Correct Targeting of Plant ARF GTPases Relies on Distinct Protein Domains

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1 Traffic 2008; 9: Blackwell Munksgaard # 2007 The Authors Journal compilation # 2007 Blackwell Publishing Ltd doi: /j x Correct Targeting of Plant ARF GTPases Relies on Distinct Protein Domains Loren A. Matheson 1, Sarabjeet S. Suri 1, Sally L. Hanton 1, Laurent Chatre 1 and Federica Brandizzi 1,2, * 1 Department of Biology, 112 Science Place, University of Saskatchewan, Saskatoon, Saskatchewan S7N 5E2, Canada 2 Department of Energy, Plant Research Laboratory, Michigan State University, East Lansing, MI 48824, USA *Corresponding author: Federica Brandizzi, brandizz@msu.edu Indispensable membrane trafficking events depend on the activity of conserved small guanosine triphosphatases (GTPases), anchored to individual organelle membranes. In plant cells, it is currently unknown how these proteins reach their correct target membranes and interact with their effectors. To address these important biological questions, we studied two members of the ADP ribosylation factor (ARF) GTPase family, ARF1 and ARFB, which are membrane anchored through the same N-terminal myristoyl group but to different target membranes. Specifically, we investigated how ARF1 is targeted to the Golgi and post-golgi structures, whereas ARFB accumulates at the plasma membrane. While the subcellular localization of ARFB appears to depend on multiple domains including the C-terminal half of the GTPase, the correct targeting of ARF1 is dependent on two domains: an N-terminal ARF1 domain that is necessary for the targeting of the GTPase to membranes and a core domain carrying a conserved MxxE motif that influences the relative distribution of ARF1 between the Golgi and post-golgi compartments. We also established that the N-terminal ARF1 domain alone was insufficient to maintain an interaction with membranes and that correct targeting is a protein-specific property that depends on the status of the GTP switch. Finally, an ARF1 ARFB chimera containing only the first 18 amino acids from ARF1 was shown to compete with ARF1 membrane binding loci. Although this chimera exhibited GTPase activity in vitro, it was unable to recruit coatomer, a known ARF1 effector, onto Golgi membranes. Our results suggest that the targeting of ARF GTPases to the correct membranes may not only depend on interactions with effectors but also relies on distinct protein domains and further binding partners on the Golgi surface. Key words: ARF1, ARFB, protein transport, secretory pathway, targeting Received 19 September 2007, revised and accepted for publication 1 November 2007, uncorrected manuscript published online 6 November 2007, published online 27 November 2007 Intracellular trafficking is regulated by the activity of small guanosine triphosphatases (GTPases) that act as molecular switches to regulate complex protein protein interactions. These GTPases control the specificity of vesicle and membrane fusion and largely influence the identity of secretory organelles (1). Low molecular weight GTPases, including Rabs, ADP ribosylation factors (ARFs) and ARFlike proteins, are ubiquitous among eukaryotes and often highly conserved among kingdoms (2). It is well established that membrane anchoring occurs through lipid modifications (3), but the correct targeting of these small proteins remains to be fully understood. Although the primary structure of the ARF family of proteins is highly conserved, ARFs are known to target different compartments in the same cell. In mammalian cells, ARF1 binds the Golgi apparatus, while the closely related ARF6 targets the plasma membrane (4,5). Furthermore, ARF homologues are known to target different compartments in different cell systems (6,7). A key example of this is provided by ARF1. In mammalian cells, ARF1 binds Golgi membranes, while in plant cells, the GTPase targets the Golgi apparatus and additional structures. These appear to be post-golgi structures that bud from the Golgi (8) and that may be involved in endocytosis (9,10). Recent studies have unraveled the underlying mechanisms for ARF1 membrane targeting in mammalian cells. An analysis based on sequence comparison and generation of protein chimeras of the mammalian ARF1 and ARF6 has shown that an MxxE amino acid motif (Methionine-xx-Glutamic acid in position ) is crucial for Golgi targeting of ARF1 in mammalian cells (4). How ARF1 is distributed to Golgi bodies and ARF1 post-golgi compartments in plant cells is yet to be understood, but the different subcellular distribution of ARF1 between plant and mammalian cells suggests that the targeting mechanisms of the GTPase may indeed be different in the two systems. The dual distribution of the plant ARF1 may imply that the GTPase necessitates signal(s) not only to target the Golgi apparatus but also to discriminate the Golgi from post-golgi structures. A plant plasma membrane-resident ARF GTPase similar to mammalian ARF6 has yet to be identified (5). In this study, we have identified an Arabidopsis plasma membrane ARF and asked how ARF GTPases are targeted to the correct membranes in plant cells. We established that the N-terminal region of ARF1 comprising amino acids 1 18 is necessary to target the ARF1 membranes and that the integrity of the MxxE motif is crucial for the steady-state partitioning of plant ARF1 between the Golgi 103

