RESUMO
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) 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.
Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Proteínas de Membrana Transportadoras/metabolismo , Sinais Direcionadores de Proteínas , Subunidades mu do Complexo de Proteínas Adaptadoras/genética , Subunidades mu do Complexo de Proteínas Adaptadoras/metabolismo , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Clatrina/metabolismo , Citosol/metabolismo , Endocitose/genética , Retículo Endoplasmático/genética , Retículo Endoplasmático/metabolismo , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Fatores de Troca do Nucleotídeo Guanina/genética , Proteínas de Membrana Transportadoras/genética , Mutação , Fenilalanina/genética , Plantas Geneticamente Modificadas , Sinais Direcionadores de Proteínas/genética , Transporte ProteicoRESUMO
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.
Assuntos
Proteínas Adaptadoras de Transporte Vesicular/metabolismo , Proteínas de Arabidopsis/metabolismo , Proteínas de Membrana Transportadoras/metabolismo , Subunidades mu do Complexo de Proteínas Adaptadoras/genética , Subunidades mu do Complexo de Proteínas Adaptadoras/metabolismo , Proteínas Adaptadoras de Transporte Vesicular/genética , Proteínas de Arabidopsis/genética , Membrana Celular/metabolismo , Endocitose/genética , Endocitose/fisiologia , Proteínas de Membrana Transportadoras/genéticaRESUMO
Plant cells possess much of the molecular machinery necessary for receptor-mediated endocytosis (RME), but this process still awaits detailed characterization. In order to identify a reliable and well-characterized marker to investigate RME in plant cells, we have expressed the human transferrin receptor (hTfR) in Arabidopsis protoplasts. We have found that hTfR is mainly found in endosomal (Ara7- and FM4-64-positive) compartments, but also at the plasma membrane, where it mediates binding and internalization of its natural ligand transferrin (Tfn). Cell surface expression of hTfR increases upon treatment with tyrphostin A23, which inhibits the interaction between the YTRF endocytosis signal in the hTfR cytosolic tail and the mu2-subunit of the AP2 complex. Indeed, tyrphostin A23 inhibits Tfn internalization and redistributes most of hTfR to the plasma membrane, suggesting that the endocytosis signal of hTfR is functional in Arabidopsis protoplasts. Co-immunoprecipitation experiments show that hTfR is able to interact with a mu-adaptin subunit from Arabidopsis cytosol, a process that is blocked by tyrphostin A23. In contrast, treatment with brefeldin A, which inhibits recycling from endosomes back to the plasma membrane in plant cells, leads to the accumulation of Tfn and hTfR in larger patches inside the cell, reminiscent of BFA compartments. Therefore, hTfR has the same trafficking properties in Arabidopsis protoplasts as in animal cells, and cycles between the plasma membrane and endosomal compartments. The specific inhibition of Tfn/hTfR internalization and recycling by tyrphostin A23 and BFA, respectively, thus provide valuable molecular tools to characterize RME and the recycling pathway in plant cells.