. Scale bar: 10 μm.