2 Matheson et al. apparatus and the ARF1 post-golgi structures. In contrast, the plasma membrane ARFB relies on interplay between the N-terminal lipid anchor and the C-terminal portion of the GTPase for correct targeting. Furthermore, we found that ARF1 must contain other sequence determinants that specify ARF1 effector recruitment. These data demonstrate that despite high sequence conservation, the targeting determinants of plant GTPases are not equal to those of other systems and that correct targeting alone is not the exclusive factor that dictates the function of ARF GTPases. S2), similar to that of the mammalian ARF6 (11). Like mammalian ARF6 as well as ARF1, the integrity of the myristoylation signal was found to be indispensable for the targeting of ARFB to the plasma membrane (Figure S2). Therefore, on the basis of these properties, ARFB appeared to be an ideal tool for direct comparisons with ARF1 to (i) identify the domain(s) of ARF1 required for its subcellular localization in plant cells and (ii) test whether redistribution of a GTPase to non-native target organelles influences its interaction with effectors. Results Identification of a plasma membrane ARF family member, ARFB To determine the domains of ARF1 responsible for its subcellular localization to Golgi bodies (8) and post-golgi structures in plants (9,10), we first sought to identify a plasma membrane-resident ARF GTPase (5,11 13). We selected a member of the Arabidopsis ARF family described by Vernoud et al. (7) that exhibited high similarity to the plasma membrane-localized human ARF6, ARFB1a, herein named ARFB for simplicity (49% identity and 73% similarity to hsarf6; Figure 1A). We first sought to determine the localization of this novel GTPase. Confocal laser scanning microscopy analyses revealed that a yellow fluorescent protein (YFP) fusion of ARFB (ARFB:YFP) was distributed at cell boundaries in tobacco leaf epidermal cells (Figure 1Bi). As tonoplast and plasma membrane are separated by a thin layer of cytosol at the cell boundaries, we analyzed tobacco leaf protoplasts expressing ARFB:YFP and observed spherical shapes indicative of plasma membrane localization (Figure 1Bii). In protoplasts, tonoplast localization is characterized by irregular shapes imposed by organelles, which delimitate the tonoplast inwards (14,15). This was not observed. However, to further ensure that ARFB was in fact targeted to the plasma membrane and not to the tonoplast, ARFB:YFP was coexpressed with the tonoplast marker BobTIP26-1:green fluorescent protein (GFP) (16). This experiment showed that the two markers did not overlap (Figure 1C). Finally, we coexpressed ARFB:YFP with plasma membrane Hþ- ATPase 4 (PMA4):GFP, a plasma membrane marker (17), and cytosolic red fluorescent protein (crfp). Confocal image analysis of live cells coexpressing these constructs indicated that the distribution of ARFB:YFP overlapped that of PMA4:GFP and, to a lesser extent, that of crfp (Figure 1D), confirming a plasma membrane localization (see also Figure S1 for additional localization analyses). Analysis of the GTPase activity and of the localization of ARFB to the plasma membrane revealed that this is an active GTPase that undergoes GDP/GTP exchange at the plasma membrane and that the localization is insensitive to the protein trafficking inhibitor brefeldin A (BFA) (Figure The conserved MxxE motif influences the distribution of the GTPase between Golgi and post-golgi structures To find the membrane-targeting determinants of plant ARF1, we generated different protein chimeras of ARFB and ARF1 (see Figure 2A for schematic). We first fused the N-terminal portion of ARF1 comprising amino acids 1 98 to the C-terminal portion of ARFB comprising amino acids The resultant chimera (ARF1-B), which lacked the MxxE motif of ARF1 (Figure 2A), was fused to YFP (ARF1-B:YFP) for subsequent localization analyses in comparison to the wild-type ARF1 fusion with GFP [ARF1:GFP; (8)]. The control based on coexpression of ARF1 fused to either YFP or GFP showed perfect colocalization; all the ARF1 punctate structures were labeled with comparable levels of both YFP and GFP (Figure 2B). In contrast, confocal image analyses of tobacco leaf epidermal cells expressing ARF1-B:YFP showed a different distribution. Although ARF1-B:YFP localized to punctate structures (Figure 2C), coexpression analyses with ARF1:GFP showed that the distribution of the two proteins was not uniform across all punctae. There was a subpopulation of structures labeled by both proteins and another population of structures predominantly labeled by ARF1:GFP. As coexpression analysis of ARF1:YFP with the known Golgi marker, a-2,6-sialyltransferase (ST):GFP (18), demonstrated that ARF1:YFP was distributed to the Golgi apparatus and to additional structures that bud from the Golgi (8,10), it was pertinent to test if the observed heterogeneity between ARF1- and ARF1-B-labeled structures was because of a quantitative shift from Golgi to post-golgi structures in the hybrid molecule. Coexpression of either ARF1:YFP (Figure 3A) or ARF1-B:YFP (Figure 3C) with ST:GFP revealed that extra Golgi structures were indeed labeled with greater intensity by the chimera compared with the wild-type ARF1 fusion. Intensity measurements between the fluorescence signals of the ARF1- B:YFP distributed on the Golgi and on post-golgi structures confirmed that these differences were quantitative. This resulted in a clear shift from a predominantly Golgi localization in the case of ARF1:YFP to a predominantly post- Golgi localization for the hybrid ARF1-B:YFP (Figure 3E). In conclusion, the replacement of the C-terminal half of ARF1 by the corresponding sequence of ARFB did not result 104 Traffic 2008; 9:

3 Targeting Mechanisms of ARF GTPases Figure 1: ARFB contains a similar IxxD motif to human ARF6 and localizes to the plasma membrane. A) Sequence alignment comparing ARFB (atarfb) with human ARF6 (hsarf6) as well as human ARF1 (hsarf1) and Arabidopsis ARF1 (atarf1). Note the presence of an IxxD motif in ARFB and ARF6 but not in ARF1 (red box). The myristoylation site position G2 is also indicated (cyan box). B) Live cell imaging of either (i) a tobacco leaf epidermal cell or (ii) tobacco leaf protoplast expressing ARFB:YFP shows a peripheral distribution of YFP fluorescence. Inset in (Bi) shows the cortical region of the cell in the plane of focus. C) To exclude the possibility that ARFB:YFP localized to the vacuole, ARFB:YFP was coexpressed with a marker for the tonoplast, BobTIP26-1:GFP. Inset demonstrates that there is no colocalization of ARFB:YFP and BobTIP26-1:GFP. Empty arrowheads indicate tonoplast, and a full arrowhead refers to plasma membrane. D) Plasma membrane distribution of the ARFB:YFP fluorescent fusion was determined with colocalization experiments in tobacco leaf epidermal cells coexpressing the plasma membrane marker (PMA4:GFP) and the cytosol marker (crfp). Inset shows a magnified view of the colocalization between PMA4:GFP and ARFB and partial overlap between ARFB and crfp. In the merged image, an arrow indicates cytosol and arrowhead refers to plasma membrane. Because of the close apposition of the plasma membrane and tonoplast, the cytosol appears sheety. Scale bars ¼ 5 mm. in plasma membrane localization, but resulted in a shift from Golgi to post-golgi localization. A close comparison of the sequences of ARF1 and ARFB showed that the first 18 amino acids were the most divergent between the N-termini of the two proteins (Figure 1A). Therefore, we hypothesized that this 18 amino acid region contained determinants that were responsible for ARF targeting and contains signals for either plasma membrane localization or Golgi/post-Golgi retention. To Traffic 2008; 9:

4 Matheson et al. Figure 2: The first 18 amino acids of ARF1 are needed to redirect ARFB to the Golgi apparatus and additional ARF1-labeled structures. A) Schematic representation of ARFB and ARF1 chimeras. B) Confocal images of cells expressing ARF1:YFP alone or in combination with ARF1:GFP. Note the homogeneous fluorescence distribution intensity across the organelles. Arrow indicates the presence of autofluorescence because of a chloroplast. C) Confocal images of cells expressing ARF1-B:YFP alone or in combination with ARF1:GFP. The two constructs colocalize to punctate structures, although the distribution of ARF1-B:YFP on some organelles (arrowheads) was lower in comparison to others. D) Images of M1-B:YFP coexpressed with ARF1:GFP demonstrate that the two constructs colocalize, although the levels of fluorescence intensity of M1-B:YFP were not homogeneous among organelles with some structures being brighter (arrowheads) than others. Scale bars ¼ 5 mm. test this, we fused amino acids 1 18 of ARF1 (M1 as this section contains a myristoylation signal) to amino acids of ARFB and expressed the protein (M1-B; see Figure 2A for schematic) fused to YFP (M1-B:YFP) in tobacco leaf epidermal cells (Figure 2D). When expressed alone, M1-B:YFP was predominantly localized at punctate 106 Traffic 2008; 9:

5 Targeting Mechanisms of ARF GTPases Figure 3: Legend on next page. Traffic 2008; 9:

6 Matheson et al. structures and was not detected at the plasma membrane. Coexpression analyses with either ARF1:GFP (Figure 2D) or ST:GFP (Figure 3D) revealed a similar punctate distribution as observed for the first hybrid ARF1-B (Figures 2C and 3C). The results show that M1-B:YFP was distributed predominantly to post-golgi structures in addition to weaker staining of the Golgi apparatus. This shift in localization was comparable to the observations with the other hybrid molecule ARF1-B as shown by statistical analysis (Figure 3E). Therefore, the first 18 amino acids of ARF1 appeared to be sufficient to redistribute the ARFB chimera from the plasma membrane to the ARF1 structures, except for a different steady-state distribution between Golgi and post-golgi structures. It has been shown in mammalian cells that the targeting of ARF1 to Golgi membranes is strongly influenced by the presence of an MxxE motif in position of the amino acid sequence (4). Fluorescent protein chimeras between human ARF1 and ARF6 that included the ARF1 MxxE motif localized to the cis Golgi; however, specific replacement of the MxxE region in ARF1 by the corresponding ARF6 region resulted in mistargeting to the trans Golgi (4). The MxxE motif is conserved in plant ARF1 [Figure 1A, (5)], while the amino acids in the corresponding position are IxxD in both mammalian ARF6 and plant ARFB (Figure 1A). The hybrids ARF1-B or M1-B no longer contained the MxxE motif. Therefore, to test the involvement of the MxxE motif in ARF1 targeting, we mutated the MLNE sequence in ARF1 to ILTD, the corresponding residues of ARFB (amino acids 110 ILTD 113 ). Coexpression analysis of ARF1 ILTD :YFP with ST:GFP confirmed a predominant accumulation of the mutant on the post-golgi structures (Figure 3B). Statistical analysis revealed a similar quantitative shift, as observed for the two hybrids (Figure 3E). Together, the results suggest that the MxxE motif plays either a role in Golgi retention of the GTPase or stimulates recycling from the post-golgi compartments to the Golgi apparatus. Plasma membrane targeting of ARFB depends on N-terminal and C-terminal domains of the GTPase It should be noted that no plasma membrane localization was observed for the two hybrids ARF1-B:YFP or M1- B:YFP or the ARF1 ILTD :YFP mutant. This suggests that either signals responsible for ARFB targeting to the plasma membrane are missing or domain(s) within the first 18 amino acids of ARF1 prevent targeting to the plasma membrane in a dominant fashion. To distinguish between these two scenarios, we generated the reciprocal YFP chimeras containing either the first 98 amino acids of ARFB fused to the C-terminal half of ARF1 (ARFB-1:YFP) or merely the first 18 amino acids of ARFB to the remainder of ARF1 (MB-1) (see Figure 2A for schematic). Both fusions contained the MxxE motif and the ARFB myristoylation motif for membrane anchoring. Interestingly, expression of these two hybrids resulted in cytosolic fluorescence (Figure 4), despite the presence of the intact myristoylation site. Also, coexpression with either ARF1:GFP (Figure 4A,C) or ST:GFP (Figure 4B,D) confirmed that the chimeras did not disrupt normal Golgi or post-golgi structure morphology. The myristoylation motif of ARFB is crucial for membrane targeting as a fluorescent fusion of ARFB with a mutation of glycine to alanine in position 2 is cytosolic (Figure S2A). However, within the context of an ARF1 molecule, the ARFB myristoylation site was unable to mediate membrane anchoring. The results suggest that the N-terminus of ARFB did not contain a transplantable plasma membrane sorting signal. The cytosolic distribution of the hybrid molecules must be because of their inability to anchor into membranes. We speculate that membrane anchoring cannot be attributed to the lipid modifications alone and that the N-terminal myristoylation site of ARFB requires information from the C-terminal half of ARFB to mediate membrane anchoring and plasma membrane targeting. Furthermore, the presence of the MxxE motif alone did not guarantee Golgi localization as the MB-1 chimera failed to target the Golgi and post-golgi structures. Figure 3: The presence of the first 18 amino acids of ARF1 ensures membrane targeting of the chimeras, and the MxxE motif influences the distribution of the GTPases between the Golgi and the post-golgi structures. A) Confocal images of cells coexpressing ARF1:YFP and ST:GFP. B) Confocal images of a tobacco leaf epidermal cell coexpressing ARF1 ILTD :YFP and ST:GFP. The colocalization of the GFP and YFP fluorescence in the merged images indicates that mutation of the MxxE signal to IxxD does not affect the distribution of ARF1. C) Confocal images of ARF1-B:YFP coexpressed with ST:GFP show that the first half of ARF1 is sufficient to recruit the chimera to the Golgi apparatus and additional punctate structures. D) The first 18 amino acids from ARF1 were fused with amino acids of ARFB, tagged with YFP and expressed in tobacco leaf cells. Images of M1-B:YFP coexpressed with ST:GFP demonstrate that the first 18 amino acids of ARF1 are sufficient to recruit the fusion to the Golgi apparatus and additional structures. Scale bars ¼ 5 mm. Arrows indicate individual Golgi stacks and arrowheads refer to additional punctate structures. E) The distribution of fluorescence intensity on the Golgi apparatus and post-golgi structures was quantified for each of the following constructs: ARF1:YFP, ARF1 ILTD :YFP, ARF1- B:YFP and M1-B:YFP as described in the Materials and Methods section. The sum of the Golgi and post-golgi structure intensities was set to 100%, and the relative distributions for each construct are presented in histogram format. The distribution of ARF1:YFP was such that there was greater fluorescence intensity on the Golgi stacks than on post-golgi structures. The opposite was true of the three other proteins tested, ARF1 ILTD :YFP, ARF1-B:YFP and M1-B:YFP, which had an augmented distribution on post-golgi structures relative to the Golgi apparatus (p < 0.05 between ARF1:YFP and ARF1-B:YFP, ARF1:YFP and M1-B:YFP as well as ARF1:YFP and ARF1 ILTD :YFP; 20 Golgi stacks and 20 post-golgi structures were quantified from 10 different cells). 108 Traffic 2008; 9:

7 Targeting Mechanisms of ARF GTPases Figure 4: The C-terminal portion of ARF1 is insufficient to target chimeras to the Golgi apparatus and post-golgi structures. Images of a cell expressing ARFB-1:YFP alone or coexpressed with either ARF1:GFP (A) or ST:GFP (B) demonstrate that ARFB-1:YFP is cytosolic, while ARF1:GFP remains on intact punctate structures. MB-1:YFP was also cytosolic whether expressed alone, with ARF1:GFP (C) or with ST:GFP (D). Therefore, the C-terminal half of ARF1 is insufficient to recruit the fusion to punctate structures. Scale bars ¼ 5 mm. The first 18 amino acids of ARF1 can only mediate targeting when present within the context of an ARF-related GTPase The experiments thus far have excluded the possibility of a transplantable plasma membrane sorting signal in the N-terminal 18 amino acids of ARFB. However, targeting of ARF1 to Golgi bodies and post-golgi structures appears to be dependent on the first 18 amino acids of this GTPase because these can redirect a hybrid containing mostly the ARFB sequence (M1-B) from the plasma membranes to internal structures. For the remainder of this work, we focused on ARF1 sorting and explored the factors that may be involved in the localization of this GTPase. To do so, we tested whether the first 18 amino acids of ARF1 alone were sufficient to target any cytosolic protein to the Golgi apparatus by fusing the 1 18 amino acid sequence of Figure 5: The first 18 amino acids of ARF1 are insufficient to provide targeting and residence on the correct membranes. A) Schematic representation of ARF :YFP and ARF :GFP. B) Confocal image of cells expressing ARF :YFP either alone or with ST:GFP. The cytosolic appearance of ARF demonstrates that the first 18 amino acids alone are insufficient to target and reside on membranes. Similarly, ARF :GFP alone or coexpressed with ST:YFP (C) was also incapable of residing on membranes. Additionally, the punctate nature of the Golgi apparatus remained intact when coexpressed with these two constructs. Scale bars ¼ 5 mm. Traffic 2008; 9:

8 Matheson et al. ARF1 to YFP (ARF :YFP; see Figure 5A for schematic) and expressed the construct in tobacco leaf epidermal cells. The protein fusion was distributed in the cytosol (Figure 5B), suggesting that the N-terminal domain of ARF1 does not constitute a transplantable sorting signal but may form a signal patch with the rest of the GTPase. We then wanted to exclude the possibility that the distribution of ARF :YFP was because of a secondary effect caused by loss of Golgi apparatus integrity. This may occur, for example, through saturation of membrane binding sites by ARF :YFP; consequently, Golgi membranes may be redistributed into the endoplasmic reticulum (ER) because of the loss of activity of endogenous ARF1 on Golgi membranes. Therefore, as a control, we coexpressed ST:GFP with ARF :YFP and found that, in the presence of ARF :YFP, the expected localization of ST:GFP to the Golgi apparatus was unaffected (Figure 5B). This confirms that the cytosolic distribution of ARF :YFP was not a result of the disruption of Golgi membrane integrity. To ensure that other specific domains of ARF1 were insufficient for targeting of the GTPase to the Golgi apparatus, we generated a construct containing the sequence of ARF1 without the first 18 amino acids fused to GFP (ARF :GFP; see Figure 5A for schematic). Similar to ARF :YFP, ARF :GFP was distributed in the cytosol of tobacco leaf epidermal cells (Figure 5C). Coexpression of ARF :GFP with ST:YFP showed that the expression of ARF :GFP did not affect the distribution of Golgi membranes. This shows that the N-terminally truncated GTPase does not interact with membrane proteins on the Golgi apparatus or post-golgi structures to mediate myristoylation-independent membrane association. Therefore, although determinants within the first 18 amino acids of ARF1 can redirect ARFB from the plasma membrane to Golgi and post-golgi structures, additional properties of ARF1 are required to ensure its correct targeting. Activation of the GTPase is necessary to ensure its residency on the plant Golgi apparatus It has been demonstrated that residence of ARF1 on the Golgi apparatus depends on its activation state, whereby inactive ARF1 is cytosolic and the active form is on membranes (8,9,19). Therefore, we postulated that activation of the GTPase may be necessary for it to reside on the membranes. To test this, we investigated whether the localization of M1-B:YFP was dependent on the activation state of the GTPase. In the M1-B chimera, we generated mutations that are known to affect the GTPase activity of ARF1 [active form ¼ Q71L and inactive form ¼ T31N (11,20)] (see Figure 6A for schematic). We then ensured that the mutations affected the ability of the chimera to hydrolyze GTP. To do so, a GTP hydrolysis assay was performed on recombinant wild-type M1-B as well as M1-B Q71L and M1-B T31N using wild-type ARF1, ARF1 Q71L and ARF1 T31N as controls. Results indicated that wild-type M1-B was capable of GTP hydrolysis and that both M1-B Q71L and M1-B T31N had a decreased ability to hydrolyze GTP, as did the ARF1 mutants, ARF1 Q71L and ARF1 T31N (Figure 6B). We determined the localization of the active and inactive mutant forms of M1-B. The GTP and GDP M1-B mutants were fused to YFP and expressed in tobacco leaf epidermal cells. We found that the active mutant, M1-B Q71L :YFP, localized to punctate structures and the cytosol (Figure 6C). These corresponded to the Golgi apparatus and additional structures (Figure 6C). Interestingly, the presence of this mutant caused partial redistribution of ST:GFP to the ER (Figure 6C). The inactive mutant, M1-B T31N :YFP, instead, was mostly cytosolic and did not appear to affect the integrity of Golgi membranes (Figure 6D). These data indicate that M1-B cycles between a cytosolic pool and the membranes of the organelles targeted by ARF1. These findings also suggest that the first 18 amino acids of ARF1 can overrule other signals present in wildtype ARFB that are required for its targeting to the plasma membrane. Importantly, our results not only support the hypothesis that the first 18 amino acids of ARF1 are necessary for the targeting of the ARF1 to the structures normally targeted by the GTPase but also that they are insufficient for the localization of the GTPase on these organelles. Similarly, the activation of the GTPase is insufficient to target membranes but is a requirement for the localization of the GTPase. M1-B:YFP competes with ARF1 for binding sites on membranes We wanted to explore the possibility that M1-B:YFP is recruited to membranes by competing for binding sites with ARF1. It has been shown that the binding of plant ARF1 to subcellular structures is a saturable process as the appearance of a fluorescent protein fusion of ARF1 in the cytosol was found to rise along with increasing levels of expression of the protein (8). We, therefore, wanted to exploit the limited availability of the ARF1-binding sites on membranes to test whether overexpression of an active mutant of M1-B would displace ARF1 from membranes to the cytosol. If this were to happen, it would be consistent with the hypothesis of a competition between ARF1 and M1-B for binding sites on the membranes. It has been shown that a GTP-locked ARF1 mutant has a longer residence on membranes in comparison to wildtype ARF1 (8). Therefore, we postulated that if M1-B competed with ARF1 for binding sites on membranes, an M1-B mutant form that would cycle on and off membranes more slowly in comparison to the wild-type M1-B would be an ideal tool to saturate ARF1-binding sites on membranes. To test this, we first determined whether GTP-restricted M1-B cycled more slowly on and off membranes than wildtype M1-B using fluorescence recovery after photobleaching (FRAP) experiments (Figure 7). We found that upon 110 Traffic 2008; 9:

9 Targeting Mechanisms of ARF GTPases Figure 6: M1-B Q71L :YFP competes for the ARF1-binding sites on membranes. A) Schematic representation of wild-type M1-B (M1- Bwt), M1-B Q71L and M1-B T31N. B) GTPase activity of recombinant GST fusions of wild-type ARF1 (ARF1wt) and M1-Bwt and their respective constitutively active and inactive mutants was measured by quantifying the ability of the GTPases to hydrolyze GTP. The GTPase activity was estimated as a measurement of the inorganic phosphate release as recorded by absorbance at 650 nm; GST alone was used as background and subtracted. The activity of each mutant is presented as a ratio relative to its respective wild-type activity. The mutant forms of ARF1 and M1-B had reduced activity with respect to the wild-type protein. C) Confocal images of tobacco leaf epidermal cells expressing M1-B Q71L :YFP either alone or with ST:GFP. M1-B Q71L :YFP colocalized with ST:GFP; however, there was a partial redistribution of ST:GFP in the ER as a result of the activity of the M1-B Q71L mutant. D) Confocal images of cells expressing M1-B T31N :YFP either alone or coexpressed with ST:GFP. The inactive form of M1-B localizes to both the cytosol and the boundaries of the cell, while the Golgi apparatus remains intact. This suggested that M1-B cycles between a plasma membrane/cytosolic pool and the membranes targeted by ARF1. Insets show the cortical region of the cell in the plane of focus. Scale bars ¼ 5 mm. Traffic 2008; 9:

10 Matheson et al. Figure 7: M1-B:YFP and M1-B: Q71L :YFP have different residency times on membranes. A) Fluorescence recovery after photobleaching experiments on a cortical section of tobacco leaf epidermal cells expressing M1-B:YFP or M1-B Q71L :YFP. Samples were treated with the actin-depolymerizing agent latrunculin B to stop Golgi movement (29). Time measured from the photobleaching event (bleach) is expressed in seconds in the subsequent frames. Arrowheads indicate bleached punctate structures. Scale bars ¼ 2 mm. B) Representative half-time recovery curves of the Golgi fluorescence of cells expressing M1-B:YFP ( seconds, n ¼ 10) or M1-B: Q71L :YFP ( seconds, n ¼ 13). Halftime is defined as the time required for the fluorescence in the photobleached region to recover to 50% of the recovery asymptote, and n is the number of bleached punctate structures. photobleaching, the fluorescence of both wild-type M1-B and M1-B Q71L recovered on the membranes (Figure 7), indicating an exchange of the bleached pools of the GTPases on the membranes with their unbleached cytosolic pools. However, the half-time recoveries of wild-type M1-B ( seconds, n ¼ 10) and M1-B Q71L ( seconds, n ¼ 13) were significantly different (p < 0.05). These data are consistent with our hypothesis that the residence of M1-B Q71L on membranes is longer than that of wild-type M1-B. The data suggest that M1-B Q71L could potentially be used to compete with ARF1-binding sites and displace ARF1 from membranes. To test this, we coexpressed M1- B Q71L :YFP with ARF1:GFP in tobacco leaf epidermal cells. As expected, we found that M1-B Q71L :YFP was distributed to the same structures targeted by ARF1 under conditions of low M1-B Q71L :YFP expression (Figure 8A). However, in conditions of overexpression of M1-B Q71L :YFP, we found that the targeting of ARF1:GFP to punctate structures was reduced with an increase of the cytosolic pool of ARF1:GFP (Figure 8A). To quantify this effect, we measured the fluorescence of the ARF1:GFP punctate structures relative to the intensity of the cytosolic fluorescence in the presence of M1-B Q71L :YFP (Figure 8B), as described by Matheson et al. (10) (see also Materials and Methods section). The relative punctate structure fluorescence intensity (RPSF) was then calculated individually for ARF1:GFP and M1- B Q71L :YFP, and the numbers for each punctate structure were paired. The M1-B Q71L :YFP readings were separated into high and low values as defined by the median fluorescence intensity value. The cutoff RPSF was arbitrarily set to 68%, i.e. cells expressing low levels of M1- B Q71L :YFP had a RPSF between 50 and 68%. Cells with an M1-B Q71L :YFP RPSF >68% were considered high expressors. Sixty punctate structures from at least 20 different cells were quantified for each group. The corresponding ARF1:GFP values were averaged and graphed according to the RPSF of M1-B Q71L :YFP. With this approach, we confirmed the initial confocal imaging observations on a displacement of ARF1:GFP in conditions of increased expression levels of M1-B Q71L :YFP (Figure 8A). Therefore, these findings strongly suggest that M1-B Q71L :YFP competes with ARF1 for binding sites on membranes. 112 Traffic 2008; 9:

11 Targeting Mechanisms of ARF GTPases Figure 8: M1-B Q71L :YFP induces the release of ARF1:GFP from membranes. A) Confocal images of tobacco leaf epidermal cells coexpressing ARF1:GFP and either low or high levels of M1-B Q71L :YFP. Scale bars ¼ 5 mm. B) The fluorescence of the ARF1 punctate structures was quantified relative to the intensity of the cytosolic fluorescence (10). This was performed when ARF1:GFP was coexpressed with either low or high levels of M1-B Q71L :YFP. Expression levels of M1-B Q71L :YFP were defined for each cell and sorted into two groups as described in the Materials and Methods and in the Results sections. The RPSF is given as a percentage of the ratio between fluorescence intensity values (arbitrary units) measured in the punctate structures and the sum of intensity values for the cytosol and punctate structure (see also Materials and Methods). The trend indicates that as M1-B Q71L :YFP expression goes up, the fluorescence intensity of ARF1:GFP on punctate structures goes down (p < 0.05; n ¼ 60, where n is the number of punctate structures quantified in each data set from 20 cells). M1-B is incapable of functionally replacing ARF1 on Golgi membranes The overlapping subcellular distribution of M1-B and the evidence that M1-B could displace ARF1 from its membrane binding sites prompted us to determine whether M1-B could functionally replace ARF1. The evidence that the inactive mutant M1-B T31N :YFP did not affect the integrity of Golgi membranes (Figure 6D), different from the reported effect of the inactive ARF1 mutant (8), argues against this hypothesis. However, to test this further, we followed two independent approaches. First, we tested if M1-B GTP mutant could inhibit the secretion of the soluble cargo a-amylase, as has previously been described for ARF1 Q71L (8,21). Second, we tested whether M1-B was capable of recruiting the known ARF1 effector complex, COPI, to the Golgi apparatus (6,10). To proceed with the first approach, tobacco leaf protoplasts were cotransfected with the secretory marker a-amylase (22) and a dilution series of either wild-type M1-B:YFP or M1-B Q71L :YFP. As a comparison, we used a dilution series of wild-type ARF1:GFP and ARF1 Q71L :GFP cotransfected with a-amylase. We found that secretion was reduced in the presence of the M1-B Q71L and ARF1 Q71L mutants (Figure 9), supporting the hypothesis that M1-B Q71L can inhibit secretion as has been reported for the equivalent ARF1 mutant (8,21). These data, however, do not distinguish whether the effect on inhibition of secretion is because of displacement of ARF1 from the Golgi (Figure 8) or a retardation of COPI vesicle uncoating. To discriminate the two possibilities, we proceeded with the second approach, measuring the recruitment efficiency of a fluorescent protein fusion of the COPI component ecop (8,10) in cells coexpressing either M1-B or M1-B Q71L compared with those expressing ARF1 or ARF1 Q71L. To do so, we expressed ecop:yfp, which is localized to the Golgi apparatus and cytosol in plant cells (8,10), and assessed its recruitment onto Golgi membranes in the presence of ARF1 and M1-B GTPases. This was carried out by normalizing the fluorescence intensity of the punctate structures labeled by ecop:yfp to cytosolic areas of identical size, as described in Matheson et al. (10). We found that the fluorescence intensity of ecop:yfp at the Golgi increased when the marker was coexpressed with ARF1 Q71L in comparison to the fluorescence intensity of ecop:yfp coexpressed with wild-type ARF1 (Figure 10 A,B; p < 0.05; n ¼ 100 Golgi stacks from 38 cells). This Traffic 2008; 9:

12 Matheson et al. Figure 9: M1-B Q71L :YFP affects secretion of bulk flow cargo. A) Histogram depicting the secretion index of a-amylase (ratio of extracellular and intracellular activities, 22) in tobacco leaf protoplasts expressing dilution series of ARF1:GFP, M1-B:YFP, ARF1 Q71L :GFP or M1- B Q71L :YFP. B) Histograms showing total a-amylase activity for the samples in A. Note that there is a decrease in both the secretion index and total activity with both GTP-restricted mutants relative to the wild-type proteins. Error bars ¼ standard error of the mean for three independent repetitions. indicated that ARF1 recruits COPI to the Golgi, as expected (6). In contrast, we found decreased levels of relative Golgi fluorescence intensity of ecop:yfp in cells where M1-B Q71L was targeted to the Golgi in comparison to the wild-type chimera (Figure 10C,D; p < 0.05; n ¼ 120 Golgi stacks from 50 cells). The results indicated that, unlike ARF1 Q71L, M1-B Q71L is incapable of recruiting ecop to intact Golgi membranes and that the reduction of amylase secretion is most likely because of displacement of ARF1 from the Golgi membranes. These data are also consistent with the redistribution of ARF1:GFP to the cytosol in the presence of M1-B Q71L :YFP in a dose-dependent manner (Figure 8). As M1-B Q71L does not functionally replace ARF1 Q71L, our data not only strongly support that the membrane environment is crucial for determining the specificity of the activity of ARF1 as previously suggested (10) but also indicate that sequence determinants must play a role in determining the specificity of the effector interaction with the GTPase. Discussion Distinct mechanisms for membrane targeting cause different localizations of plant ARF GTPases with a high degree of sequence identity Our knowledge of the trafficking of cargo proteins and receptors in the secretory pathway has dramatically increased in the past few years, particularly with respect to signals that allow proteins to travel within the membrane and lumen of secretory organelles [reviewed by Matheson et al. (23)]. However, how the cell achieves correct targeting of essential proteins like ARF GTPases that are localized beyond secretory membranes is largely unknown. Here, we have demonstrated that two ARF GTPases that share a high degree of sequence similarity (Figure 1), the known ARF1 and the novel plasma membrane-targeted ARFB, are distributed on different cellular compartments (Figures 1 3). The targeting determinants appear to differ significantly between the two GTPases. The targeting of ARF1 depends on interplay between a domain comprised within 114 Traffic 2008; 9:

13 Targeting Mechanisms of ARF GTPases Figure 10: M1-B Q71L is incapable of efficiently recruiting coatomer onto Golgi membranes. A) Confocal images of tobacco leaf epidermal cells coexpressing ecop:yfp and either ARF1:GFP or ARF1 Q71L :GFP. B) The fluorescence of the punctate structures labeled by ecop:yfp was quantified relative to the intensity of the cytosolic fluorescence in the presence of wild-type ARF1 or ARF1 Q71L. The RPSF is given as a percentage of the ratio between fluorescence intensity values (arbitrary units) measured in the punctate structures and the sum of intensity values for the cytosol and punctate structure (10). The trend indicates that ARF Q71L :GFP functions to recruit ecop:yfp as shown by the significant increase in relative fluorescence intensity on punctate structures (p < 0.05; n ¼ 100 Golgi stacks from 38 cells). C) Confocal images of tobacco leaf epidermal cells coexpressing ecop:yfp and either M1-B:GFP or M1-B Q71L :GFP. D) The fluorescence of the punctate structures labeled by ecop:yfp was quantified relative to the intensity of the cytosolic fluorescence in the presence of wildtype M1-B or the active form of M1-B. In contrast to the result in C, M1-B Q71L was incapable of recruiting ecop:yfp as demonstrated by the significant increase in cytosolic fluorescence intensity labeled by ecop:yfp (p < 0.05; n ¼ 120 Golgi stacks from 50 cells). Scale bar ¼ 5 mm, n is the number of punctate structures quantified for each data set. the first 18 amino acids of the protein and GTPase activation. This was demonstrated by the evidence that (i) the first 18 amino acids of ARF1 are sufficient to distribute a fusion to most of the ARFB sequence (M1-B:YFP) to the structures normally targeted by ARF1 (Figure 2) and that (ii) a fusion of the first 18 amino acids of ARFBtomostoftheARF1sequence(MB-1:YFP)failedto target the ARF1 membranes (Figure 4). The latter piece of evidence also excludes the possibility that the N-myristoylation motif is the determining factor for correct membrane targeting of ARF1 as this is a common signal in ARF1 and ARFB (Figure 1). Although determinants within the first 18 amino acids of ARF1 downstream from the myristoylation motif are sufficient for targeting ARF1 to the Golgi apparatus and the ARF1-labeled organelles, a fusion of these amino acids to a fluorescent protein, M1:YFP, failed to associate with Golgi membranes (Figure 5). Therefore, our results indicate that factors additional to the N-terminus domain are necessary for ensuring the residency of ARF1 on membranes. The evidence that a fluorescent protein fusion to ARF1 amino acids is cytosolic (Figure 5) but that the wild-type M1-B:YFP chimera and its active mutant target the ARF1 membranes (Figures 3 and 6) is consistent with a model that activation is the additional essential requisite for ARF1 membrane localization (8,9,24). Therefore, our data argue that the binding of ARF1 to membranes is ruled by a two-step process: receptor-mediated membrane targeting followed by dynamic residency. In this view, amino acids 1 18 would be sufficient for a receptor-mediated recognition of ARF1 on target membranes. Subsequent activation of ARF1 mediated by a guanine exchange factor (GEF) would ensure the residency of ARF1 on membranes until an inactivation-induced dissociation step takes place. ARFB targeting requirements appear substantially different from those of ARF1. In clear contrast with ARF1, the first 18 amino acids of ARFB are insufficient to distribute a fluorescent chimera of those amino acids and most of the ARF1 sequence (MB-1) to the plasma membrane Traffic 2008; 9:

14 Matheson et al. (Figure 4). In addition, amino acids of ARFB do not contain a signal that can overrule the targeting effect of the N-terminal 18 amino acids of ARF1 (Figure 2). This suggests that a combination of multiple domains of ARFB at the N- and C-termini may be necessary for the GTPase to target the plasma membrane. Finally, activation of ARFB is not a requisite to target membranes as the inactive mutant of ARFB was found at the plasma membrane similar to its wild-type counterpart (Figure S2). Our work suggests that targeting of similar GTPases to different organelles is controlled by a combination of mechanisms specified by multiple sequence attributes rather than one interchangeable sequence motif. Such a mechanism may be interpreted as an efficient quality control for preventing deleterious effects that may result from GTPase mistargeting to non-native membranes. The MxxE motif of ARF1 influences the steady-state distribution of the GTPase Here, we have shown that the steady-state distribution of ARF1 between the Golgi apparatus and the post-golgi structures in plant cells is strongly influenced by the MxxE motif (Figures 2 and 3). When this signal was either mutated to the corresponding ARFB amino acids IxxD or eliminated in certain ARF1 ARFB chimeras, the distribution of the proteins shifted from the Golgi apparatus to the post-golgi compartments in comparison to the distribution of wild-type ARF1. These data suggest that ARF1 may interact with one receptor or multiple receptors localized on the Golgi apparatus and post-golgi structures. Mutation of the MxxE motif appears to induce a reduction in the affinity of ARF1 for Golgi-resident surface receptors and increases the affinity for post-golgi-localized proteins. A similar scenario has been postulated for the mammalian ARF1. Similar to the plant ARF1, the targeting of mammalian ARF1 to Golgi membranes is strongly influenced by the presence of an MxxE motif in position of the amino acid sequence (4). It was shown that fluorescent protein chimeras between ARF1 and ARF6 that included the MxxE motif of ARF1 localized to the cis Golgi; however, replacement of the MxxE region in ARF1 by the corresponding ARF6 region resulted in localization to the trans Golgi. It has been shown that ARF1 localizes on the Golgi and post-golgi compartments that bud from the Golgi (8). It is therefore possible that analogous to mammalian ARF1, the mutation of the MxxE motif in plant ARF1 causes a shift of the mutant proteins to the trans Golgi and immediate post-golgi compartments. The evidence that, in clear contrast with the ARF1 mutant lacking an intact MxxE motif, wild-type ARF1 was capable of an interaction with the SNARE membrin in mammalian cells prompted the suggestion that membrin could be the receptor for ARF1 to the Golgi and that the redistribution of the ARF1 mutant was because of its interaction with alternative factors, such as ARF GEFs at the trans Golgi (4). At present, the nature of the ARF1 receptor on plant membranes is unknown. Membrin has been localized on the cis Golgi in tobacco leaf epidermal cells (25,26). If in plants, analogous to mammalian cells, the ARF1 receptor is indeed membrin, then there must be at least an additional receptor for ARF1 on the post-golgi structures. M1-B competes for the binding sites of ARF1 on membranes but does not recruit coatomer We have shown that overexpression of the active mutant of M1-B leads to displacement of ARF1:GFP from the Golgi apparatus (Figure 8). It is possible that the observed depletion of ARF1 from Golgi membranes could be because of either a direct or an indirect effect of the M1- B fusion. If the effect was direct, depletion of ARF1 from Golgi membranes may be linked to saturation of ARF1- binding sites as a result of overexpression of the M1-B hybrid. This scenario postulates that M1-B interacts with the ARF1 receptor on Golgi membranes. In conditions of low expression, M1-B has preferential binding at the trans Golgi and trans Golgi network. In conditions of overexpression, M1-B may also titrate ARF1-binding sites at the cis- and medial-golgi and displace ARF1 from the Golgi membrane. An alternative scenario postulates that M1-B does not bind the ARF1-binding sites on the Golgi membranes. Depletion of ARF1 from membranes may be linked to inhibition of the ARF1 ARFB GTP mutant in retrograde traffic of components from post-golgi compartments to the Golgi that are needed for Golgi integrity. Therefore, the depletion of ARF1 from membranes would follow loss of integrity of ARF1-binding membranes. Overexpression of the M1-B active mutant also leads to partial redistribution of a Golgi marker into the ER and reduced secretion of a soluble cargo molecule (Figures 6 and 9). This evidence suggests that overexpression of M1-B Q71L interferes with protein export, but it is not clear whether the transport inhibition occurs at the ER Golgi interface or at the Golgi to plasma membrane step. We have shown that M1-B Q71L is released from membranes more slowly than the wild-type M1-B. This effect is most likely linked to the inability of the mutant GTPase to be deactivated. Therefore, different from the wild-type M1-B, M1-B Q71L may efficiently compete for binding sites with the ectopically expressed ARF1:GFP and with the endogenous ARF1. The differences in the dynamic cycles of wild type and M1-B Q71L on and off Golgi membranes may cause a fast saturation of the ARF1 receptor and subsequent displacement of ARF1 from the membranes. Displacement of ARF1 from membranes and M1-B Q71L - mediated collapse of secretion are in sharp contrast to the fact that M1-B Q71L fails to recruit coatomer on the Golgi. In addition, the inactive mutant of M1-B had no effect on Golgi integrity (Figure 6), while the same mutation of wildtype ARF1 exhibits a drastic BFA-like effect on constitutive secretion (21) and Golgi morphology (8), probably through titration of the ARF1 exchange factor. The different behavior of the fusion in comparison to the inactive form of ARF1 could be because of the fact that, unlike ARF1 GDP, 116 Traffic 2008; 9:

15 Targeting Mechanisms of ARF GTPases the M1-B T31N fusion is incapable of trapping the ARF1 exchange factor in an unproductive complex. The results suggest that the hybrid M1-B does not functionally replace ARF1 and that Golgi/post-Golgi targeting is dependent on mechanisms other than just effector interactions. At present, it cannot be inferred whether M1-B exploits cellular GEFs and guanine activating proteins (GAPs) to complete its cycles of GDP and GTP exchange on membranes or whether it also competes with ARF1 for those molecules. Our data show that M1-B is capable of a spontaneous GTPase activity similar to ARF1 (Figure 6). Therefore, it is also possible that M1-B completes the GDP/GTP cycle in cells independent from an interaction with GAPs and GEFs. Our functional data on the activity of a GTPase relocated to a non-native environment strongly support the view that amino acid determinants must be in place to specify the interaction of the GTPase with its effectors, despite the striking conservation of amino acid sequences among ARF GTPases. In this view, correct targeting of a GTPase to an organelle is the first step to ensure efficient activity of the protein, and sequence determinants have a subsequent role in the selection of ARF effectors. ARFB is a close isoform of human and yeast ARF6 ARF6 is a member of the ARF family that is known to regulate endocytosis and cytoskeleton remodeling in mammalian cells (12). Here, we present evidence that ARFB is a member of the ARF family that is targeted to the plasma membrane, similar to human and yeast ARF6 (Figure 1; Figures S1 and S2). An additional feature common to ARFB and ARF6 is that both proteins complete cycles of exchange of GDP/GTP at the plasma membrane (27). Our novel data on the subcellular localization of ARFB and of the factors that influence its targeting to the plasma membrane provide the opportunity to investigate whether ARFB is functionally similar to human yeast ARF6. was generated in a similar fashion but with primers specific for amino acids of ARF1. To generate the 110 ILTD 113 mutant of ARF1, we performed site-directed mutagenesis to alter the relevant codons. Similarly, site-directed mutagenesis was used to create ARF1 G2A, ARFB Q71L, ARFB T31N, M1-B Q71L and M1-B T31N. All mutants were subcloned upstream of either YFP or GFP. crfp was generated by amplification of mrfp using primers with XbaI/SacI sequences for subcloning into pvkh18-en6. Dr Marc Boutry, Université Catholique de Louvain, Belgium kindly provided the plasmid DNA encoding the plasma membrane marker, PMA4:GFP (32). Transgenic Nicotiana tabacum plants stably expressing the tonoplast marker, BobTIP26-1:GFP (16), were a generous gift of Nathalie Leborgne- Castel, Université de Bourgogne, France. ARF1 markers, ARF1:GFP and ARF1 Q71L :GFP, were previously described in Stefano et al. (8). For coatomer labeling, we used a YFP fusion with the Arabidopsis homolog of ecop, a component of the COPI coatomer (8). To enable glutathione S-transferase (GST)-tagged purification of proteins tested for GTPase activity, DNA encoding ARF1, ARF1 Q71L, ARFB, ARFB Q71L, M1-B and M1-B Q71L was subcloned in the recombinant Escherichia coli expression vector pgex (Amersham). The GST tag was placed upstream of the sequence of interest. The primer sequences used for the subcloning and mutagenesis indicated above are available upon request. Plant material and transient expression system Four-week-old Nicotiana tabacum (cv. Petit Havana) greenhouse plants grown at 258C were used for Agrobacterium tumefaciens (strain GV3101)- mediated stable DNA integration (33). The bacterial optical density (OD) 600 used for plant transformations was 0.05 for all constructs with the exception of ARF1 Q71L :GFP and ST:GFP, which were transformed at an OD 600 of 0.03 and 0.2, respectively. All laser scanning confocal experiments were carried out with wild-type plants with the exception of coexpression experiments with ecop:yfp and BobTIP26-1:GFP, which were stable transgenic tobacco plants expressing those protein fusions. For expression in protoplasts, N. tabacum plants (cv. Petit Havana) were grown under standard sterile conditions (22). Tobacco leaf protoplast preparation and subsequent DNA transfection through electroporation were performed as described by Phillipson et al. (22); the plasmid concentrations used are given in Figure 9. Equal volumes of 10-fold concentrated cell extract and culture medium were analyzed by enzyme assay. a-amylase assays and calculation of the secretion index (which represents the ratio between the extracellular and intracellular activities) were performed as previously described (22). Materials and Methods Molecular cloning Standard molecular techniques were used for subcloning. The fluorescent proteins used in this study were based on fluorescent fusions with mgfp5 (28), enhanced cyan fluorescent protein (ECFP) or enhanced yellow fluorescent protein (EYFP) (Clontech Inc.). The spectral properties of mgfp5 allow efficient spectral separation from YFP (29) and monomeric red fluorescent protein (mrfp) (30). Rat ST fused to GFP was used as an ER/Golgi marker (31). The complementary DNA of ARFB (At2g15310) was obtained as an Arabidopsis Biological Resource Center (ABRC) clone and amplified by polymerase chain reaction (PCR) primers containing the XbaI and SalI sites for subcloning upstream of either YFP or GFP in the binary vector pvkh18- En6. The ARF1/ARFB chimeras were subcloned in a similar manner, whereby each portion was amplified by PCR and fused by overlapping PCR procedures. ARF :YFP was created by PCR amplification of the DNA encoding the first 18 amino acids with primers containing the XbaIand SalI sites for subcloning upstream of YFP in pvkh18-en6. ARF :GFP Sampling, imaging and spot FRAP Transformed leaves were analyzed h after infection of the lower epidermis. Confocal imaging was performed using an inverted LSM Zeiss 510 META and a 63 water immersion objective. For imaging expression of GFP constructs, YFP constructs or both, imaging settings were used as described in Brandizzi et al. (29) to exclude fluorochrome cross talk. For simultaneous imaging of the triple labeling ((N-(3-triethylammoniumpropyl)- 4-(p-diethylaminophenylhexatrienyl)-pyridinium 2Br) (FM4-64), YFP and 4 0,6-diamidino-2-phenylindole (DAPI)), the fluorescence of FM4-64, ARFB- YFP and DAPI was detected using the 594-, 514- and 405-nm laser lines of HeNe, Argon and blue diode lasers, respectively. The imaging configuration of the microscope was set with a main dichroic HFT 405/514/594 and emission filters and nm (for YFP and DAPI, respectively) and nm spectral dye separation settings of the LSM 510 META (for FM4-64). Simultaneous imaging was carried out using the linesequential multitrack scanning mode configuration of the microscope. Spot FRAP experiments for fluorochrome photobleaching and half-time computation were performed as described by Brandizzi et al. (29). Segments of transformed leaves (5 mm 2 ) were submerged in 25 mm latrunculin B (Calbiochem; stock solution, 10 mm in dimethyl sulphoxide) for 1 h Traffic 2008; 9: