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Clathrin-Mediated Constitutive Endocytosis of PIN Auxin Efflux Carriers in Arabidopsis
Current Biology 17, 520–527, March 20, 2007 ª2007 Elsevier Ltd All rights reserved
DOI 10.1016/j.cub.2007.01.052
Report Clathrin-Mediated Constitutive Endocytosis of PIN Auxin Efflux Carriers in Arabidopsis Pankaj Dhonukshe,1 Fernando Aniento,2 Inhwan Hwang,3 David G. Robinson,4 Jozef Mravec,1 York-Dieter Stierhof,1 and Jirˇ´ı Friml1,* 1 Center for Plant Molecular Biology (ZMBP) Auf der Morgenstelle 3 University of Tu¨bingen D-72076 Tu¨bingen Germany 2 Departamento de Bioquı´mica y Biologı´a Molecular Facultad de Farmacia Universidad de Valencia Avda. Vicente Andre´s Estelle´s, s/n E-46100 Burjassot, Valencia Spain 3 Division of Molecular and Life Sciences Pohang University of Science and Technology Pohang, 790-784 Korea 4 Department of Cell Biology Heidelberg Institute for Plant Sciences University of Heidelberg 69120 Heidelberg Germany
Summary Endocytosis is an essential process by which eukaryotic cells internalize exogenous material or regulate signaling at the cell surface [1]. Different endocytic pathways are well established in yeast and animals; prominent among them is clathrin-dependent endocytosis [2, 3]. In plants, endocytosis is poorly defined, and no molecular mechanism for cargo internalization has been demonstrated so far [4, 5], although the internalization of receptor-ligand complexes at the plant plasma membrane has recently been shown [6]. Here we demonstrate by means of a green-to-red photoconvertible fluorescent reporter, EosFP [7], the constitutive endocytosis of PIN auxin efflux carriers [8] and their recycling to the plasma membrane. Using a plant clathrin-specific antibody, we show the presence of clathrin at different stages of coated-vesicle formation at the plasma membrane in Arabidopsis. Genetic interference with clathrin function inhibits PIN internalization and endocytosis in general. Furthermore, pharmacological interference with cargo recruitment into the clathrin pathway blocks internalization of PINs and other plasma-membrane proteins. Our data demonstrate that clathrin-dependent endocytosis is operational in plants and constitutes the predominant pathway for the internalization of numerous plasmamembrane-resident proteins including PIN auxin efflux carriers.
*Correspondence: [email protected]
Results and Discussion Constitutive Endocytosis and Recycling of PIN Proteins In Vivo Recent reports have suggested that the internalization of certain transporters, receptors, and extracellularpeptide ligands occurs at the plant plasma membrane (for detailed reviews, see [4, 5]). The presumptive constitutive internalization and recycling of PIN auxin efflux carriers [9] is of particular interest because this process is considered to be important for regulation of the directionality and throughput of intercellular auxin signaling [10, 11] and thus plays a central role in many plant developmental processes including patterning and tropisms [12–14]. The finding that PIN constitutively cycles is based on the pharmacological inhibition of de novo protein synthesis and protein trafficking to the plasma membrane and is supported by the immunocytochemical visualization of PIN proteins [9]. To gain further insights into PIN cycling, we followed the subcellular dynamics of PIN2 in root epidermis cells of Arabidopsis lines expressing a functional PIN2-Green Fluorescent Protein (PIN2-GFP) fusion [15, 16]. In these lines, because of the high signal intensity, PIN2-GFP exhibited less pronounced polarity of its localization. This strategy, however, allowed the detection of PIN2-GFP intracellularly, in addition to its known plasma-membrane localization, in control cells (Figure 1A) as well as in cells treated with the proteinsynthesis inhibitor cycloheximide (CHX) (see Figure S1A in the Supplemental Data available online). The intracellular PIN2-GFP punctate structures were dynamic and moved with a velocity of about 8 mm/min (see superimposition of two successive frames color-coded in red and green; 6 s interval between frames). To test whether the intracellular PIN2-GFP localizes to the endocytic pathway, we treated the PIN2-GFP-expressing Arabidopsis seedlings with the fluorescent endocytic tracer FM4-64 [17–19]. Quantification of intracellular signals of FM4-64 and PIN2-GFP showed that 39% (64%) of the internalized FM4-64 overlaps with the PIN2-GFP punctae, whereas 93% (66%) of intracellular PIN2GFP colocalizes with FM4-64 (Figure 1C). These results indicate that PIN2-GFP remains both at the plasma membrane and on endosomal compartments, thus supporting the concept of constitutive PIN endocytic cycling. Although single-confocal-section time-lapse microscopy does not deliver a complete picture of the situation, a closer inspection of PIN2-GFP fluorescence at the plasma membrane suggested its sequential internalization as follows: (1) accumulation of PIN2-GFP at specific plasma-membrane domains, (2) its enhanced sequestration into a vesicle-like structures, and (3) its subsequent internalization (Figure 1B). To further confirm the internalization of PIN2 from the plasma membrane and its retargeting to the plasma membrane, we engineered a green-to-red photoconvertible version of PIN2 by using the photoconvertible
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Figure 1. Real-Time Dynamics of PIN2 in Arabidopsis Root Cells (A) PIN2-GFP overlay of frame 1 (green) on frame 2 (+6 s, red) shows dynamics of PIN2-labeled endosomes. (B) Presumptive internalization of the PIN2-GFP-labeled vesicle from the plasma membrane. White arrows show sequential internalization events. (C) Colocalization (yellow arrows in merged channel) of PIN2-GFP (green arrows in green channel) and endocytic tracer FM4-64 (red arrows in red channel). (D–G) Tracking of PIN2-EosFP internalization from the plasma membrane to endosomes by UV-induced green-to-red photoconversion; internalization induced by BFA treatment. (H–K) Tracking of PIN2-EosFP recycling from endosomes to the plasma membrane by UV-induced green-to-red photoconversion; recycling induced by BFA removal.
fluorescent protein EosFP [7]. EosFP remained inferior to GFP in terms of sensitivity; therefore, we had to select highly fluorescent transgenic Arabidopsis lines where PIN2-EosFP exhibits less polarized distribution because of its overexpression. On the other hand, with the ability of UV-induced green-to-red photoconversion of EosFP at the desired location in the cell, we were able to track the translocation of PIN2-EosFP from one place to another. In order to monitor the endocytosis of PIN2EosFP, we photoconverted it from green-to-red at the selected plasma-membrane area and followed its dynamics (Figures 1D and 1E). To better visualize the endocytosed PIN2-EosFP upon photoconversion, we inhibited the recycling of proteins from endosomes to the plasma membrane by using Brefeldin A (BFA) [9].
