Recycling of Solanum Sucrose Transporters Expressed in Yeast, Tobacco, and in Mature Phloem Sieve Elements

Molecular Plant Advance Access published October 5, 2010 Molecular Plant

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Pages 1–11, 2010

RESEARCH ARTICLE

Recycling of Solanum Sucrose Transporters Expressed in Yeast, Tobacco, and in Mature Phloem Sieve Elements Johannes Lieschea,b, Hong-Xia Hea, Bernhard Grimma, Alexander Schulzb and Christina Ku¨hna,1

ABSTRACT The plant sucrose transporter SUT1 (from Solanum tuberosum, S. lycopersicum, or Zea mays) exhibits redoxdependent dimerization and targeting if heterologously expressed in S. cerevisiae (Kru¨gel et al., 2008). It was also shown that SUT1 is present in motile vesicles when expressed in tobacco cells and that its targeting to the plasma membrane is reversible. StSUT1 is internalized in the presence of brefeldin A (BFA) in yeast, plant cells, and in mature sieve elements as confirmed by immunolocalization. These results were confirmed here and the dynamics of intracellular SUT1 localization were further elucidated. Inhibitor studies revealed that vesicle movement of SUT1 is actin-dependent. BFA-mediated effects might indicate that anterograde vesicle movement is possible even in mature sieve elements, and could involve components of the cytoskeleton that were previously thought to be absent in SEs. Our results are in contradiction to this old dogma of plant physiology and the potential of mature sieve elements should therefore be re-evaluated. In addition, SUT1 internalization was found to be dependent on the plasma membrane lipid composition. SUT1 belongs to the detergent-resistant membrane (DRM) fraction in planta and is targeted to membrane raft-like microdomains when expressed in yeast (Kru¨gel et al., 2008). Here, SUT1–GFP expression in different yeast mutants, which were unable to perform endocytosis and/or raft formation, revealed a strong link between SUT1 raft localization, the sterol composition and membrane potential of the yeast plasma membrane, and the capacity of the SUT1 protein to be internalized by endocytosis. The results provide new insight into the regulation of sucrose transport and the mechanism of endocytosis in plant cells. Key words:

Transporters; membrane proteins; phloem physiology; protein targeting; potato.

INTRODUCTION Recent advances have provided new insights into how sucrose transport in higher plants is regulated. Sucrose transporter expression and activity are tightly regulated at various levels (Ku¨hn and Grof, 2010). The transcription of SUTs from Solanaceous species follows a circadian rhythm (Chincinska et al., 2008) and transcript abundance is regulated at the transcriptional and post-transcriptional levels (He et al., 2008). The oligomerization behavior of SUT1 proteins like Solanum tuberosum SUT1 (StSUT1) (from Solanum tuberosum) and Solanum lycopersicum SUT1 (SlSUT1) (from Solanum lycopersicon) is redox-dependent, since oxidative agents, like H2O2, decrease the monomer-to-dimer ratio in yeast as well as in plants (Kru¨gel et al., 2008). Redox reagents are also able to affect StSUT1 targeting in yeast, suggesting that the plasma membrane targeting of the transporter somehow depends on its quarternary structure. The secretion pathway of proteins destined for the plasma membrane (PM) or the cell wall is

known for many years. It includes the synthesis at the rough endoplasmic reticulum (ER), transfer to the Golgi apparatus, and, after maturation, transfer to the PM. The transfer happens in small transport vesicles. PM-bound proteins are incorporated in the vesicle membrane and reach the PM when the transport vesicle fuses with the PM. In contrast, the reverse way, namely the endocytosis of membrane-intrinsic proteins followed by degradation or recycling to the PM, has been a matter of debate until recent years. The best characterized proteins undergoing endocytosis are the auxin carriers PIN1, PIN2, and AUX1 from Arabidopsis 1 To whom correspondence should be addressed. E-mail Christina.kuehn@ biologie.hu-berlin.de, fax +49-30-2093-6103, tel. +49-30-2093-6337.

ª The Author 2010. Published by the Molecular Plant Shanghai Editorial Office in association with Oxford University Press on behalf of CSPP and IPPE, SIBS, CAS. doi: 10.1093/mp/ssq059 Received 1 June 2010; accepted 6 September 2010

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a Humboldt University, Institute of Biology, Plant Physiology, Philippstrabe 13, Building 12, 10115 Berlin, Germany b Plant Physiology and Anatomy Laboratory, Department of Plant Biology, Faculty of Life Sciences, Copenhagen University, Thorvaldsensvej 40, DK-1871 Frederiksberg C, Denmark

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RESULTS

Figure 1. Transient Expression of StSUT1–GFP after Co-Infiltration with Sub-Cellular Marker Proteins in Tobacco Leaves.

