Trafficking of the human transferrin receptor in plant cells: effects of tyrphostin A23 and brefeldin A.
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.
Ortiz-Zapater
Elena
E
Departamento de Bioquímica y Biología Molecular, Facultad de Farmacia, Universidad de Valencia, Avda Vicente Andrés Estellés s/n, 46100-Burjassot (Valencia), Spain.
Soriano-Ortega
Esther
E
Marcote
María Jesús
MJ
Ortiz-Masiá
Dolores
D
Aniento
Fernando
F
D033983:Adaptor Protein Complex mu Subunits / Q000378:metabolism; D017360:Arabidopsis / Q000235:genetics* / Q000378:metabolism*; D020126:Brefeldin A / Q000494:pharmacology*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005786:Gene Expression Regulation; D006801:Humans; D030821:Plants, Genetically Modified; D011500:Protein Synthesis Inhibitors / Q000494:pharmacology; D021381:Protein Transport / Q000187:drug effects; D011523:Protoplasts / Q000378:metabolism; D011990:Receptors, Transferrin / Q000235:genetics* / Q000378:metabolism*; D020032:Tyrphostins / Q000494:pharmacology*
D033983:Adaptor Protein Complex mu Subunits; D004791:Enzyme Inhibitors; D011500:Protein Synthesis Inhibitors; D011990:Receptors, Transferrin; D020032:Tyrphostins; C114975:tyrphostin A51; D020126:Brefeldin A; C060641:tyrphostin A23