Trafficking of the human transferrin receptor in plant cells: effects of tyrphostin A23 and brefeldin  A

Abstract

SummaryPlant 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 l2-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. Coimmunoprecipitation experiments show that hTfR is able to interact with a l-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.

Single-Molecule Analysis of PIP2;1 Dynamics and Partitioning Reveals Multiple Modes of Arabidopsis Plasma Membrane Aquaporin Regulation

“…TyrA23 can specifically prevent the interaction between cargo motifs destined for endocytosis and the m2 subunit of the clathrin binding AP-2 adaptor complex (Banbury et al, 2003). A recent report showed that TyrA23 efficiently inhibited interaction between the human transferrin receptor and a m-adaptin subunit and then blocked the internalization of the human transferrin receptor in Arabidopsis protoplasts (Ortiz-Zapater et al, 2006). Thus, it is widely used to inhibit clathrin-dependent endocytosis without causing discernible morphological changes (Dhonukshe et al, 2007;Robinson et al, 2008).…”

The Plant Defense Elicitor Cryptogein Stimulates Clathrin-Mediated Endocytosis Correlated with Reactive Oxygen Species Production in Bright Yellow-2 Tobacco Cells

“…The fact that tyrphostin A23 alone does not inhibit FM4-64 uptake of untreated cells (this study; Ortiz-Zapater et al, 2006;Dhonukshe et al, 2007), indicates that constitutive endocytotic traffic can continue in the presence of this inhibitor. This suggests that, although constitutive and cryptogein-induced endocytosis appears to be mediated by morphologically identical CCPs at the plasma membrane, the regulatory mechanisms involved could be different.…”

“…Some tyrphostin members such as tyrphostin A23 have also been used to inhibit endocytosis: specific inhibition of clathrin-mediated endocytosis due to the interaction between tyrphostin A23 and the subunit m2 of the AP-2 complex, one component of the membrane coat associated with clathrin, has been reported (Banbury et al, 2003). This inhibitor has been used at concentrations of 500 or 350 mM in mammalian cells to inhibit internalization of the Tf-R (Banbury et al, 2003) or the prion protein (Taylor et al, 2005), and in plant protoplasts to inhibit human Tf-R internalization (Ortiz-Zapater et al, 2006). More recently, tyrphostin A23 was utilized at a concentration of 30 mM to inhibit internalization of the PIN auxin efflux carrier and different plasma membrane proteins in Arabidopsis roots (Dhonukshe et al, 2007).…”

“…The authors used for each tyrphostin a concentration 10 times higher than their specific IC 50 (50% inhibition of initial activity) calculated for the EGFR kinase activity (Gazit et al, 1989). In plant cells, tyrphostins A23 and A51 were used at the concentration of 350 mM to inhibit human Tf-R uptake in Arabidopsis protoplasts (Ortiz-Zapater et al, 2006). These two compounds were tested at a lower concentration (30 mM) for their effect on the uptake of several membrane proteins, but the authors underlined that tyrphostins may also affect other processes than the one studied (Dhonukshe et al, 2007).…”

“…Moreover, clathrin-dependent endocytosis has been shown very recently to be operational in plants and used as a predominant pathway, as in animal cells, for internalization of plasma membrane proteins, such as the auxin carrier PIN1 in Arabidopsis (Arabidopsis thaliana), and for spindle and phragmoplast formation as well as for endocytosis in tobacco (Nicotiana tabacum) Bright Yellow-2 (BY-2) cells (Dhonukshe et al, 2007;Tahara et al, 2007). In addition, endocytosis of the human Tf-R, known to be clathrin-dependent in animal cells, is inhibited by an RME inhibitor after expression in plant protoplasts, suggesting the existence of clathrin machinery for RME in plant cells (Ortiz-Zapater et al, 2006). Nevertheless, even if recent evidence for the involvement of vesicle trafficking in the plant immune response against pathogens has been reported (Robatzek, 2007), data are lacking concerning the nature of endocytosis in a plant defense context.…”