StSUT1–GFP Transiently Expressed in Nicotiana benthamiana Localizes to the Plasma Membrane, Endoplasmic Reticulum, and Motile Vesicles

(A–C) Co-expression of StSUT1–GFP with the ER marker HDEL-mCherry. (D–F) StSUT1–GFP-expressing tissue was incubated for 5 min with the PM marker FM4-64. (G–I) StSUT1–GFP-expressing tissue was incubated for 40 min with the endocytosis marker FM4-64. Co-localization is detectable in endocytic vesicles (arrows). (J–L) Co-expression of StSUT1–GFP with Sialyltransferase-mRFP. (A, D, G, J) StSUT1–GFP fluorescence in green; (B, E, H, K) marker fluorescence in red; (C, F, I, L) overlay of the two proceeding images, yellow fluorescence indicates co-localization (arrows); StSUT1–GFP co-localized with the PM marker FM4-64 as well as with the ER marker HDEL, but not with the Golgi marker ST. Scale bars: 20 lm (A–F), 10 lm (G–I).

Intracellular localization of plant proteins can conveniently be studied by transientexpression offluorescentGFP-fusion proteins in Agrobacterium-infiltrated tobacco sourceleaves (Andrewsand Curtis, 2005). StSUT1 is a 54-kDa membrane-intrinsic protein that was localized to the phloem cells in immunohistochemical experiments (Ku¨hnet al., 1997). A geneconstruct in which GFP is fused to theC-terminalendofStSUT1expressedundercontroloftheCaMV 35S promoter reveals the intracellular localization of the SUT1 protein. It has been detected at the PM and the ER, as well as in small motile vesicles (Kru¨gel et al., 2008). The vesicles have a size of about 200–500 nm. The localization of the StSUT1–GFP fusion protein at the PM and endoplasmatic reticulum (ER) membrane was confirmed by the use of established markers. HDEL is an ER retention signal for soluble proteins (Gomord et al., 1997). When a GFP variant is combined with a signal peptid and HDEL, it specifically labels the ER. Co-expression with StSUT1 shows the overlap in signal (see Figure 1A–1C). The styryl dye FM4-64 can be used as a PM marker because it labels exclusively the PM for at least the first 10 min after application (Bolte et al., 2004). Figure 1D–1F shows the overlapping signal of StSUT1–GFP and the PM-

marker FM4-64 after 5 min of incubation, proving the expected PM targeting of StSUT1. Figure 1G–1I shows co-localization of StSUT1–GFP with the marker FM4-64 after 40 min of incubation in endocytic vesicles. Another co-expression experiment demonstrated that the motile vesicles are not Golgi bodies. Sialyltransferase fused to mRFP is a specific marker for Golgi bodies (Boevink et al., 1998) and Figure 1J–1L shows them to be distinct from the StSUT1–GFP-containing structures. The experiment confirms the expected PM localization of StSUT1, essential for its predicted role as phloem loader. In addition, it is also targeted to the ER and to intracellular vesicles.

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thaliana. They were found to be constantly internalized from and recycled to the PM in Arabidopsis root cells (Geldner et al., 2001, 2003; Kleine-Vehn et al., 2006). This endocytic cycling is essential for the polar distribution of the carriers and, thereby, for the polar auxin transport. The mechanism of endocytosis in plants is not fully elucidated but there are indications that it is highly conserved in all eukaryotic cells. One well characterized pathway in animal and yeast involves clathrin as coat protein of endocytic vesicles. Clathrin was recently identified in plant cells and the dependence of PIN2 endocytosis on clathrin was demonstrated (Kleine-Vehn et al., 2006). Possible clathrin-independent pathways are less well investigated, but there is evidence, mostly based on studies in yeast, that membrane microdomains play an important role. It is thought that the special composition of lipids and sterols in these so-called membrane rafts is essential for the internalization step in the absence of clathrin. In plants, sterols were found to be essential for endocytosis of auxin transporters AUX1 and PIN2 (Kleine-Vehn et al., 2006; Men et al., 2008). StSUT1 was detected in the detergent-resistant membrane fraction of the plant PM, a sign for raft association (Kru¨gel et al., 2008). The observations presented above encouraged us to further investigate the dynamics of the intracellular localization of SUT1. Here, we provide evidence for a membrane raft-dependent internalization of SUT1. The dynamics of raft formation indicate a novel mode of protein activity regulation. Additional data showing selective targeting of StSUT1 in planta demonstrate the relevance of experiments with SUT1–GFP expressed in heterologous systems.

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Endocytosis of SUT1 In Planta

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In order to answer the question of whether the SUT1–GFP-positive vesicles belong to the exocytosis or to endocytosis pathways, the translational inhibitor cycloheximide was used (Figure 2A–2E). Treatment with cycloheximide did not interrupt vesicle formation or movement. After 4–5 h, the predicted half-life of the SUT1 protein (Ku¨hn et al., 1997), the fluorescence signal started to disappear from the plasma membrane and SUT1–GFP appears in a mobile vesicle population with increasing diameter (Figure 2). This can be taken as a hint that vesicle formation is related to protein turnover or recycling of the SUT1 protein. Earlier, it was shown that brefeldin A (BFA) treatment leads to StSUT1–GFP accumulation in so-called BFA compartments (Kru¨gel et al., 2008). BFA is a fungal toxin that inhibits the function of ARF–GTPases (Jackson and Casanova, 2000), which are needed for the formation of transport vesicles at the Golgi apparatus (Geldner et al., 2003; Richter et al., 2007). In the concentration used, the effect of BFA was an inhibition of protein transport to the PM but not endocytosis, and the formation of BFA compartments comprising the trans-Golgi network and the trans-most Golgi stacks (Lam et al., 2007). This is exactly the pattern that was described for other proteins that show constant turnover, like the auxin carrier PIN1 (Geldner et al., 2001) or the boron transporter BOR1 (Takano et al., 2005).