Accordingly, photoconverted red PIN2-EosFP, along with its unconverted green form, internalized from the plasma membrane and accumulated in intracellular endosomal aggregates—the so-called BFA compartments (Figures 1F and 1G)—thereby directly demonstrating PIN2 endocytosis. In a complementary experiment, we allowed PIN2-EosFP to first internalize in response to BFA and then specifically photoconverted BFA-compartment-localized PIN2-EosFP. After BFA removal, photoconverted red PIN2-EosFP disappeared from the intracellular aggregates and appeared back at the plasma membrane (Figures 1H–1K), demonstrating its targeting from the endosomes to the cell surface. In summary, these results provide in vivo evidence for the trafficking of PIN2 to and from the plasma membrane.
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Clathrin Is Localized at Different Stages of Coated-Vesicle Formation at the Plasma Membrane in Arabidopsis The detection of constitutive PIN2-GFP internalization raises the question about the mechanism by which PIN proteins are internalized. No mechanism of endocytosis has been identified in plants so far, but several different types have been documented in yeast and animals, all of which involve the regulated formation of vesicles at the plasma membrane [1]. The major and best characterized is clathrin-mediated endocytosis, which utilizes the geometric constraints resulting from the binding of clathrin triskelions to the plasma membrane at the site of endocytic-vesicle formation [20, 21]. In plants, coated-like pits have been detected at the plasma membrane on many occasions [22], and internalization of a typical clathrin-specific cargo—the human transferrin receptor (hTfR)—has recently been demonstrated in plant protoplasts [23]. Plant genomes also contain homologs of components of the clathrin-related machinery, including clathrin heavy and light chains, adaptins, and possible accessory proteins [4, 24]. To address the possible role of clathrin in PIN internalization, we performed immunogold electron microscopy on Arabidopsis root cells by using an antiserum raised against a plant clathrin heavy-chain peptide [25]. In western blots from bacterial extracts, this antibody detected a single band of 45 kDa representing the Histagged clathrin peptide, whereas in the plant extracts it detected a band of 190 kDa corresponding to the predicted size of plant clathrin heavy chain (Figure 2G). On the basis of the typical morphology of clathrin-coated structures, we detected all of the morphological intermediates of clathrin-coated pit formation at the plasma membrane from their initiation through vesicle excision, internalization, and loss of clathrin coat (Figure 2A). The sizes of the clathrin-coated vesicles in Arabidopsis were typically smaller (30 nm in diameter without the clathrin coat) than those in mammalian cells (approximately 100 nm) [1]. Using a novel protocol for ultrastructural localization (see Supplemental Experimental Procedures), we found clathrin to be mainly present on internalizing vesicles at the plasma membrane (see arrows in Figure 2B), but also at the trans side of the Golgi apparatus (Figure 2C). Closer observation clearly revealed the association of immunogold in a semicircular or circular manner reminiscent of the coming together of individual clathrin triskelions to form clathrin-coated vesicles at the plasma membrane (Figures 2D and 2E). This localization pattern was further confirmed by immunofluorescence observations, which detected clathrin at the plasma membrane, in the cytosol, and at the transGolgi-related structures (Figure 2H). The extent of clathrin at the plasma membrane was weaker as compared to its intracellular signal, a result that may reflect the shorter resident time at the plasma membrane as compared to the intracellular structures. At the plasma membrane, clathrin partially colocalized with PINs (Figures 2I and 2J). The intracellular clathrin signal partially colocalized with the trans-Golgi marker ST-YFP (Figure 2K) [26] and slightly less with the trans-Golgi network (TGN) marker V-ATPase subunit VHA-a1 (a1) [27] (Figure 2L). This confirms the presence of clathrin in Arabidopsis root cells at intracellular locations similar to those in
mammalian cells [3], emphasizing its prominent localization to coated pits at the plasma membrane. Thus, clathrin is perfectly positioned to mediate internalization of plant plasma-membrane proteins including PINs. Genetic Interference with the Clathrin Machinery Impairs PIN Internalization and Endocytosis in General We next addressed whether functionally competent clathrin is required for the internalization of PIN proteins. Studies in mammals have shown that the overexpression of the C-terminal part of clathrin heavy chain (termed ‘‘clathrin Hub’’) binds to and titers away the light chains, thus making them unavailable for clathrin cage formation [28]. This leads to strong dominant-negative effects on clathrin-mediated processes. We employed the same strategy to examine the functional role of clathrin in Arabidopsis. Therefore, we prepared GFP- and RFP-tagged Arabidopsis clathrin Hub1 constructs and overexpressed them under the control of a strong viral 35S promoter in Arabidopsis protoplasts. To test for an effect on endocytosis, we monitored the endocytic uptake of FM4-64 [19, 29, 30] in protoplasts showing an observable amount of cytoplasm, in which it is easier to follow endocytosis. Around 80% of the protoplasts (n = 52) were viable and showed FM4-64 uptake into endosomal compartments (Figure 3A) that aggregated after application of BFA (Figure 3B). After expression of GFP-Hub1, which as expected was localized in the cytosol (green in Figure 3D), inhibition of FM4-64 internalization was observed both in the absence (Figure 3C) and in the presence of BFA (Figure 3D, compare FM4-64 distribution with Figure 3B) in about 75% of the protoplasts (n = 46), indicating a defect in endocytosis. These results point to the existence of clathrin-mediated endocytosis in plants. They also show that the uptake of general endocytic tracers, e.g., FM4-64, largely depends on clathrin, suggesting that clathrin-dependent endocytosis accounts for most, if not all, of the internalization at the plant plasma membrane. To test whether PINs are cargoes for clathrin-mediated endocytosis, we monitored the internalization of the PIN1 and PIN2 in Arabidopsis protoplasts. Consistent with the results from Arabidopsis root cells, the localization of PIN1-GFP was detected both at the plasma membrane and as intracellular punctae (Figure 3E) in approximately 78% protoplasts (n = 48). Among the protoplasts bearing intracellular PIN1-GFP punctae, 82% of the intracellular PIN1-GFP signal colocalized with FM4-64 (Figure 3F), confirming its localization along the endocytic pathway. BFA treatment resulted, as expected, in the internalization of PIN1GFP from the plasma membrane and its accumulation in BFA compartments (Figure 3G) in 80% of protoplasts (n = 40). However, after overexpression of RFP-Hub1 (red in Figure 3H), both the incidence of PIN1-GFP in endocytic compartments and its BFA-induced aggregation were impaired (Figure 3H, compare PIN1-GFP distribution with Figure 3G) in about 76% of protoplasts (n = 42). Similar results were also obtained with PIN2 (Figures 3I–3L) and another plasma-membrane protein— the water channel PIP2 [31] (Figures 3M–3P). It seems that Hub1 expression affects preferentially PIN endocytosis and not so much the exocytosis, because the
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Figure 2. Visualization of clathrin and clathrin-coated pits at the plasma membrane of Arabidopsis cells (A) Transmission electron microscope (TEM) images of subsequent stages of clathrin coated-pit formation through vesicle fission and, finally, the loss of clathrin coat from the internalized vesicle (yellow arrow). (Samples were high-pressure frozen, freeze substituted, and embedded in epoxy resin.) The scale bar represents 100 nm. (B–F) Immunogold-TEM analysis of root epidermis cells with ultrathin thawed cryo-sections. Clathrin labeling with silver-enhanced Nanogold reveals localization of clathrincoated vesicles both at the plasma membrane (B, D, and E) and at the trans side of the Golgi apparatus (C). The plasma membrane is shown by ‘‘PM’’ and the Golgi apparatus by ‘‘G,’’ and yellow arrows show coated vesicles. For clarity, the forming coated pit (red half circle in [D]) and coated vesicles (red circles in [E]) are marked by the dotted lines. Control shows no gold labeling in the absence of primary antibody (F). Scale bars represent 500 nm (B), 250 nm (C), and 100 nm (D and E). (G) Western blot showing the specificity of the clathrin antibody (1: Hisx6-Clathrin, Coomassie staining; 2: Hisx6-Clathrin, clathrin antibody; 3: Arabidopsis extracts, clathrin antibody; and 4: Arabidopsis extracts, control serum). (H–L) Immunolocalization of clathrin (red) with PIN1-GFP (green in [I]), PIN2-GFP (green in [J]), ST-YFP (green in [K]), and a1-GFP (green in [L]). The insets in (H) and (I) and white arrowheads in (H)–(J) and (L) show localization of clathrin at the plasma membrane where it shows partial colocalization with plasma-membrane proteins such as PIN1 (I) and PIN2 (J). Root epidermis and cortex (H, J, K, and L) and stele (I) cells are shown. DAPI-stained nuclei are in blue.
photobleached PIN2-EosFP recovered at the plasma membrane after Hub1 expression too (see Figures S1B and S1E). These findings underline the importance of the clathrin-mediated endocytic pathway for the internalization of plasma-membrane-localized proteins in plants in general, and they identify PIN proteins as prominent endogenous substrates of clathrin-dependent endocytosis. Pharmacological Interference with Cargo Recruitment into the Clathrin Pathway Inhibits PIN Internalization In yeast and animals, an established way to inhibit the clathrin-dependent internalization of cell-surface cargo
molecules is the pharmacological interference with endocytic-cargo recruitment. The tyrosine analog Tyrphostin A23 is a well-characterized inhibitor of the recruitment of endocytic cargo into the clathrin-mediated pathway both in vivo and in vitro [32]. It specifically prevents the interaction between the m2 subunit of the clathrin-binding AP-2 adaptor complex and endocytic motifs present in the cytoplasmic domain of plasmamembrane proteins destined for endocytosis such as the hTfR [32]. A close structural analog, Tyrphostin A51, does not elicit these effects and is routinely used as a negative control [32]. Despite the fact that the molecular target of Tyrphostin A23 in plants has not been unequivocally demonstrated, a recent report
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Figure 3. Dominant-Negative Clathrin Hub1 Expression Inhibits Endocytosis in Arabidopsis Protoplasts (A and B) Uptake of endocytic tracer FM4-64 into the endocytic pathway (A) and its accumulation in BFA compartments after BFA treatment (B). (C and D) Expression of Hub1 (C) or GFP-Hub1 (green in [D]) impairs FM4-64 uptake (C) and its accumulation in BFA compartments (D). (E and F) Localization of PIN1-GFP (green) at the plasma membrane and at the endosomal compartments colocalizing with FM4-64 (red). See the inset in (F) for the magnified view. (G and H) Internalization of PIN1-GFP (green) into BFA compartments following BFA treatment (G) is impaired by expression of RFP-Hub1 (red) (H). (I and J) Localization of PIN2-EosFP (green) at the plasma membrane and at the endosomal compartments colabeled with FM4-64 (red). (K and L) Internalization of PIN2-EosFP (green) into BFA compartments following BFA treatment (K) is inhibited by expression of RFP-Hub1 (red) (L). (M) Localization of PIP2-GFP at the plasma membrane and at the intracellular compartments. (N and O) Internalization of PIP2-GFP (green) into BFA compartments following BFA treatment (N) is impaired by expression of RFP-Hub1 (red) (O). (P) DIC image showing the area occupied by the cytosol (yellow arrow) and vacuole (v). The scale bar represents 20 mm.
shows that Tyrphostin A23 efficiently inhibited interaction between hTfR and a m-adaptin subunit from Arabidopsis cytosol and blocked the internalization of the hTfR in Arabidopsis protoplasts [23]. We therefore tested the effects of Tyrphostin A23 and A51 on the
internalization of PIN proteins in Arabidopsis root cells. We visualized constitutive PIN2 cycling by detecting the PIN2-GFP protein both at the plasma membrane and at the endocytic compartments (Figures 4A and 4B). When applied to Arabidopsis seedlings, Tyrphostin
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Figure 4. Tyrphostin A23 Inhibits Internalization of Cargoes from the Plasma Membrane of Arabidopsis Root Cells (A–D) (A) and (B) show PIN2-GFP (green) at the plasma membrane and at the intracellular compartments colocalizing with FM4-64 (red). (C and D) Tyrphostin A23 inhibits appearance of intracellular PIN2-GFP (green) but allows FM4-64 (red) uptake. Note that the inset in (B) shows a magnified view of cointernalization of PIN2-GFP with FM4-64 to endocytic compartment and the inset in (D) shows internalization of only FM4-64 but not that of PIN2-GFP. (E and F) Internalization of PIN2-GFP (green) into FM4-64-labeled (red) BFA compartments after BFA treatment. (G and H) Tyrphostin A23 inhibits PIN2-GFP (green) BFA-dependent internalization but allows FM4-64 (red) uptake into BFA compartments. (I–K) Polar localization of PIN1 (red) and staining of TGN by a1 (green) in control (I). Internalization of PIN1 into a1-labeled BFA compartments following BFA treatment (J) is inhibited by Tyrphostin A23 (K). (L) Tyrphostin A23 does not interfere with recovery of PIN1 (red) at the plasma membrane and recovery of normal a1-labeled TGN (green) distribution after removal of BFA. (M and N) PIN2 (red) internalization into GNOM-labeled (green) BFA compartments following BFA treatment (M) is inhibited by Tyrphostin A23 (N). (O and P) Internalization of PM-ATPase (green) into ARF1-labeled (red) BFA compartments following BFA treatment (O) is inhibited by Tyrphostin A23 (P). (Q and R) Internalization of PIP2-GFP into BFA compartments following BFA treatment (Q) is inhibited by Tyrphostin A23 (R). (S and T) Internalization of LTi6b-GFP into BFA compartments following BFA treatment (S) is inhibited by Tyrphostin A23 (T). (U) Tyrphostin A23 does not interfere with recovery of LTi6b-GFP at the plasma membrane after removal of BFA. DAPI-stained nuclei are in blue.
A23 (Figures 4C and 4D), but not A51 (Figure S2A), efficiently inhibited the intracellular appearance of PIN2 but still allowed detectable FM4-64 uptake (Figures 4C and 4D). This is consistent with the idea that the interaction between a clathrin adaptor complex and cargo molecules at the plasma membrane is required for the
sorting of cargo proteins into clathrin-coated vesicles, but is not essential for clathrin-coated vesicle formation per se [33]. Indeed, Tyrphostin A23 disrupted the recruitment of PIN2 but nevertheless allowed endocytic vesicles (still incorporating FM4-64-labeled membrane) to form and internalize (Figures 4C and 4D).
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The inhibition of PIN2 internalization by Tyrphostin A23 was even more apparent in the presence of BFA. In the control situation, BFA caused PIN2 to accumulate into the BFA compartments, where it colocalized with FM4-64 (Figures 4E and 4F). In the presence of Tyrphostin A23, only FM4-64 accumulated in BFA compartments, whereas PIN2 was retained at the plasma membrane (Figures 4G and 4H). Similar results were also obtained for other plasma-membrane-resident proteins, which were previously suggested to undergo BFA-sensitive endocytic cycling, such as PM-ATPase, PIP2 water channel, and the low-temperature-induced plasmamembrane protein Lti6b [19, 26, 34]. Tyrphostin A23, but not A51, inhibited the BFA-induced internalization of all these proteins (Figures 4O–4T and Figures S2B– S2D). The action of Tyrphostin A23 in Arabidopsis was reversible (Figures S3A–S3C) and appeared to be specific for endocytic-cargo recruitment at the plasma membrane because it did not show any apparent effects on other subcellular trafficking processes at concentrations that already efficiently inhibited cargo internalization at the plasma membrane. For example, after Tyrphostin A23 treatment, the appearance of the TGN/ endosomes (as visualized by a1 [27], GNOM [35], and ARF1 [15] markers) was largely unchanged, and in the presence of BFA these markers accumulated into the BFA compartments (Figures 4I–4P). In addition, when Tyrphostin A23 was washed away with BFA, both plasma-membrane-localized proteins and TGN/endosomes accumulated into the BFA compartments similar to the ones observed in case of BFA treatment alone (Figures S3A–S3C). In addition, Tyrphostin A23 did not visibly interfere with exocytosis because, after BFA washout in the presence of Tyrphostin A23, endosomes and the Golgi apparatus recovered and plasma-membrane proteins, including PINs, were recycled from the BFA compartments back to the plasma membrane (Figures 4L and 4U and Figure S3D). In addition, in the presence of Tyrphostin A23, recovery of Lti6B-GFP [26] was observed at the plasma membrane when it was photobleached both from the cytosol and from the plasma membrane (see Figure S4), indicating that Tyrphostin A23 does not completely inhibit the forward traffic toward the plasma membrane. It cannot be entirely excluded that Tyrphostin A23 may also affect other processes; however, its strong effects on internalization of PINs and other cargoes implicate the importance of clathrin-dependent endocytosis for the internalization of many plasma-membrane-resident proteins in plants. Conclusions Endocytosis is of paramount importance for the function of both unicellular and multicellular organisms. Despite numerous recent demonstrations that various plant plasma-membrane proteins are internalized from the cell surface [4, 6, 9, 19, 26, 30, 34], a molecular mechanism for endocytosis in plants has not been demonstrated. Through several approaches including live-cell imaging, electron microscopy, and genetic and pharmacological studies, we show here that an endocytic mechanism utilizing the coat protein clathrin is operational in plants. In addition, we have identified several endogenous cargoes for clathrin-dependent endocytosis, prominent among them the plant-specific,
constitutively cycling PIN protein family of auxin efflux carriers. Interference with clathrin-dependent endocytosis impairs the internalization of many plant plasmamembrane proteins and inhibits endocytosis in general, suggesting that the clathrin-dependent internalization machinery is the major endocytic mechanism in plant cells. We feel that an important topic for future studies will be, in addition to the identification of ancillary, regulatory elements of the internalization machinery, a full appreciation of the diverse functions of endocytosis in plants. In particular, elucidating the role of clathrindependent PIN internalization in regulating auxin transport will be of fundamental importance to our understanding of how this evolutionarily conserved mechanism is utilized in mediating plant-specific physiological and developmental roles. Supplemental Data Supplemental Data include Experimental Procedures and four figures and are available with this article online at: http://www. current-biology.com/cgi/content/full/17/6/520/DC1/. Acknowledgments We thank J. Wiedenmann, G. Ju¨rgens, C. Luschnig, B. Scheres, M. Grebe, W. Michalke, D. Ehrhardt, and K. Schumacher for sharing published material. We thank C. Brancato for assistance with Arabidopsis protoplast transformations. This work was supported by a Volkswagenstiftung (P.D. J.F., J.M.), EMBO Young Investigator Program (J.F.), the Deutsche Forschungsgemeinschaft (D.G.R.), SFB 446 (Y.-D.S.), and the Ministerio de Educacio´n y Ciencia, grant BFU2005-00071 (F.A.). Received: November 21, 2006 Revised: January 18, 2007 Accepted: January 18, 2007 Published online: February 15, 2007 References 1. Alberts, B., Johnson, A., Lewis, J., Raff, M., Roberts, K., and Walter, P. (2002). Molecular Biology of the Cell, 4th Edition (New York: Garland Science). 2. Conner, S.D., and Schmid, S.L. (2003). Regulated portals of entry into the cell. Nature 422, 37–44. 3. Kirchhausen, T. (2000). Clathrin. Annu. Rev. Biochem. 69, 699– 727. 4. Murphy, A.S., Bandyopadhyay, A., Holstein, S.E., and Peer, W.A. (2005). Endocytotic cycling of PM proteins. Annu. Rev. Plant Biol. 56, 221–251. 5. Russinova, E., and de Vries, S.C. (2005). Receptor-mediated endocytosis in plants. In Plant Endocytosis, Volume 1, J. Samaj, F. Baluska, and D. Menzel, eds. (Heidelberg: Springer Verlag), pp. 103–115. 6. Robatzek, S., Chinchilla, D., and Boller, T. (2006). Ligandinduced endocytosis of the pattern recognition receptor FLS2 in Arabidopsis. Genes Dev. 20, 537–542. 7. Wiedenmann, J., Ivanchenko, S., Oswald, F., Schmitt, F., Rocker, C., Salih, A., Spindler, K.D., and Nienhaus, G.U. (2004). EosFP, a fluorescent marker protein with UV-inducible greento-red fluorescence conversion. Proc. Natl. Acad. Sci. USA 101, 15905–15910. 8. Petrasek, J., Mravec, J., Bouchard, R., Blakeslee, J.J., Abas, M., Seifertova, D., Wisniewska, J., Tadele, Z., Kubes, M., Covanova, M., et al. (2006). PIN proteins perform a rate-limiting function in cellular auxin efflux. Science 312, 914–918. 9. Geldner, N., Friml, J., Stierhof, Y.D., Jurgens, G., and Palme, K. (2001). Auxin transport inhibitors block PIN1 cycling and vesicle trafficking. Nature 413, 425–428. 10. Friml, J. (2003). Auxin transport - shaping the plant. Curr. Opin. Plant Biol. 6, 7–12.
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DOI 10.1016/j.cub.2007.01.052
Report Clathrin-Mediated Constitutive Endocytosis of PIN Auxin Efflux Carriers in Arabidopsis Pankaj Dhonukshe,1 Fernando Aniento,2 Inhwan Hwang,3 David G. Robinson,4 Jozef Mravec,1 York-Dieter Stierhof,1 and Jirˇ´ı Friml1,* 1 Center for Plant Molecular Biology (ZMBP) Auf der Morgenstelle 3 University of Tu¨bingen D-72076 Tu¨bingen Germany 2 Departamento de Bioquı´mica y Biologı´a Molecular Facultad de Farmacia Universidad de Valencia Avda. Vicente Andre´s Estelle´s, s/n E-46100 Burjassot, Valencia Spain 3 Division of Molecular and Life Sciences Pohang University of Science and Technology Pohang, 790-784 Korea 4 Department of Cell Biology Heidelberg Institute for Plant Sciences University of Heidelberg 69120 Heidelberg Germany
Summary Endocytosis is an essential process by which eukaryotic cells internalize exogenous material or regulate signaling at the cell surface [1]. Different endocytic pathways are well established in yeast and animals; prominent among them is clathrin-dependent endocytosis [2, 3]. In plants, endocytosis is poorly defined, and no molecular mechanism for cargo internalization has been demonstrated so far [4, 5], although the internalization of receptor-ligand complexes at the plant plasma membrane has recently been shown [6]. Here we demonstrate by means of a green-to-red photoconvertible fluorescent reporter, EosFP [7], the constitutive endocytosis of PIN auxin efflux carriers [8] and their recycling to the plasma membrane. Using a plant clathrin-specific antibody, we show the presence of clathrin at different stages of coated-vesicle formation at the plasma membrane in Arabidopsis. Genetic interference with clathrin function inhibits PIN internalization and endocytosis in general. Furthermore, pharmacological interference with cargo recruitment into the clathrin pathway blocks internalization of PINs and other plasma-membrane proteins. Our data demonstrate that clathrin-dependent endocytosis is operational in plants and constitutes the predominant pathway for the internalization of numerous plasmamembrane-resident proteins including PIN auxin efflux carriers.
*Correspondence: [email protected]
Results and Discussion Constitutive Endocytosis and Recycling of PIN Proteins In Vivo Recent reports have suggested that the internalization of certain transporters, receptors, and extracellularpeptide ligands occurs at the plant plasma membrane (for detailed reviews, see [4, 5]). The presumptive constitutive internalization and recycling of PIN auxin efflux carriers [9] is of particular interest because this process is considered to be important for regulation of the directionality and throughput of intercellular auxin signaling [10, 11] and thus plays a central role in many plant developmental processes including patterning and tropisms [12–14]. The finding that PIN constitutively cycles is based on the pharmacological inhibition of de novo protein synthesis and protein trafficking to the plasma membrane and is supported by the immunocytochemical visualization of PIN proteins [9]. To gain further insights into PIN cycling, we followed the subcellular dynamics of PIN2 in root epidermis cells of Arabidopsis lines expressing a functional PIN2-Green Fluorescent Protein (PIN2-GFP) fusion [15, 16]. In these lines, because of the high signal intensity, PIN2-GFP exhibited less pronounced polarity of its localization. This strategy, however, allowed the detection of PIN2-GFP intracellularly, in addition to its known plasma-membrane localization, in control cells (Figure 1A) as well as in cells treated with the proteinsynthesis inhibitor cycloheximide (CHX) (see Figure S1A in the Supplemental Data available online). The intracellular PIN2-GFP punctate structures were dynamic and moved with a velocity of about 8 mm/min (see superimposition of two successive frames color-coded in red and green; 6 s interval between frames). To test whether the intracellular PIN2-GFP localizes to the endocytic pathway, we treated the PIN2-GFP-expressing Arabidopsis seedlings with the fluorescent endocytic tracer FM4-64 [17–19]. Quantification of intracellular signals of FM4-64 and PIN2-GFP showed that 39% (64%) of the internalized FM4-64 overlaps with the PIN2-GFP punctae, whereas 93% (66%) of intracellular PIN2GFP colocalizes with FM4-64 (Figure 1C). These results indicate that PIN2-GFP remains both at the plasma membrane and on endosomal compartments, thus supporting the concept of constitutive PIN endocytic cycling. Although single-confocal-section time-lapse microscopy does not deliver a complete picture of the situation, a closer inspection of PIN2-GFP fluorescence at the plasma membrane suggested its sequential internalization as follows: (1) accumulation of PIN2-GFP at specific plasma-membrane domains, (2) its enhanced sequestration into a vesicle-like structures, and (3) its subsequent internalization (Figure 1B). To further confirm the internalization of PIN2 from the plasma membrane and its retargeting to the plasma membrane, we engineered a green-to-red photoconvertible version of PIN2 by using the photoconvertible
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Figure 1. Real-Time Dynamics of PIN2 in Arabidopsis Root Cells (A) PIN2-GFP overlay of frame 1 (green) on frame 2 (+6 s, red) shows dynamics of PIN2-labeled endosomes. (B) Presumptive internalization of the PIN2-GFP-labeled vesicle from the plasma membrane. White arrows show sequential internalization events. (C) Colocalization (yellow arrows in merged channel) of PIN2-GFP (green arrows in green channel) and endocytic tracer FM4-64 (red arrows in red channel). (D–G) Tracking of PIN2-EosFP internalization from the plasma membrane to endosomes by UV-induced green-to-red photoconversion; internalization induced by BFA treatment. (H–K) Tracking of PIN2-EosFP recycling from endosomes to the plasma membrane by UV-induced green-to-red photoconversion; recycling induced by BFA removal.
fluorescent protein EosFP [7]. EosFP remained inferior to GFP in terms of sensitivity; therefore, we had to select highly fluorescent transgenic Arabidopsis lines where PIN2-EosFP exhibits less polarized distribution because of its overexpression. On the other hand, with the ability of UV-induced green-to-red photoconversion of EosFP at the desired location in the cell, we were able to track the translocation of PIN2-EosFP from one place to another. In order to monitor the endocytosis of PIN2EosFP, we photoconverted it from green-to-red at the selected plasma-membrane area and followed its dynamics (Figures 1D and 1E). To better visualize the endocytosed PIN2-EosFP upon photoconversion, we inhibited the recycling of proteins from endosomes to the plasma membrane by using Brefeldin A (BFA) [9].
Accordingly, photoconverted red PIN2-EosFP, along with its unconverted green form, internalized from the plasma membrane and accumulated in intracellular endosomal aggregates—the so-called BFA compartments (Figures 1F and 1G)—thereby directly demonstrating PIN2 endocytosis. In a complementary experiment, we allowed PIN2-EosFP to first internalize in response to BFA and then specifically photoconverted BFA-compartment-localized PIN2-EosFP. After BFA removal, photoconverted red PIN2-EosFP disappeared from the intracellular aggregates and appeared back at the plasma membrane (Figures 1H–1K), demonstrating its targeting from the endosomes to the cell surface. In summary, these results provide in vivo evidence for the trafficking of PIN2 to and from the plasma membrane.
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Clathrin Is Localized at Different Stages of Coated-Vesicle Formation at the Plasma Membrane in Arabidopsis The detection of constitutive PIN2-GFP internalization raises the question about the mechanism by which PIN proteins are internalized. No mechanism of endocytosis has been identified in plants so far, but several different types have been documented in yeast and animals, all of which involve the regulated formation of vesicles at the plasma membrane [1]. The major and best characterized is clathrin-mediated endocytosis, which utilizes the geometric constraints resulting from the binding of clathrin triskelions to the plasma membrane at the site of endocytic-vesicle formation [20, 21]. In plants, coated-like pits have been detected at the plasma membrane on many occasions [22], and internalization of a typical clathrin-specific cargo—the human transferrin receptor (hTfR)—has recently been demonstrated in plant protoplasts [23]. Plant genomes also contain homologs of components of the clathrin-related machinery, including clathrin heavy and light chains, adaptins, and possible accessory proteins [4, 24]. To address the possible role of clathrin in PIN internalization, we performed immunogold electron microscopy on Arabidopsis root cells by using an antiserum raised against a plant clathrin heavy-chain peptide [25]. In western blots from bacterial extracts, this antibody detected a single band of 45 kDa representing the Histagged clathrin peptide, whereas in the plant extracts it detected a band of 190 kDa corresponding to the predicted size of plant clathrin heavy chain (Figure 2G). On the basis of the typical morphology of clathrin-coated structures, we detected all of the morphological intermediates of clathrin-coated pit formation at the plasma membrane from their initiation through vesicle excision, internalization, and loss of clathrin coat (Figure 2A). The sizes of the clathrin-coated vesicles in Arabidopsis were typically smaller (30 nm in diameter without the clathrin coat) than those in mammalian cells (approximately 100 nm) [1]. Using a novel protocol for ultrastructural localization (see Supplemental Experimental Procedures), we found clathrin to be mainly present on internalizing vesicles at the plasma membrane (see arrows in Figure 2B), but also at the trans side of the Golgi apparatus (Figure 2C). Closer observation clearly revealed the association of immunogold in a semicircular or circular manner reminiscent of the coming together of individual clathrin triskelions to form clathrin-coated vesicles at the plasma membrane (Figures 2D and 2E). This localization pattern was further confirmed by immunofluorescence observations, which detected clathrin at the plasma membrane, in the cytosol, and at the transGolgi-related structures (Figure 2H). The extent of clathrin at the plasma membrane was weaker as compared to its intracellular signal, a result that may reflect the shorter resident time at the plasma membrane as compared to the intracellular structures. At the plasma membrane, clathrin partially colocalized with PINs (Figures 2I and 2J). The intracellular clathrin signal partially colocalized with the trans-Golgi marker ST-YFP (Figure 2K) [26] and slightly less with the trans-Golgi network (TGN) marker V-ATPase subunit VHA-a1 (a1) [27] (Figure 2L). This confirms the presence of clathrin in Arabidopsis root cells at intracellular locations similar to those in
mammalian cells [3], emphasizing its prominent localization to coated pits at the plasma membrane. Thus, clathrin is perfectly positioned to mediate internalization of plant plasma-membrane proteins including PINs. Genetic Interference with the Clathrin Machinery Impairs PIN Internalization and Endocytosis in General We next addressed whether functionally competent clathrin is required for the internalization of PIN proteins. Studies in mammals have shown that the overexpression of the C-terminal part of clathrin heavy chain (termed ‘‘clathrin Hub’’) binds to and titers away the light chains, thus making them unavailable for clathrin cage formation [28]. This leads to strong dominant-negative effects on clathrin-mediated processes. We employed the same strategy to examine the functional role of clathrin in Arabidopsis. Therefore, we prepared GFP- and RFP-tagged Arabidopsis clathrin Hub1 constructs and overexpressed them under the control of a strong viral 35S promoter in Arabidopsis protoplasts. To test for an effect on endocytosis, we monitored the endocytic uptake of FM4-64 [19, 29, 30] in protoplasts showing an observable amount of cytoplasm, in which it is easier to follow endocytosis. Around 80% of the protoplasts (n = 52) were viable and showed FM4-64 uptake into endosomal compartments (Figure 3A) that aggregated after application of BFA (Figure 3B). After expression of GFP-Hub1, which as expected was localized in the cytosol (green in Figure 3D), inhibition of FM4-64 internalization was observed both in the absence (Figure 3C) and in the presence of BFA (Figure 3D, compare FM4-64 distribution with Figure 3B) in about 75% of the protoplasts (n = 46), indicating a defect in endocytosis. These results point to the existence of clathrin-mediated endocytosis in plants. They also show that the uptake of general endocytic tracers, e.g., FM4-64, largely depends on clathrin, suggesting that clathrin-dependent endocytosis accounts for most, if not all, of the internalization at the plant plasma membrane. To test whether PINs are cargoes for clathrin-mediated endocytosis, we monitored the internalization of the PIN1 and PIN2 in Arabidopsis protoplasts. Consistent with the results from Arabidopsis root cells, the localization of PIN1-GFP was detected both at the plasma membrane and as intracellular punctae (Figure 3E) in approximately 78% protoplasts (n = 48). Among the protoplasts bearing intracellular PIN1-GFP punctae, 82% of the intracellular PIN1-GFP signal colocalized with FM4-64 (Figure 3F), confirming its localization along the endocytic pathway. BFA treatment resulted, as expected, in the internalization of PIN1GFP from the plasma membrane and its accumulation in BFA compartments (Figure 3G) in 80% of protoplasts (n = 40). However, after overexpression of RFP-Hub1 (red in Figure 3H), both the incidence of PIN1-GFP in endocytic compartments and its BFA-induced aggregation were impaired (Figure 3H, compare PIN1-GFP distribution with Figure 3G) in about 76% of protoplasts (n = 42). Similar results were also obtained with PIN2 (Figures 3I–3L) and another plasma-membrane protein— the water channel PIP2 [31] (Figures 3M–3P). It seems that Hub1 expression affects preferentially PIN endocytosis and not so much the exocytosis, because the
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Figure 2. Visualization of clathrin and clathrin-coated pits at the plasma membrane of Arabidopsis cells (A) Transmission electron microscope (TEM) images of subsequent stages of clathrin coated-pit formation through vesicle fission and, finally, the loss of clathrin coat from the internalized vesicle (yellow arrow). (Samples were high-pressure frozen, freeze substituted, and embedded in epoxy resin.) The scale bar represents 100 nm. (B–F) Immunogold-TEM analysis of root epidermis cells with ultrathin thawed cryo-sections. Clathrin labeling with silver-enhanced Nanogold reveals localization of clathrincoated vesicles both at the plasma membrane (B, D, and E) and at the trans side of the Golgi apparatus (C). The plasma membrane is shown by ‘‘PM’’ and the Golgi apparatus by ‘‘G,’’ and yellow arrows show coated vesicles. For clarity, the forming coated pit (red half circle in [D]) and coated vesicles (red circles in [E]) are marked by the dotted lines. Control shows no gold labeling in the absence of primary antibody (F). Scale bars represent 500 nm (B), 250 nm (C), and 100 nm (D and E). (G) Western blot showing the specificity of the clathrin antibody (1: Hisx6-Clathrin, Coomassie staining; 2: Hisx6-Clathrin, clathrin antibody; 3: Arabidopsis extracts, clathrin antibody; and 4: Arabidopsis extracts, control serum). (H–L) Immunolocalization of clathrin (red) with PIN1-GFP (green in [I]), PIN2-GFP (green in [J]), ST-YFP (green in [K]), and a1-GFP (green in [L]). The insets in (H) and (I) and white arrowheads in (H)–(J) and (L) show localization of clathrin at the plasma membrane where it shows partial colocalization with plasma-membrane proteins such as PIN1 (I) and PIN2 (J). Root epidermis and cortex (H, J, K, and L) and stele (I) cells are shown. DAPI-stained nuclei are in blue.
photobleached PIN2-EosFP recovered at the plasma membrane after Hub1 expression too (see Figures S1B and S1E). These findings underline the importance of the clathrin-mediated endocytic pathway for the internalization of plasma-membrane-localized proteins in plants in general, and they identify PIN proteins as prominent endogenous substrates of clathrin-dependent endocytosis. Pharmacological Interference with Cargo Recruitment into the Clathrin Pathway Inhibits PIN Internalization In yeast and animals, an established way to inhibit the clathrin-dependent internalization of cell-surface cargo
molecules is the pharmacological interference with endocytic-cargo recruitment. The tyrosine analog Tyrphostin A23 is a well-characterized inhibitor of the recruitment of endocytic cargo into the clathrin-mediated pathway both in vivo and in vitro [32]. It specifically prevents the interaction between the m2 subunit of the clathrin-binding AP-2 adaptor complex and endocytic motifs present in the cytoplasmic domain of plasmamembrane proteins destined for endocytosis such as the hTfR [32]. A close structural analog, Tyrphostin A51, does not elicit these effects and is routinely used as a negative control [32]. Despite the fact that the molecular target of Tyrphostin A23 in plants has not been unequivocally demonstrated, a recent report
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Figure 3. Dominant-Negative Clathrin Hub1 Expression Inhibits Endocytosis in Arabidopsis Protoplasts (A and B) Uptake of endocytic tracer FM4-64 into the endocytic pathway (A) and its accumulation in BFA compartments after BFA treatment (B). (C and D) Expression of Hub1 (C) or GFP-Hub1 (green in [D]) impairs FM4-64 uptake (C) and its accumulation in BFA compartments (D). (E and F) Localization of PIN1-GFP (green) at the plasma membrane and at the endosomal compartments colocalizing with FM4-64 (red). See the inset in (F) for the magnified view. (G and H) Internalization of PIN1-GFP (green) into BFA compartments following BFA treatment (G) is impaired by expression of RFP-Hub1 (red) (H). (I and J) Localization of PIN2-EosFP (green) at the plasma membrane and at the endosomal compartments colabeled with FM4-64 (red). (K and L) Internalization of PIN2-EosFP (green) into BFA compartments following BFA treatment (K) is inhibited by expression of RFP-Hub1 (red) (L). (M) Localization of PIP2-GFP at the plasma membrane and at the intracellular compartments. (N and O) Internalization of PIP2-GFP (green) into BFA compartments following BFA treatment (N) is impaired by expression of RFP-Hub1 (red) (O). (P) DIC image showing the area occupied by the cytosol (yellow arrow) and vacuole (v). The scale bar represents 20 mm.
shows that Tyrphostin A23 efficiently inhibited interaction between hTfR and a m-adaptin subunit from Arabidopsis cytosol and blocked the internalization of the hTfR in Arabidopsis protoplasts [23]. We therefore tested the effects of Tyrphostin A23 and A51 on the
internalization of PIN proteins in Arabidopsis root cells. We visualized constitutive PIN2 cycling by detecting the PIN2-GFP protein both at the plasma membrane and at the endocytic compartments (Figures 4A and 4B). When applied to Arabidopsis seedlings, Tyrphostin
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Figure 4. Tyrphostin A23 Inhibits Internalization of Cargoes from the Plasma Membrane of Arabidopsis Root Cells (A–D) (A) and (B) show PIN2-GFP (green) at the plasma membrane and at the intracellular compartments colocalizing with FM4-64 (red). (C and D) Tyrphostin A23 inhibits appearance of intracellular PIN2-GFP (green) but allows FM4-64 (red) uptake. Note that the inset in (B) shows a magnified view of cointernalization of PIN2-GFP with FM4-64 to endocytic compartment and the inset in (D) shows internalization of only FM4-64 but not that of PIN2-GFP. (E and F) Internalization of PIN2-GFP (green) into FM4-64-labeled (red) BFA compartments after BFA treatment. (G and H) Tyrphostin A23 inhibits PIN2-GFP (green) BFA-dependent internalization but allows FM4-64 (red) uptake into BFA compartments. (I–K) Polar localization of PIN1 (red) and staining of TGN by a1 (green) in control (I). Internalization of PIN1 into a1-labeled BFA compartments following BFA treatment (J) is inhibited by Tyrphostin A23 (K). (L) Tyrphostin A23 does not interfere with recovery of PIN1 (red) at the plasma membrane and recovery of normal a1-labeled TGN (green) distribution after removal of BFA. (M and N) PIN2 (red) internalization into GNOM-labeled (green) BFA compartments following BFA treatment (M) is inhibited by Tyrphostin A23 (N). (O and P) Internalization of PM-ATPase (green) into ARF1-labeled (red) BFA compartments following BFA treatment (O) is inhibited by Tyrphostin A23 (P). (Q and R) Internalization of PIP2-GFP into BFA compartments following BFA treatment (Q) is inhibited by Tyrphostin A23 (R). (S and T) Internalization of LTi6b-GFP into BFA compartments following BFA treatment (S) is inhibited by Tyrphostin A23 (T). (U) Tyrphostin A23 does not interfere with recovery of LTi6b-GFP at the plasma membrane after removal of BFA. DAPI-stained nuclei are in blue.
A23 (Figures 4C and 4D), but not A51 (Figure S2A), efficiently inhibited the intracellular appearance of PIN2 but still allowed detectable FM4-64 uptake (Figures 4C and 4D). This is consistent with the idea that the interaction between a clathrin adaptor complex and cargo molecules at the plasma membrane is required for the
sorting of cargo proteins into clathrin-coated vesicles, but is not essential for clathrin-coated vesicle formation per se [33]. Indeed, Tyrphostin A23 disrupted the recruitment of PIN2 but nevertheless allowed endocytic vesicles (still incorporating FM4-64-labeled membrane) to form and internalize (Figures 4C and 4D).