Endocytosis of StSUT1 in Sieve Elements In contrast to all other PM proteins undergoing endocytosis in plants, such as PIN1 (Geldner et al., 2001), AUX1 (Kleine-Vehn et al., 2006), BOR1 (Takano et al., 2005), and KAT1 (Hurst et al., 2004) or membrane receptor proteins such as BRI1 (Russinova et al., 2004), StSUT1, and SlSUT1 are localized in enucleate sieve elements (SEs) (Ku¨hn et al., 1997; Hackel et al., 2006; Kru¨gel et al., 2008). It was shown by immunolocalization that the accumulation of SUT1 in BFA compartments can also be induced in the mature SEs of the potato leaf phloem (Kru¨gel et al., 2008; Liesche et al., 2008). Pre-treatment of potato leaf tissue with BFA prior to fixation and embedding resulted in punctuate structures in the sieve element (SE) that labeled with the SUT1-specific antibody. The same result could be obtained in SEs of potato stem phloem (Figure 3). StSUT1 localized to the PM and vesicles in SEs that were treated with BFA prior to fixation (Figure 3C–3F), whereas it was only detected at the SE PM when the stem tissue was not pre-treated with BFA (Figure 3A and 3B). Together with the effect of BFA on StSUT1–GFP expressed in tobacco epidermis cells (Kru¨gel et al., 2008), this localization indicates that the structures observed here are the same BFA-induced compartments. The effect of BFA on SEs was consistent in loading and transport phloem.

The Movement of SUT1-Containing Vesicles Is Actin-Dependent The actin cytoskeleton is generally seen as essential for the internalization step of endocytosis and trafficking of internal

Figure 2. StSUT1–GFP Expressed in Tobacco Epidermis Cells Treated with Protein Synthesis Inhibitor Cycloheximide. Treatment causes depletion of transporters at the PM and accumulation in putative lytic vacuoles (arrows). Images were taken after incubation for 2.5, 4, 5, 6 h, and overnight. Scale bars: 20 lm.

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tested by treatment with Oryzalin, which de-polymerizes tubulin. Here, we demonstrated that the intracellular movement of StSUT1 vesicles was stopped by application of Cytochalasin D (Figure 4A) and Latrunculin B (Figure 4B), but was unaffected by Oryzalin (Figure 4C). It is therefore inferred that the intracellular trafficking of SUT1 is dependent on actin but not tubulin (see also supplemental movies 1 and 2).

Internalization of SUT1 Depends on Association with Membrane Rafts

BFA treatment causes formation of vesicles (arrows) in sieve elements that are not present in the water control; sieve elements (SE) can be identified by their sieve plate (SP) connections; StSUT1-specific antibody-binding is visualized by help of FITCcoupled secondary antibodies on longitudinal stem sections; scale bars: 10 lm.

vesicles regardless of which other components are involved. The dependence on actin can be demonstrated by application of specific inhibitors. Cytochalasin D inhibits de- and re-polymerization of actin fibers, whereas Latrunculin B leads to de-polymerization. Involvement of microtubules can be

Membrane Raft-Dependent Internalization of SUT1 Expressed in Yeast Cells The small size of membrane rafts generally restrains their investigation in plant cells (Opekarova´ et al., 2010). Yeast cells, however, can be used as experimental systems for the analysis of plant proteins because their membrane domains are believed to have similar attributes to plant membrane rafts (Malinska et al., 2003; Grossmann et al., 2006, 2007; Opekarova´ et al., 2010).

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Figure 3. Immunohistochemical Detection of StSUT1 on Samples Treated with Water as Control (A, B) and BFA (C–F) before Fixation.

Membrane rafts are defined as ‘small (10–200nm), highly dynamic, sterol- and sphingolipid-enriched domains that compartmentalize cellular processes’ (Pike, 2006). We have previously shown that SlSUT1–GFP is targeted to raft-like microdomains in yeast and can be detected in the detergentresistant membrane (DRM) fraction of plant plasma membranes (Kru¨gel et al., 2008). Although there is still controversy around the exact composition, definition, and function of such membrane domains, there is consensus on the existence of membrane subdomains, especially the sterol-enriched membrane patches usually referred to as raft-like microdomains. Raft-like microdomains were previously linked to endocytosis (Kleine-Vehn et al., 2006; Men et al., 2008). The use of Cyclodextrins provides a convenient, precise, and reproducible method for modulating the cholesterol content of tissue culture cells (Christian et al., 1997). Depletion of phytosterols from the plant plasma membrane by inhibiting sterol biosynthesis with methyl-b-Cyclodextrin (mbCD) leads to disruption of membrane rafts (Roche et al., 2008). Here, mbCD treatment prevented the formation of SUT1 vesicles in tobacco cells (Figure 4D), suggesting a role for sterols in the internalization of SUT1 from the PM. To test whether SUT1 endocytosis depends on clathrin, the transformed tobacco leaf tissue was incubated in Tyrphostin A23. Tyrphostin does not block endocytic events, but specifically inhibits cargo recruitment for clathrin-coated vesicles (Banbury et al., 2003). In contrast to mbCD, formation of SUT1 vesicles was not affected by Tyrphostin. Likewise, it did not alter the effect of BFA (Figure 4E). The typical compartments containing SUT1 were formed as was previously shown for treatment with BFA (Kru¨gel et al., 2008). These results indicate that SUT1 might not be internalized via the clathrindependent pathway, but instead by an alternative mechanism involving lipid rafts.

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Figure 4. StSUT1–GFP in Infiltrated Tobacco Leaves after Treatment with Cytochalasin B (A), latrunculin B (B), or oryzalin (C). Three pictures were taken from the same region at intervals of 180 s and overlayed. In (A) and (B), rings mark vesicles that remain in the same position over the total imaging period. In (C), two vesicles that are in different positions due to their movement are highlighted by arrows. Cytochalasin B and Latrunculin stop vesicle movement by actin filament disruption, whereas Oryzalin has no effect on vesicle movement. Scale bars: 20 lm. Movies of the same treatments can be downloaded from supplementary material. (D) StSUT1–GFP after treatment with 2% MbCD for 20 min. (E) StSUT1–GFP after treatment with 30 lM Tyrphostin A23 and 50 lM BFA for 1 h; all figures are maximum projections of z image stacks; scale bar: 20 lm.

Here, we expressed SlSUT1, which is a close homolog of StSUT1, in Saccharomyces cerevisiae cells. SlSUT1–GFP accumulated in raft-like microdomains when expressed in the complementation mutant SUSY7 upon treatment with the oxidizing agents H2O2 or L-cysteine (Figure 5A). In contrast, SISUT1–GFP did not accumulate in raft-like microdomains in the Derg6 mutant, regardless of pre-treatment with H2O2, but was instead evenly distributed over the PM (Figure 5B). The SUSY7 strain lacks an external invertase and can therefore only grow on

SlSUT1–GFP is concentrated in raft-like microdomains in the presence of oxidizing agents. No such concentration is observed when the same construct used in (A) was expressed in the mutant yeast strains Derg6 upon H2O2 treatment (B). SlSUT1–GFP expression in the yeast mutant Dend3 shows constitutive concentration in raftlike microdomains without any pretreatment (C). The yeast wildtype strain BY4741 has been used as a control strain (not shown). The lipid marker filipin reveals the concentration of lipids in membrane patches in Dend3 (E) and the homogenous distribution in Derg6 (D), similar to SlSUT1–GFP distribution. Images in (D) and (E) show true colors of blue for filipin. Scale bars: 4 lm.

sucrose medium when it is transformed with a sucrose transporter (Riesmeier et al., 1992). Derg6 cells, however, are defective in ergosterol biosynthesis, which is the major component of yeast membrane rafts and analogous to cholesterol in plant and animal cells (Bagnat et al., 2000; Iwaki et al., 2008). To test whether raft localization is a pre-requisite of endocytosis, we expressed the same SUT1–GFP fusion construct in the yeast mutant strain Dend3, which is defective in endocytosis. The end3 gene encodes a protein that is required for the internalization step of endocytosis and for the organization of the cortical actin cytoskeleton (Benedetti et al., 1994). When expressed in this mutant, the SlSUT1–GFP fusion protein accumulated in membrane patches instead of being evenly distributed at the PM as was observed in Derg6 cells (Figure 5C). The same localization has previously been shown for the yeast hexose transporter Hxt2 and the endocytosis marker FM4-64 in a wild-type strain when endocytosis was blocked with azide and fluoride (Walther et al., 2006). These patches, which they termed eisosomes, were characterized as static sites for internalization of membrane proteins and lipids. It can be

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Figure 5. SlSUT1–GFP Expressed in Yeast Strain SUSY7 (A) after pretreatment with H2O2 as previously shown (Kru¨gel et al., 2008).

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SUT1 Raft Association Depends on the Membrane Potential It is already understood that the membrane potential governs the lateral segregation of plasma membrane proteins and lipids in yeast (Grossmann et al., 2007). For the hexose transporter HUP1 expressed in yeast, application of the substrate glucose was also shown to destroy raft localization. The even distribution of HUP1 over the PM in yeast is accompanied by a loss of glucose transport activity. The effect is not osmotic, since sorbitol is not able to destroy raft-like structures. Glucose, as well as the substrate sucrose, abolished the accumulation of SlSUT1–GFP in membrane rafts (Figure 6B and 6C). Both substrates are taken up by the yeast cells via a proton symport mechanism so the import of sugars might therefore affect the membrane potential of yeast cells. At the same concentrations, sorbitol was not able to destroy the raft-like distribution of the SlSUT1–GFP fusion protein if expressed in the Dend3 mutant (Figure 6D). In order to determine whether the membrane potential of yeast cells plays a role in SUT1 distribution, various inhibitors have been tested. The protonophore carbonylcyanide 3-chlorophenylhydrazone (CCCP), which is known to abolish the proton gradient over the membrane, has a similar effect on SlSUT1–GFP distribution (Figure 6E).

DISCUSSION The Dynamic Localization of SUT1 in the Cell

Figure 6. SlSUT1–GFP Expression in the Yeast Mutant Dend3. (A, E, F) Single scans of SlSUT1–GFP in Dend3. (B–D) Maximum projections of z image stacks. (A) untreated Dend3 yeast cells expressing SlSUT1–GFP; (B) Dend3 cells treated with 100 mM sucrose for 10 min; (C) Dend3 cells treated with 220 mM glucose for 20 min; (D) Dend3 cells treated with 100 mM sorbitol for 45 min. (E) Dend3 cells treated with 50 lM CCCP for 30 min. (F) Dend3 cells treated with 100 lM methyl-b-cyclodextrin (MbCD) for 30 min. Scale bars: 4 lm.

The results presented above illustrate the dynamic localization of SUT1 in the cell. The data from the different experiments have been combined in a hypothetical model (Figure 7), which, in the case of the phloem-specific SUT1 protein, might involve both SEs and companion cells and the transport between these cell types. The presence of StSUT1–GFP in motile vesicles and the BFA sensitivity of its targeting shown by Kru¨gel et al. (2008) indicated a functional relevance of SUT1’s localization. BFA causes a reorganization of endosomal compartments prohibiting secreted proteins of reaching the PM but not endocytosis. Long inhibition of protein synthesis with cycloheximide resulted in accumulation of SUT1 in lytic vacuoles. The constant presence of SUT1 in vesicles and the effect of BFA indicate that the endocytosis leads not only to SUT1 degradation, but also to a constant recycling of the protein. This process has been described before for the auxin transporter PIN1 (Geldner et al., 2001). As for PIN1, endocytic cycling of SUT1 might be of high importance for the regulation of transport activity.

StSUT1 Shows Endocytosis to Be Possible in Sieve Elements Endocytosis of the sucrose transporter StSUT1 from potato was demonstrated here using a GFP-fusion protein and a transient tobacco expression system together with immunolocalization experiments. SUT1 is shown to be internalized, and its purpose

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concluded that in the endocytosis-deficient mutant Dend3, SlSUT1–GFP accumulates at these internalization sites. The fluorescent dye filipin binds specifically to sterols and has been used to detect these lipids in cell membranes (Grebe et al., 2003). Filipin staining confirmed that eisosomes are enriched in sterols. In Dend3 cells, sterols exhibited the same pattern of distribution as observed for SlSUT1–GFP (Figure 5C and 5E), while in the ergosterol mutant Derg6, both SlSUT1– GFP and sterols were distributed uniformly at the plasma membrane (Figure 5B and 5D). It was shown here, by application of mbCD, that the formation of raft-like domains containing SlSUT1–GFP is steroldependent. The patchy distribution in Dend3 cells changed to a homogeneous signal at the PM in less than 15 min after mbCD application (Figure 6F). The same uniform distribution was observed for SlSUT1–GFP expressed in yeast mutant Derg6 that is deficient in ergosterol biosynthesis (Figure 5B). Ergosterols are a major component of yeast raft-like microdomains (Bagnat et al., 2000; Iwaki et al., 2008). In the same way, Grossmann and co-workers demonstrated the lipid and ergosterol dependence of accumulation of various proton symporters at sites of endocytosis (Grossmann et al., 2006, 2007). We conclude that endocytosis of SlSUT1 in yeast cells depends on the lipid composition of the yeast plasma membrane and on the cortical actin cytoskeleton.

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Part of these processes might occur in companion cells, since not all organelles shown to be involved by the specific inhibitors in this study are present in SEs (such as Golgi and lytic vacuoles). SUT1 (magenta) follows the secretion pathway from the ER through Golgi apparatus and trans-Golgi network to the PM. The transport vesicles are moving along actin strands and their movement is therefore sensitive to Cytochalasin D and Latrunculin B. BFA causes a reorganization of endosomal compartments, prohibiting secreted proteins from reaching the PM, but not endocytosis. At the PM, SUT1 localizes differentially to sterol-enriched membrane rafts (blue) and ‘normal’ membrane domains. This is dependent on the redox state and the membrane potential, as H2O2 induces raftassociation whereas CCCP, sucrose, and glucose prevent the relocation. Disruption of the sterol-enriched domains prevents internalization. Long inhibition of protein synthesis with cycloheximide results in accumulation of SUT1 in lytic vacuoles. The constant presence of SUT1 in vesicles and the effect of BFA indicate that the endocytosis does not only lead to SUT1 degradation, but also feeds a constant recycling.

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of cytoskeletal components in mature sieve elements, a prerequisite for endocytic events, is under question. The high throughput phloem sap analysis also found actin to be present in SEs of Brassica napus (Giavalisco et al., 2006) and Cucurbita maxima (Walz et al., 2004; Lin et al., 2009). These studies also found actin de-polymerizing factors, which are regulators of microfilament assembly. In addition, transcripts of myosins, motor proteins needed for transport along actin fibers, were found in Ricinus communis phloem exudates (Doering-Saad et al., 2006). Kulikova et al. (2003) detected high amounts of actin in phloem exudates of Cucurbita pepo immunologically and identified actin filaments in the sap by electron microscopy. Moreover, proteinaceous structures were detected by electron microscopy in the parietal layer of SEs in Solanum lycopersicum and Vicia faba phloem (Ehlers et al., 2000). The authors describe clamp-like structures with a size similar to that of actin microfilaments. They cite inadequate fixation procedures as the reason for the structures not having been previously reported. Thompson and Wolniak (2008) highlighted internal membranes in SEs using a membrane-anchored fluorescent protein expressed under control of a companion-cell-specific promoter. Internal membranes can also be observed on electron micrographs of mature SEs when the tissue was pre-treated with EDTA prior to fixation (Liesche et al., 2008). Judged by the size of 100–200 nm in diameter, they could well be transport vesicles of the secretory and/or endocytic pathway. Vesicle-like structures have previously been observed on electron microscopic images of SEs from Streptanthus (Sjo¨lund, 1997), Vicia faba (van Bel et al., 2002), and in minor vein SEs from Hedera helix and Ajuga reptans (Hoffmann-Thoma et al., 2001). Endocytosis of StSUT1 could also be of importance for the trafficking of proteins from CCs into SEs, as illustrated in a hypothetical model by Ku¨hn and Grof (2010).

Endocytosis of SUT1 Is Dependent on Sterol-Enriched Membrane Rafts does not seem to be primarily protein degradation, but a constant recycling. SUT1 has a predicted half time of only 4–5 h (Ku¨hn et al., 1997; Ransom-Hodgkins et al., 2003). Therefore, it is not possible that the protein is synthesized already in immature SEs, but has to be synthesized in companion cells (CCs) and thereafter transferred to the SE PM. This implicates that endocytosis is possible in mature phloem sieve elements. SEs were long thought to function only as a conduit for phloem bulk flow. Recent reports suggesting the operation of many metabolic processes in SEs led to a change in this view (van Bel et al., 2002; Lin et al., 2009). The confirmation of these cells being the main site for sucrose loading in Solanaceaous species and the demonstration of capability for endocytosis further substantiates the new paradigm of the SE as an important link in signal transduction between the different plant organs. Because SEs are subject to extensive structural changes during maturation to optimize translocation, the existence

At the PM, a differential localization of SUT1 in sterol-enriched membrane rafts and ‘normal’ membrane domains has been shown previously in yeast (Kru¨gel et al., 2008). The localization can be influenced by reducing and oxidizing reagents, providing a direct link to the regulation of sucrose transport activity. Here, we demonstrate that internalization of StSUT1 depends on the formation of raft-like structures. It was shown earlier that BFA has no effect in the yeast ergosterol mutant Derg6, whereas internalization of SUT1–GFP was observed in the control strain SUSY7 in response to BFA treatment (Kru¨gel et al., 2008). Furthermore, no vesicles were detected in infiltrated tobacco leaves after application of mbCD, which inhibits raft formation by cholesterol depletion of the plasma membrane (Figure 4D). The yeast mutant Dend3, defective in actin polymerization, shows constitutive raft localization of SUT1–GFP, even without any pre-treatment of the cells. MbCD reverses the constitutive raft association of SUT1–GFP and leads to homogenous

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Figure 7. Hypothetical Model Showing the Posttranslational Movement of SUT1 Protein, Involving ER, Golgi, Vesicles, and Plasma Membrane.

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Depolarization of the PM Resolves Raft-Like Microdomains The activity of proton symporters depends on the membrane potential. StSUT1 does not show any uptake activity when the membrane is depolarized, which can be achieved by using the protonophore Carbonyl cyanide 3-chlorophenylhydrazone (CCCP) (Kru¨gel et al., 2008). It was discovered here that not only activity of StSUT1, but also localization in patches is dependent on the membrane potential. Depolarization with CCCP leads to a homogenous membrane distribution of StSUT1 in the yeast mutant strain Dend3 (Figure 6E). Similar experiments performed by Grossmann and colleagues (2007) demonstrated that ergosterols and transport proteins dissolve from the patches to redistribute at the membrane uniformly in response to depolarization. These observations provide a link between change in localization and transport activity, but a direct connection is not likely. The dissolving of raft-like microdomains might result from the less ordered state of the depolarized membrane (Grossmann et al., 2007). The addition of sucrose and high concentrations of glucose has the same effect on StSUT1–GFP localization as CCCP (Figure 6B, 6C, and 6E). The phenomenon is no effect of change in osmolarity caused by addition of sucrose to the medium. Sorbitol, another osmotically active sugar, does not affect the protein localization (Figure 6D). Grossmann describes a similar effect of substrate on HUP1 as a consequence of transient depolarization of the PM (Grossmann et al., 2007). Therefore, this phenomenon is most likely not a direct effect of substrate–protein interaction, but a secondary effect of membrane depolarization. The link between lipid-enriched membrane rafts and endocytosis in yeast is strengthened by the observation that end3p, the factor that is essential for endocytosis, interacts with DRM structures (Benedetti et al., 1994). These observations prove that in yeast, ergosterol- and lipid-dependent microdomains are functionally linked to endocytosis and that StSUT1–GFP can be found at these internalization sites. The data from yeast analysis and the presence of StSUT1 in plant DRMs (Kru¨gel

et al., 2008) strongly suggest the involvement of membrane rafts in the endocytic process. The results of this work have important implications for the research on the mechanism and physiological role of endocytosis in plants as well as on sieve element function. Future studies on raft-dependent regulation of endocytosis in the plant system will lead to a more conclusive picture of the processes outlined here.

METHODS Transformation of Agrobacterium tumefaciens Cloning of StSUT1–GFP was described previously (Kru¨gel et al., 2008). HDEL-mCherry (TAIR) was kindly provided by Alexis Kasaras and ST-mRFP by Karl Oparka. Agrobacterium tumefaciens strain pGV2260 was used for transformation. An aliquot of electro-competent cells was thawed on ice. After addition of 2 ng of the vector and careful mixing, the cell-DNA suspension was filled into a pre-cooled electroporation cuvette (slit 1 mm). The transformation was carried out in an electroporator using the following settings: voltage: 1800 V, capacitance: 50 lF, resistance: 129 X. The cells were re-suspended in 400 ll YEP medium immediately after pulsing, then transferred to a reaction tube and incubated for 1 h at 28
C. An aliquot was plated out on YEP medium containing appropriate antibiotics as selection markers and incubated at 28
C for 2 d.

Infiltration of Tobacco Leaf Cells Transformation was basically carried out as described by Sparkes et al. (2006). After testing for optimal efficiency, the following protocol was used. The Agrobacterium cultures were inoculated in the morning in 4 ml YEP medium (see above), containing appropriate selection markers, and incubated on a rotary shaker (280 rpm, 30
C) for 4–5 h. With an OD600 of about 0.1, the cultures were then centrifuged for 20 min with 3000 g at 16
C and, afterwards, the pelleted cells resolved in 8 ml infiltration buffer (10 mM magnesiumchloride, 10 mM MES pH 5.7, 100 lM acetosyringone). After incubation for at least 2 h, the suspension was injected into the lower side of the leaves using a syringe without needle. The leaves were used for confocal analysis the third and fourth days after infiltration. Nicotiana benthamiana was cultivated under greenhouse conditions.

Confocal Analysis of Transformed Tobacco Leaf Cells Small slices were cut out of infiltrated leaf areas and immediately analyzed using a Leica TCS SP2 CLSM (Leica Microsystems GmbH, Wetzlar). The detection settings were chosen according to the fluorophores. Excitation of GFP and YFP with argon laser was at 488 nm, of mRFP, FM4-64, and mCherry with heliumneon laser at 543 nm. Reagents were applied by infiltration using a syringe without needle on the lower side of a transformed leaf. Concentration and incubation time before imaging of the reagents

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distribution of the protein at the plasma membrane. The effect of mbCD on the SUT1 distribution in yeast and in plant cells strongly suggests an interdependence of SUT1 association to raft-like structures and its capacity to be internalized. Experiments with Tyrphostin A23 argue for a clathrin-independent endocytosis of the SUT1 protein (Figure 4E). The presented results are also relevant for the understanding of the mechanisms of endocytosis in plant cells. Until now, only the highly conserved pathway of clathrin-dependent endocytosis was well characterized in plants. SUT1 co-localizes with an ER marker and the PM marker FM4-64, which undergoes endocytosis in plants. No co-localization with a Golgi marker was detectable (Figure 2I). It was concluded from inhibitor studies affecting vesicle trafficking that SUT1 targeting to the plasma membrane is reversible and that the protein can be internalized in an actin-dependent manner. Inhibitor of tubulin had no effect on SUT1 vesicle trafficking (Figure 4C).

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applied: Brefeldin A (Sigma) 50 lM, 1 h; Cycloheximide (Fluka) 10 lM, 2–12 h; Cytochalasin D (Sigma) 200 lM, 30 min; Latrunculin B (Sigma) 15–25 lM, 30 min; Wortmannin (Sigma) 40 lM, 90 min; Oryzalin (Fluka) 20 lM, 2 h; Methyl-b-Cyclodextrin (Sigma) 30 lM, 0.5–2 h; Tyrphostin A 23 (Sigma) 30 lM, 1 h; FM4-64 (Invitrogen) 50 lM, 0–2 h.

Yeast Transformation

Confocal Analysis of Yeast Cells Overnight cultures of yeast cells were centrifuged for 4 min at 3500 rpm. The pellet, resolved in 500 ll water and again centrifuged with the same parameters, was dissolved in 100 ll of water or solution. From this, 20 ll were brought on slides and analyzed using the Leica TCS SP2 with GFP-specific detection settings and excitation at 488 nm. Filipin was imaged using a fluorescence microscope (Carl Zeiss, Jena) with attached microcamera (Olympus, Tokyo) and DAPI-specific filter settings. Reagents were applied with the following concentrations and incubation times before imaging: Methyl-b-Cyclodextrin (Sigma) 100 lM, 30 min; CCCP (Sigma) 50 lM, 30 min; Sucrose (Carl Roth) 1–220 mM, 10 min–1 h; Glucose (Duchefa Biochemie) 220 mM, 20 min; Sorbitol (Applichem) 100 mM, 45 min; H2O2 (Merck) 10 mM, 30 min DTT 10 mM EDTA 100 mM; Filipin (Sigma) 50 lg ml 1, 30 min.

Immunocytochemistry Samples for immunolocalization experiments were taken from Solanum tuberosum cv. Desire´e grown in the greenhouse. For fixation, slender stripes were cut out of the plant material with a defatted razor blade and transferred to fixation solution (4% paraformaldehyde, 0.1% glutaraldehyde, in 0.1 M phosphate buffer pH 7.3) in small glass tubes with snap-on lids. Vacuum infiltration for up to 30 min enhanced tissue penetration of the fixative. Samples were then incubated in fresh fixative overnight at 4
C. For embedding in LR-White, dehydration was carried out with the following steps. On washing with phosphate buffer followed the ethanol succession with

Recycling of Sucrose Transporter SUT1

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30%, 50%, 70%, two times 96% ethanol, with 30-min incubation on ice for each step. Then, samples were incubated on a rotary shaker in 96% ethanol:LR-White (1:1) solution for 1 h, then two times for 1 h in LR-White and lastly in fresh LR-White overnight. Specimens were prepared by adjusting the plant material in LR-White-filled gelatin capsules, which were then polymerized by baking for 2 d at 55
C. After trimming the specimen with a defatted razor blade, 0.8 lm thin sections were prepared using an ultra-microtome (Reichert und Jung Ultracut E, Leica). For immunolocalization experiments on thin sections, a fast and simple procedure was used. In the first step, accessible tissue surface was increased by 10 min etching with 0.1 M HCl. Then, the sections were blocked for 30 min with 2% BSA. After washing with ddH2O, the first antibody was applied and the samples incubated overnight at 4
C. To limit the amount of antibody serum needed, sections were circled in using a fat pen. Affinity-purified peptide antibody against StSUT1 (Kru¨gel et al., 2008) was used in a 1:10 dilution for immunolocalization. Incubation followed washing, two times with PBS-T and once with PBS for 5 min each. Then, samples were incubated with the second antibody for 45 min at room temperature in the dark. As second antibody, a FITC-conjugated antimouse antibody (Sigma) diluted 1:50 in PBS was used. After three washing steps, a drop of 50% glycerol in PBS was added on the sections to stabilize the fluorescent molecule and the cover slip put on. Imaging at the CLSM was conducted with FITC-specific detection parameters and excitation at 488 nm.

SUPPLEMENTARY DATA Supplementary Data are available at Molecular Plant Online.

FUNDING Financial support came from DFG to C.K. (SFB 429).

ACKNOWLEDGMENTS We thank Karl Oparka (Edinburgh), Thierry Berge´s (Poitiers), Guido Grossmann (Regensburg), Doris Rentsch (Bern), and Alexis Kasaras (FU Berlin) for providing material and mutants, Aleksandra Hackel for excellent technical assistance, and Damian Drew (Copenhagen) for proofreading. We thank Karl Oparka for helpful discussion and support. No conflict of interest declared.

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Yeast strains SUSY7 (Riesmeier et al., 1992), Derg6 (YML008c), Dpil 1 (Mat a; his3D1, leu2D0, met15D0, ura3D0YGR086c:: kanMX4; Walther et al., 2006), Dend3 (systematic name: YNL084c), and the wild-type BY4741 (MATa; his3D1, leu2D0, met15D0, ura3D0) were transformed with StSUT1–YFP in vector pDR196 as follows. After growing the yeast strains overnight to a concentration of OD600 0.8, cells were centrifuged for 5 min with 2000 rpm. Concentrated cells are solved in 1 ml mix 1 (1 ml lithium acetate, 0.5 ml 103 TE, 5 ml 2 M sorbitol, 3.5 ml water) and incubated for 10 min at room temperature. Then, 3 ll of insert DNA and 5 ll salmon sperm were solved in 40 ll of yeast cells in mix 1. After addition of 230 ll Mix 2 (1.5 ml lithium acetate, 1.5 ml 103 TE, 10 ml PEG (60%), 2 ml water), the whole mix was incubated for 30 min at 30
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