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The inhibition of PIN2 internalization by Tyrphostin A23 was even more apparent in the presence of BFA. In the control situation, BFA caused PIN2 to accumulate into the BFA compartments, where it colocalized with FM4-64 (Figures 4E and 4F). In the presence of Tyrphostin A23, only FM4-64 accumulated in BFA compartments, whereas PIN2 was retained at the plasma membrane (Figures 4G and 4H). Similar results were also obtained for other plasma-membrane-resident proteins, which were previously suggested to undergo BFA-sensitive endocytic cycling, such as PM-ATPase, PIP2 water channel, and the low-temperature-induced plasmamembrane protein Lti6b [19, 26, 34]. Tyrphostin A23, but not A51, inhibited the BFA-induced internalization of all these proteins (Figures 4O–4T and Figures S2B– S2D). The action of Tyrphostin A23 in Arabidopsis was reversible (Figures S3A–S3C) and appeared to be specific for endocytic-cargo recruitment at the plasma membrane because it did not show any apparent effects on other subcellular trafficking processes at concentrations that already efficiently inhibited cargo internalization at the plasma membrane. For example, after Tyrphostin A23 treatment, the appearance of the TGN/ endosomes (as visualized by a1 [27], GNOM [35], and ARF1 [15] markers) was largely unchanged, and in the presence of BFA these markers accumulated into the BFA compartments (Figures 4I–4P). In addition, when Tyrphostin A23 was washed away with BFA, both plasma-membrane-localized proteins and TGN/endosomes accumulated into the BFA compartments similar to the ones observed in case of BFA treatment alone (Figures S3A–S3C). In addition, Tyrphostin A23 did not visibly interfere with exocytosis because, after BFA washout in the presence of Tyrphostin A23, endosomes and the Golgi apparatus recovered and plasma-membrane proteins, including PINs, were recycled from the BFA compartments back to the plasma membrane (Figures 4L and 4U and Figure S3D). In addition, in the presence of Tyrphostin A23, recovery of Lti6B-GFP [26] was observed at the plasma membrane when it was photobleached both from the cytosol and from the plasma membrane (see Figure S4), indicating that Tyrphostin A23 does not completely inhibit the forward traffic toward the plasma membrane. It cannot be entirely excluded that Tyrphostin A23 may also affect other processes; however, its strong effects on internalization of PINs and other cargoes implicate the importance of clathrin-dependent endocytosis for the internalization of many plasma-membrane-resident proteins in plants. Conclusions Endocytosis is of paramount importance for the function of both unicellular and multicellular organisms. Despite numerous recent demonstrations that various plant plasma-membrane proteins are internalized from the cell surface [4, 6, 9, 19, 26, 30, 34], a molecular mechanism for endocytosis in plants has not been demonstrated. Through several approaches including live-cell imaging, electron microscopy, and genetic and pharmacological studies, we show here that an endocytic mechanism utilizing the coat protein clathrin is operational in plants. In addition, we have identified several endogenous cargoes for clathrin-dependent endocytosis, prominent among them the plant-specific,
constitutively cycling PIN protein family of auxin efflux carriers. Interference with clathrin-dependent endocytosis impairs the internalization of many plant plasmamembrane proteins and inhibits endocytosis in general, suggesting that the clathrin-dependent internalization machinery is the major endocytic mechanism in plant cells. We feel that an important topic for future studies will be, in addition to the identification of ancillary, regulatory elements of the internalization machinery, a full appreciation of the diverse functions of endocytosis in plants. In particular, elucidating the role of clathrindependent PIN internalization in regulating auxin transport will be of fundamental importance to our understanding of how this evolutionarily conserved mechanism is utilized in mediating plant-specific physiological and developmental roles. Supplemental Data Supplemental Data include Experimental Procedures and four figures and are available with this article online at: http://www. current-biology.com/cgi/content/full/17/6/520/DC1/. Acknowledgments We thank J. Wiedenmann, G. Ju¨rgens, C. Luschnig, B. Scheres, M. Grebe, W. Michalke, D. Ehrhardt, and K. Schumacher for sharing published material. We thank C. Brancato for assistance with Arabidopsis protoplast transformations. This work was supported by a Volkswagenstiftung (P.D. J.F., J.M.), EMBO Young Investigator Program (J.F.), the Deutsche Forschungsgemeinschaft (D.G.R.), SFB 446 (Y.-D.S.), and the Ministerio de Educacio´n y Ciencia, grant BFU2005-00071 (F.A.). Received: November 21, 2006 Revised: January 18, 2007 Accepted: January 18, 2007 Published online: February 15, 2007 References 1. Alberts, B., Johnson, A., Lewis, J., Raff, M., Roberts, K., and Walter, P. (2002). Molecular Biology of the Cell, 4th Edition (New York: Garland Science). 2. Conner, S.D., and Schmid, S.L. (2003). Regulated portals of entry into the cell. Nature 422, 37–44. 3. Kirchhausen, T. (2000). Clathrin. Annu. Rev. Biochem. 69, 699– 727. 4. Murphy, A.S., Bandyopadhyay, A., Holstein, S.E., and Peer, W.A. (2005). Endocytotic cycling of PM proteins. Annu. Rev. Plant Biol. 56, 221–251. 5. Russinova, E., and de Vries, S.C. (2005). Receptor-mediated endocytosis in plants. In Plant Endocytosis, Volume 1, J. Samaj, F. Baluska, and D. Menzel, eds. (Heidelberg: Springer Verlag), pp. 103–115. 6. Robatzek, S., Chinchilla, D., and Boller, T. (2006). Ligandinduced endocytosis of the pattern recognition receptor FLS2 in Arabidopsis. Genes Dev. 20, 537–542. 7. Wiedenmann, J., Ivanchenko, S., Oswald, F., Schmitt, F., Rocker, C., Salih, A., Spindler, K.D., and Nienhaus, G.U. (2004). EosFP, a fluorescent marker protein with UV-inducible greento-red fluorescence conversion. Proc. Natl. Acad. Sci. USA 101, 15905–15910. 8. Petrasek, J., Mravec, J., Bouchard, R., Blakeslee, J.J., Abas, M., Seifertova, D., Wisniewska, J., Tadele, Z., Kubes, M., Covanova, M., et al. (2006). PIN proteins perform a rate-limiting function in cellular auxin efflux. Science 312, 914–918. 9. Geldner, N., Friml, J., Stierhof, Y.D., Jurgens, G., and Palme, K. (2001). Auxin transport inhibitors block PIN1 cycling and vesicle trafficking. Nature 413, 425–428. 10. Friml, J. (2003). Auxin transport - shaping the plant. Curr. Opin. Plant Biol. 6, 7–12.
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