A member of the mitogen-activated protein 3-kinase family isinvolved in the regulation of plant vacuolar glucose uptake

Karina Wingenter1, Oliver Trentmann1, Irina Winschuh1, Imke I. Hormiller2, Arnd G. Heyer2, Jorg Reinders3,

Alexander Schulz4, Dietmar Geiger4, Rainer Hedrich4,5 and H. Ekkehard Neuhaus1,*

1Plant Physiology, University of Kaiserslautern, Erwin-Schrodinger Straße, D-67653 Kaiserslautern, Germany,2Botany Department, University of Stuttgart, Pfaffenwaldring 57, D-70569 Stuttgart, Germany,3Institute of Functional Genomics, University of Regensburg, Josef-Engert Straße 9, D-93053 Regensburg, Germany,4Julius-von-Sachs Institute, Molecular Plant Physiology and Biophysics, Julius-Maximilians University Wurzburg,

Julius-von-Sachs Platz 2, D-97082 Wurzburg, Germany, and5King Saud University, Riyadh, Saudi Arabia

Received 11 July 2011; revised 5 August 2011; accepted 9 August 2011; published online 26 September 2011.

*For correspondence (fax +49 631 205 2600; e-mail neuhaus@rhrk.uni-kl.de).

SUMMARY

Vacuolar solute accumulation is an important process during plant development, growth and stress

responses. Although several vacuolar carriers have been identified recently, knowledge regarding the

regulation of transport is still limited. Solute accumulation may be controlled by various factors, such as

alterations in carrier abundance or activity. Phosphorylation via kinases is a well-known principle for activation

or deactivation of proteins. Several phosphorylated proteins have been identified in the tonoplast proteome;

however, kinases that catalyse the phosphorylation of tonoplast proteins are currently unknown. The

tonoplast monosaccaride transporter from Arabidopsis (AtTMT1) and its homologue from barley have multiple

phosphorylation sites in their extremely large loops. Here we demonstrate that the loop of AtTMT1 interacts

with a mitogen-activated triple kinase-like protein kinase (VIK), that an aspartate-rich loop domain is required

for effective interaction, and that the presence of VIK stimulates glucose import into isolated vacuoles.

Furthermore, the phenotype of VIK loss-of-function plants strikingly resembles that of plants lacking AtTMT1/

2. These data suggest that VIK-mediated phosphorylation of the AtTMT1 loop enhances carrier activity and

consequently vacuolar sugar accumulation. As many phosphorylated proteins have been identified in the

tonoplast, differential phosphorylation may be a general mechanism regulating vacuolar solute import.

Keywords: glucose, vacuole, MAP 3-kinase, major facilitator superfamily, transport, Arabidopsis.

INTRODUCTION

Photoautotrophic plants are able to use sunlight for CO2

fixation and its conversion into sugars. Sugars are important

substrates in energy production and anabolic reactions, and

are basic components of cell walls, starch and other bio-

polymers. Accordingly, cellular sugar distribution and its

storage in various compartments or tissues must be

adjusted to the physiological demands of the plant. In

addition to temporary storage in the chloroplasts or vacu-

oles of photosynthetic ‘source’ tissues, sugars are also

delivered via long-distance phloem transport to developing

or heterotrophic ‘sink’ tissues (Martinoia et al., 2007).

Usually, sugar is deposited in the form of insoluble starch in

plastids, whereas vacuoles accumulate soluble sugars.

Potato plants and cereals are characterized by high plastidial

starch contents in their heterotrophic tubers or kernels.

Sugarcane and sugar beet mainly accumulate soluble sug-

ars in their vacuoles but in different tissues, namely leaves/

stems and tap roots, respectively for cane and beet. Thus,

the regulation of tissue-specific and intracellular sugar dis-

tribution is of physiological importance and also agronomic

interest. In this study, we investigate regulation of the

tonoplast monosaccaride transporter from Arabidopsis

thaliana (AtTMT1) to obtain insights into the control of sugar

provision to plant vacuoles.

The major facilitator superfamily (MFS) is the largest

transporter family, and sugar carriers (including sugar

alcohol carriers and related proteins) represent one of the

largest sub-groups within this superfamily (Chang et al.,

890 ª 2011 The AuthorsThe Plant Journal ª 2011 Blackwell Publishing Ltd

The Plant Journal (2011) 68, 890–900 doi: 10.1111/j.1365-313X.2011.04739.x

2004). The genome of the model plant Arabidopsis thaliana

encodes more than 50 putative MFS-type sugar carriers

(Buttner and Sauer, 2000). MFS-type sugar carriers comprise

diverse transporter families with a range of functions,

substrate specificities or cellular localization. Two MFS

families of vacuolar monosaccharide transporters have been

identified in Arabidopsis. Both the vacuolar glucose trans-

porter (AtVGT) family and the tonoplast monosaccharide

transporter (AtTMT) family contain three isoforms (Wormit

et al., 2006; Aluri and Buttner, 2007).

Investigations of single, double and triple AtTMT loss-of-

function mutant plants revealed that AtTMT1 and AtTMT2

represent the main tonoplast glucose importers, whereas

AtTMT3 and the AtVGTs apparently play a minor or different

role (Wormit et al., 2006; Aluri and Buttner, 2007). Mutant

plants with increased AtTMT1 abundance had altered sugar

sensing and partitioning and exhibited increased seed

yields. Therefore, AtTMT1 and AtTMT2 appear to play a

key function in sugar homeostasis of the plant cell (Wing-

enter et al., 2010).

Proteome studies identified a high amount of phos-

phorylated proteins in the tonoplast fraction (Endler et al.,

2009). Accordingly, it was assumed that phosphorylation

may regulate the activity or function of tonoplast intrinsic

carriers. Interestingly, AtTMT1, 2 and 3 contain multiple

putative phosphorylation sites (NetPhos 2.0; http://

www.cbs.dtu.dk/services/NetPhos/), and at least one site in

AtTMT1 has been shown to be phosphorylated under native

conditions (Whiteman et al., 2008). Moreover, the TMT

homologue from barley (Hordeum vulgare) has 12 proven

phosphorylation sites (Endler et al., 2009). The vast majority

of putative phosphorylation sites and all the proven phos-

phorylation sites are located in the extremely large loop that

is typical of TMTs (Wormit et al., 2006).

In a search for mechanisms regulating vacuolar glucose

import, we identified a predicted member of the mitogen-

activated protein triple kinase (MAP3K) family (MAPK

Group, 2002). We showed that this kinase physically binds

to the loop of AtTMT1 and is able to phosphorylate this

domain, and that its presence stimulates vacuolar glucose

uptake. An important (probably regulatory) role of this

kinase in vacuolar glucose import is furthermore sup-

ported by investigations of corresponding loss-of-function

mutants.

RESULTS

Plant vacuolar TMT-type sugar transporters have

a unique protein structure

TMT proteins accept glucose and fructose as substrates

(Wormit et al., 2006; Wingenter et al., 2010), and possess

several conserved domains that are also present in other

monosaccharide carriers (Henderson, 1991; Weschke et al.,

2000; Cho et al., 2010). Interestingly, members of the TMT

family are characterized by a large hydrophilic loop between

transmembrane helices six and seven (Wormit et al., 2006)

(Figure S1). This loop is approximately four to five times

larger than the corresponding domain in related monosac-

charide transporters from plants, animals, fungi or bacteria

(Henderson, 1991; Wormit et al., 2006).

TMT proteins possess a high number of predicted phos-

phorylation sites, nearly all of which are located in the loop

region (NetPhos 2.0). Furthermore, all experimentally pro-

ven phosphorylated residues of TMTs occur in the loop. The

loop of the barley TMT homologue possesses 12 phosphor-

ylated amino acid residues (Endler et al., 2009). Several of

these phosphorylation sites are conserved in TMTs of

Arabidopsis, and one of these sites has been shown to be

phosphorylated in native AtTMT1 (Whiteman et al., 2008).

In addition to these multiple phosphorylation sites, TMT

proteins also exhibit a remarkably high accumulation of

aspartate or glutamate residues in approximately the centre

of the loop sequence (Figure S1). In AtTMT1, 11 aspartate

residues are located in a segment of only 20 amino acids

(aspartate cluster). The corresponding region of AtTMT2

consists of 23 amino acids and contains 14 aspartate and

glutamate residues. Within a sequence of 17 amino acids,

AtTMT3 contains nine aspartate and glutamate residues. It is

tempting to speculate that these negatively charged resi-

dues are required for electrostatic interaction with other so

far unknown proteins.

The recombinant AtTMT1 loop physically interacts with

a putative ankyrin repeat protein kinase

To identify Arabidopsis proteins that may interact with TMT

proteins, we performed an immobilized metal-affinity

chromatography-based (IMAC-based) screen using the

AtTMT1 loop region as bait. The histidine-tagged loop was

synthesized in Escherichia coli, purified to homogeneity,

refolded (Figure S2, lane 2) and linked to nickel-chelating

Sepharose. Subsequently, a soluble protein fraction from

Arabidopsis leaves was applied to the affinity column to al-

low protein interaction with the attached loop. To investi-

gate whether significant amounts of soluble proteins

interact with the Ni-Sepharose, Arabidopsis leaf extracts

were additionally analysed by IMAC in the absence of the

attached loop. The presence of moderate concentrations of

imidazole in the binding and washing buffers avoids or at

least reduces any interaction with the Ni-Sepharose. The

histidine-tagged AtTMT1 loop protein was trapped on

Ni-Sepharose and subsequently eluted by application of

high imidazole concentrations in the elution buffer. Eluted

proteins were analysed by SDS–PAGE. More than ten pro-

teins of various sizes co-eluted with the loop (Figure S3, lane

2). It is very likely that several soluble plant proteins also

interact with the IMAC material. However, no proteins were

detectable in the eluate lacking the loop (Figure S3, lane 1).

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Mass spectrometry is a very sensitive method that allows

the detection and identification of proteins. Using this

method, more than 80 proteins were identified in the loop-

containing eluate (Table S1). These proteins comprise pro-

teins that interact with the IMAC material as well as proteins

that exhibit specific or ‘non-specific’ interaction with the

AtTMT1 loop. Interestingly, these proteins included the

mitogen-activated triple kinase (MAP3K) VH1-interacting

kinase (VIK) (At1g14000, Table S1; marked in bold) (MAPK

Group, 2002). This protein is known to interact with the VH1/

BRL2 receptor-like kinase that is involved in leaf vein

formation (Ceserani et al., 2009).

Investigation of physical interaction of AtTMT1 and VIK

in living cells

It is not clear whether VIK exhibits non-specific physical

interaction with the recombinant loop protein or with the Ni-

Sepharose, or whether VIK recognizes and modifies the

AtTMT1 in vivo. vik RNA is detectable in every analysed

tissue, with its highest expression level in leaves, and

accumulates in response to drought and cold treatment as

well as in the presence of high glucose and salt concentra-

tions (Figure 1). This expression pattern resembles that of

attmt1 RNA (Wormit et al., 2006), and therefore suggests

that both proteins are present in the same tissues and under

the same conditions, supporting a possible in vivo inter-

action of VIK and AtTMT1.

Bimolecular fluorescence complementation (BiFC) (Kerp-

pola, 2008) is used to validate protein interactions in living

cells (Geiger et al., 2009b). It is based on the association of

complementary yellow fluorescent protein (YFP) fragments

fused to the potential partner proteins. Close proximity of

the complementary fragments results in re-formation of the

fluorescent marker protein. The intensity of the emitted

fluorescence is proportional to the strength of the inter-

action. Additionally, the subcellular localization of the inter-

action can be deduced from the fluorescence distribution in

transformed cells.

Arabidopsis protoplasts were transiently transformed

with constructs tmt1::yfpNT and vik::yfpCT for synthesis of

the respective YFP fragment fusion proteins. Co-expression

of tmt1::yfpNT and vik::yfpCT resulted in a clearly visible BiFC

signal in the protoplasts (Figure 2a,c). This signal persisted

after lysis of the cells and release of the vacuoles, suggesting

tight association of the two marker protein fragments at the

tonoplast (Figure 2b,d). To validate the BiFC data, a control

experiment was performed using an alternative kinase from

Arabidopsis (MEKK1) (MAPK Group, 2002) that is assumed

not to interact with AtTMT1. Co-expression of the AtTMT1

and MEKK1 fusion proteins (mekk1::yfpCT and tmt1::yfpNT)

led to slight YFP fluorescence within protoplasts (Figure 2e).

However, the BiFC signal disappeared after induction of cell

lysis (Figure 2f). Therefore, we conclude that AtTMT1 and

MEKK1 exhibit a weak physical interaction that may be

caused by over-expression and a correspondingly high

density of the recombinant proteins. Disruption of the

plasma membrane and release of the vacuoles resulted in

dilution of proteins and dissociation of the YFP fragments.

VIK functionally interacts with the recombinant

AtTMT1 loop peptide

VIK is classified as a potential protein kinase and thus may

catalyse phosphorylation of its target proteins (MAPK Group

2002; Ceserani et al., 2009). To test whether VIK functionally

interacts with AtTMT1, we performed a phosphorylation

assay using radioactively labelled [c32P]-ATP as the phos-

phate group donor. Bovine serum albumin (BSA) also

possesses several predicted phosphorylation sites (NetPhos

2.0, http://www.cbs.dtu.dk/services/NetPhos/), but should

not act as a substrate of VIK and thus was used as a negative

control. Purified recombinant VIK (Figure S2, lane 6) was

incubated either with purified recombinant AtTMT1 loop

Figure 1. Tissue-specific and stimuli-affected expression of vik.

Quantitative RT-PCR was performed to investigate transcription of the vik

gene. Expression levels are given as relative values compared to expression

of the control gene ef1a. Each value represents the mean of three individual

experiments, each with three replicate samples, � SE.

(a) Quantitative RT-PCR for analysis of tissue-specific vik expression. mRNA

was isolated from various developmental stages of Arabidopsis grown on

soil.

(b) Quantitative RT-PCR for analysis of the effects of drought, high sugar

contents, cold and salt stress on vik expression. Plants were cultivated on soil

under various stress conditions (drought stress: watering was stopped 7 days

prior to harvest; cold stress: incubation for 48 h at 4�C; salt stress: watering

with 100 mM NaCl at days 4 and 2 prior to harvest), and the mRNA of leaves

from 5-week-old plants was isolated and analysed. For investigation of the

effects of high sugar concentrations on gene expression, seedlings were

grown for 7 days in liquid culture supplemented with 5% glucose 14 h prior to

harvesting of plant material.

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ª 2011 The AuthorsThe Plant Journal ª 2011 Blackwell Publishing Ltd, The Plant Journal, (2011), 68, 890–900

peptide (Figure 3a, left panel) or BSA (Figure 3a, right

panel). In the presence of VIK, substantial phosphotransfer

to the AtTMT1 loop (Figure 3b, left panel) but not to BSA

occurred (Figure 3b, right panel). Furthermore, the protein

kinase was also labelled with 32P (compare Figure 3b, left

and right panels).

The aspartate cluster of the loop is required for

effective functional VIK interaction

Electrostatic interaction between positively and negatively

charged domains in kinases and target proteins is suggested

to be involved in (MAP) kinase docking and substrate rec-

ognition (Tanoue et al., 2000; Liu et al., 2006). To investigate

whether the aspartate cluster in the loop domain of AtTMT1

is required for VIK interaction, we replaced all aspartates in

the negatively charged domain (Figure S1) by asparagines.

The modified loop (–Asp) and the native AtTMT1 loop (Wt)

were heterologously expressed, purified (Figure S2, lanes 2

and 4) and used as bait ligands for surface plasmon reso-

nance (SPR) analysis (Figure 4). Use of VIK as prey caused a

fast increase in the SPR signal, and use of the native loop led

to higher levels compared to the mutated protein. Addition

of buffer solution (lacking VIK) to the bound proteins

resulted in a decrease in SPR signals, reflecting dissociation

of the respective complexes. The slopes of the SPR curves

suggest that the interaction between VIK and the native loop

differs from that between VIK and the modified loop (Fig-

ure 4). No SPR signal occurred when BSA (negative control)

was used as bait.

In addition to the SPR analysis, the role of the aspartate

cluster in functional protein interaction was investigated

using a phosphorylation assay (Figure 4, inset). VIK-depen-

dent phosphorylation of the mutated loop peptide (47.039

digital light units mm)2 of 32P) was approximately twofold

lower than that of the unmodified loop (91.404 digital light

units mm)2 of 32P). The reduced phosphorylation of the

mutated loop peptide may be due to impaired VIK/substrate

interaction.

VIK-dependent phosphorylation stimulates AtTMT1

transport activity

These results show that VIK physically interacts with

AtTMT1 (Table S1, Figures 2 and 4) and that VIK phospho-

rylates the central loop peptide (Figures 3 and 4, inset). The

question arises whether VIK-mediated phosphorylation

positively or negatively affects AtTMT1 transport activity.

We performed a transport study using isolated vacuoles and

quantified [14C]-glucose uptake in the presence or absence

(a)

(c)

(e) (f)

(b)

(d)

Figure 2. Bimolecular fluorescence complementation (BiFC) between VIK and AtTMT1.

(a) Survey of Arabidopsis protoplasts expressing vik::yfpCT and tmt1::yfpNT. Fluorescence image (left) and bright-field image (right).

(b) Survey of released yellow-fluorescing vacuoles expressing vik::yfpCT and tmt1::yfpNT (left) plus the corresponding bright-field image (right).

(c) Fluorescence image (left) and bright-field image (right) of protoplasts expressing vik::yfpCT and tmt1::yfpNT. Invaginations around the chloroplasts indicate BiFC

between AtTMT1 and VIK at the vacuolar membrane.

(d) Released yellow-fluorescing vacuole expressing vik::yfpCT and tmt1::yfpNT (left) and the corresponding bright-field image (right).

(e) Fluorescence image (left) and bright-field image (right) of Arabidopsis protoplasts expressing mekk1::yfpCT and tmt1::yfpNT. Note that BiFC fluorescence is not

restricted to the vacuolar membrane, indicating an over-expression ‘artifact’ rather than a specific physical interaction between AtTMT1 and MEKK1.

(f) Vacuoles released from protoplasts expressing mekk1::yfpCT and tmt1::yfpNT.

In all images, red fluorescence represents the autofluorescence of chloroplasts. Scale bars = 100 lm (a, b) and 20 lm (c–f).

Regulation of vacuolar glucose transport 893

ª 2011 The AuthorsThe Plant Journal ª 2011 Blackwell Publishing Ltd, The Plant Journal, (2011), 68, 890–900

of purified recombinant VIK. Addition of VIK led to signifi-

cantly higher glucose uptake (0.33 nmol ll)1) into vacuoles

compared to experiments lacking recombinant kinase

(0.15 nmol ll)1) (Figure 5). Because AtTMT1 is one of the

major vacuolar glucose importers (Wormit et al., 2006a),

these data suggest that VIK-dependent phosphorylation

stimulates/activates AtTMT1. However, we cannot rule out

the possibility that other vacuolar sugar transporters are

controlled by the catalytic activity of VIK. Use of vacuoles

isolated from vik1 loss-of-function mutants will allow such

analysis.

VIK loss-of-function mutants phenocopy Arabidopsis plants

lacking the vacuolar sugar carriers AtTMT1 and 2

The absence of VIK apparently results in decreased vacuolar

sugar import (Figure 5), and therefore mutant plants lacking

VIK should phenotypically resemble AtTMT1/2 loss-of-

function mutants (Wormit et al., 2006; Wingenter et al.,

2010). Two putative vik T-DNA insertion lines (vik1 and vik2)

were screened for homozygous plants, and the absence

of functional vik mRNA in the mutants was verified by

Northern blotting (Figure S4). To allow comparison of the

data, vik1 and vik2 mutant plants were cultivated and

investigated using methods identical to those used for

AtTMT1/2 loss-of-function mutants (Wormit et al., 2006).

The vik1 and vik2 mutant plants were cultivated in liquid

medium, and biomass production was documented. In

standard medium containing moderate sucrose concentra-

tions, there was no significant difference in fresh weight

between vik mutants and wild-type (Wt) plants (Figure 6a).

However, use of high glucose concentrations resulted in

fresh weights of vik1 and vik2 mutants that were approx-

imately fivefold lower than those of Wt plants (2.4 and

2.7 mg FW, respectively, versus 12.7 mg) (Figure 6b). As

Figure 3. Phosphorylation of the recombinant AtTMT1 loop protein by VIK.

The recombinant AtTMT1 loop protein was incubated with recombinant VIK

and [c-32P]-ATP for 1 h. As a negative control, BSA was used instead of the

AtTMT1 loop.

(a) SDS–PAGE. Lane M comprises protein markers: the molecular mass (kDa)

of the marker proteins is indicated.

(b) Radioautogram.

Figure 4. Investigation of the role of the aspartate cluster using surface

plasmon resonance (SPR) analyses and phosphorylation assays.

Surface plasmon resonance (SPR) analysis was performed using the AtTMT1

loop (Wt) and the modified loop peptide (–Asp) as bait and with VIK as prey.

Recombinant loop proteins were attached to the sensor chip. Buffer contain-

ing recombinant VIK was applied and binding progressed for 60 sec. Finally,

medium without VIK was added to remove interacting VIK. The SPR signal is

given in response units (RU). Inset: Phosphotransfer to the AtTMT1 loop

lacking the aspartate cluster. Recombinantly synthesized and purified AtTMT1

loop protein (Wt) and the modified loop peptide (–Asp) were incubated for

10 sec with [c-32P]-ATP and heterologously expressed purified VIK. This

experiment was performed more than three times. A representative radioau-

togram and quantified radioactivity are shown.

Figure 5. Effects of VIK absence or presence on glucose import into vacuoles

from Arabidopsis leaves.

Vacuoles isolated from 5-week-old plants were incubated for 10 min at 22�C in

100 lM radioactively labelled glucose with or without the addition of 0.5 lg

heterologously expressed purified VIK. The data represent means of three

individual experiments, each with 9 or 10 replicates, � SE. Asterisks indicate

a significant difference between samples with or without VIK (P < 0.005)

(Student’s t test).

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ª 2011 The AuthorsThe Plant Journal ª 2011 Blackwell Publishing Ltd, The Plant Journal, (2011), 68, 890–900

high sorbitol concentrations did not result in differences in

biomass between mutant and Wt plants, the growth

repression of vik1 and vik2 mutants in the presence of

high glucose concentrations is not due to osmotic effects

(Figure S5). A similar glucose-dependent reduction in plant

growth (approximately 3.5-fold) in comparison to Wt plants

was also observed for attmt1/2 double mutants (Wingenter

et al., 2010).

During cold stress, Arabidopsis shows glucose accumu-

lation in leaves (Wanner and Junttila, 1999). This process is

impaired in AtTMT1/2 loss-of-function mutants (Wormit

et al., 2006). Leaf tissues of cold-treated vik mutants also

exhibited reduced glucose concentrations (72 and 63 lmol

glucose/g FW for vik1 and vik2, respectively) compared to Wt

plants (Figure 6c).

According to FRET (fluorescence resonance energy trans-

fer) analysis, cytosolic glucose levels in Arabidopsis are

quite low (in the micromolar range) (Chaudhuri et al., 2008).

However, impaired vacuolar glucose import leads to

increased cytosolic concentrations, which are known to

stimulate mitochondrial respiration. In fact, under standard

conditions, attmt1/2 mutants exhibited 1.4-fold higher

respiration rates than Wt plants (Wingenter et al., 2010). In

contrast to the attmt1/2 mutants, respiration rates were

similar in the vik lines and Wt plants (4.6–5.4% 14CO2

respiration) (Figure 7a). However, exposure to high external

glucose led to differences in respiration rates between Wt

(5.6% 14CO2 respiration) and vik mutant lines (7.0 and 8.3%14CO2 respiration for vik1 and vik2, respectively) (Figure 7b).

Thus, we conclude that vik mutants phenotypically

resemble plants lacking both AtTMT1 and AtTMT2 (Wormit

et al., 2006; Wingenter et al., 2010).

vik2 mutants exhibit an unbalanced cellular glucose

compartmentation

Non-aqueous fractionation is a method that is well-suited for

determination of intracellular glucose compartmentation in

plants (Wingenter et al., 2010). To confirm that VIK is in fact

involved in the regulation of vacuolar glucose import, we

performed non-aqueous fractionation of Wt and vik2 seed-

lings grown at room temperature in liquid culture (with or

without additional glucose) and quantified the glucose

concentration in various cell compartments (Figure 8). Both

genotypes contained nearly identical total concentrations of

glucose per mg fresh weight (data not shown) but exhibited

altered intracellular sugar compartmentation. Without

additional external glucose, Wt plants contained 60%

glucose in their vacuole and approximately 32% glucose in

the cytosol (Figure 8a). In contrast, vik2 plants stored <40%

of total glucose in the vacuole and showed high glucose

contents in the cytosol. The lowest amounts of total glucose

are associated with plastids. In the presence of high external

glucose concentrations, vacuoles of Wt plants accumulated

51% of the total glucose, whereas the vacuoles of vik2

mutants contained only 20% of the total glucose (Figure 8b).

Thus, cellular glucose compartmentation, particularly

vacuolar glucose accumulation, is altered in vik2 mutants.

Figure 6. Effects of glucose application on seedling fresh weight and of cold

treatment on glucose contents of wild-type (Wt) and vik T-DNA plants.

(a) Fresh weight of Wt and vik mutant plants grown for 12 days in liquid

culture medium.

(b) Fresh weight of Wt and vik mutant plants grown for 12 days in liquid

culture medium to which 5% glucose was added 14 h prior to harvesting of

the plant material.

(c) Glucose concentration in leaves of cold-treated Wt plants and vik mutants.

Plants were grown on soil for 5 weeks under standard conditions and

incubated for 18 h at 4�C. Grey bars represent Wt samples, horizontally

striped bars represent vik1 samples, and diagonally striped bars represent

vik2 samples. Asterisks indicate significant differences between for differ-

ences between the respective vik mutants and Wt (P < 0.005) (Student’s

t test). The data represent means of three individual experiments, each with

four replicate samples, � SE.

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DISCUSSION

The vacuolar glucose transporters AtTMT1 and AtTMT2 are

crucial for intracellular sugar homeostasis, sugar sensing

and carbon-based seed yield, and represent the main glu-

cose transporters in the tonoplast (Wormit et al., 2006;

Wingenter et al., 2010). However, AtTMT3 and the vacuolar

glucose transporters are of minor importance in terms of

general sugar provision to leaf vacuoles and instead fulfil

specific functions under non-standard conditions or in other

tissues (Wormit et al., 2006; Aluri and Buttner, 2007). Based

on the presence of several conserved domains, AtTMT

proteins and the vacuolar glucose transporters are members

of the group of MFS-type sugar transporters (Buttner and

Sauer, 2000). Interestingly, all TMT proteins from various

plant species are characterized by a large hydrophilic loop.

This loop region is several times longer than the corre-

sponding domain of other related sugar transporters from

various organisms (Henderson, 1991; Wormit et al., 2006).

Furthermore, it contains several clustered negatively

charged amino acids (Figure S1) and multiple putative or

proven phosphorylation sites (Whiteman et al., 2008; Endler

et al., 2009). These peculiarities of the TMT loops indicate

interactions with kinases, and suggest that they are not pri-

marily required for sugar translocation but instead exhibit

regulatory functions.

No kinases that physically or functionally interact with

vacuolar carriers are currently known. We performed an

affinity chromatography-based approach to identify loop-

interacting proteins, and identified the kinase VIK (in addi-

tion to other proteins) as a possible AtTMT1-binding partner

(Table S1). VIK is a member of the family of mitogen-

activated protein triple kinases (MAP3K) (MAPK Group

Figure 7. Respiration of [14C]-glucose in wild-type (Wt) and vik plants.

Wt and vik mutant plants were grown for 12 days in liquid culture medium

without (a) or with additional glucose (5%) application 14 h prior to the start of

the experiment (b). Dark grey bars represent Wt samples, horizontally striped

bars represent vik1 samples, and diagonally striped bars represent vik2

samples. Respiratory activity was measured as the release of [14C]-CO2 based

on imported [14C]-glucose (Jung et al., 2009). Data represent the means of

four individual experiments, each with five replicate samples, � SE. Asterisks

indicate significant differences between the respective vik T-DNA mutants and

Wt (***P < 0.005, *P < 0.5) (Student’s t test).

Figure 8. Intracellular sugar compartmentation in Arabidopsis seedlings

grown in liquid culture.

(a) Plants were grown for 7 days in standard liquid culture medium.

(b) Plants were grown for 7 days in standard liquid culture medium to which

5% glucose was added 14 h prior to harvest.

Sugar compartmentation was analysed by non-aqueous fractionation. Glu-

cose concentrations in vacuoles, the cytosol and the plastids were deter-

mined, summed and set to 100% (total glucose). The glucose contents in the

respective compartments were calculated accordingly, and are given as the

percentage of total glucose. Glucose contents in the vacuole (dark grey bars),

the cytosol (white bars) and plastids (light grey bars) are shown. Data

represent means of three independent experiments, each with three replicate

samples, � SE. n.d., not detectable.

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ª 2011 The AuthorsThe Plant Journal ª 2011 Blackwell Publishing Ltd, The Plant Journal, (2011), 68, 890–900

2002). MAP3Ks are known to catalyse phosphotransfer to

upstream kinases in signal cascades, and are of fundamental

importance during cell development. Arabidopsis possesses

approximately 60 MAP3K-type proteins; however, the phys-

iological role of most of these kinases is not or only poorly

understood (MAPK Group 2002). VIK was previously iden-

tified as an interaction partner of the receptor-like kinase

VH1/BRL2, and was accordingly proposed to be part of a

signal cascade required for accurate vasculature formation

(Ceserani et al., 2009).

Bimolecular fluorescence complementation (Kerppola,

2008) was used to verify that VIK and the transporter

AtTMT1 exhibit close physical proximity in living cells

(Figure 2). A tight in vivo interaction of VIK and AtTMT1 is

further supported by a recent proteome study that identified

VIK in the tonoplast fraction of Arabidopsis leaf vacuoles

(Whiteman et al., 2008). Moreover, transcription of both

tmt1 and vik is high in leaves, and is stimulated by high

external sugar concentrations and in response to certain

abiotic stress factors (cold, drought and salt, Figure 1)

(Wormit et al., 2006). Thus, it is assumed that the corre-

sponding proteins, AtTMT1 and VIK, accumulate and coop-

erate under these conditions.

Using a phosphorylation assay, we demonstrated that

the recombinant AtTMT1 loop acts as substrate for VIK

(Figure 3). Furthermore, replacement of negatively

charged amino acids (aspartate cluster) in the middle of

the loop region by neutral ones reduced the physical and

functional interaction with VIK (Figure 4). Interestingly,

negatively charged domains related to the TMT1 aspartate

cluster (Figure S1) are rather rare but are present in MAP

kinases such as the human enzymes ERK, p38 and JNK

(Tanoue et al., 2000). These amino acid arrangements are

known as conserved docking domains, and act as docking

motifs for both upstream and downstream partner pro-

teins (Tanoue et al., 2000). Interestingly, site-directed

mutations in conserved docking domains impair interac-

tions with partner proteins (Figure 4, inset) (Tanoue et al.,

2000; Liu et al., 2006). Thus the aspartate cluster in the

AtTMT1 loop may represent the docking site for VIK

recognition and interaction. AtTMT2, AtTMT3 and TMTs

from other plants also possess comparable negatively

charged clusters near the centre of the loop (Figure S1),

and these proteins may therefore also be targets of VIK or

other kinases.

Interestingly, VIK possesses N-terminally located ankyrin

repeats and thus is characterized as a member of MAP3K

sub-group C1, which is the only MAP3K sub-group that

harbours ankyrin repeats (MAPK Group 2002). Ankyrin

repeats mediate protein–protein interactions in very diverse

families of proteins (Li et al., 2006). Thus, investigation of the

role of these repeats in the physiological interaction with

AtTMT1 or other tonoplast proteins will be an interesting

topic for future studies.

Our data show that VIK physical interacts with the AtTMT1

transporter at the vacuolar membrane (Figure 2), and that

the loop peptide is involved in substrate recognition and

functional interaction (Table S1, Figures 3 and 4). VIK-

dependent phosphorylation results in increased activity of

AtTMT1 (and probably of further TMTs) and stimulates

glucose import into intact vacuoles (Figure 5). The positive

impact of VIK on AtTMT1 activity is additionally supported

by the fact that the phenotypes of plants lacking VIK

resemble those of mutants with reduced sugar import

capacity caused by impaired TMT activity (Wormit et al.,

2006; Wingenter et al., 2010). Detailed analysis of the

physiological properties of vik loss-of-function mutants

(Figure S4) suggests that VIK, similar to the main sugar

transporters AtTMT1 and 2, plays an important role in

intracellular sugar distribution and homeostasis (Figures 6

and 8). In the presence of high sugar concentrations, vik

mutants show reduced growth (Figure 6b). Furthermore,

cold-stress induced glucose accumulation in leaves is

impaired when VIK is absent (Figure 6c).

In many respects, vik mutants phenotypically resemble

attmt1/2 mutants. However, there is one important physio-

logical difference. Compared to Wt plants, attmt1/2 mutants

exhibited higher respiration rates under standard conditions

(Wingenter et al., 2010), whereas significantly increased

respiration rates in vik mutants required pre-exposure to

high external sugar concentrations (Figure 7). This differ-

ence in respiration is in line with the proposed role of VIK in

stimulation of vacuolar sugar transport. In attmt1/2 mutants,

cytosolic glucose levels and thus mitochondrial glucose

respiration are already enhanced under standard conditions

because the dominant vacuolar glucose importers are

missing. In contrast, vik mutants lack the interacting kinase

but possess the complete set of sugar transporters and

therefore are able to import glucose into the vacuole.

However, application of high external glucose concentra-

tions requires enhanced import capacity to allow sugar

deposition into the vacuole. High sugar concentrations

always result in enhanced respiration; however, the increase

is higher in vik mutants than in Wt plants (Figure 7b). In

summary, our results suggest (i) that VIK is activated by high

cytosolic glucose concentrations, (ii) that activated VIK

phosphorylates the loop of AtTMT1, and (iii) that the

phosphorylated transporter has a higher transport capacity,

leading to increased vacuolar sugar accumulation. Accord-

ingly, we postulate that VIK controls sugar homeostasis and

intracellular partitioning by adjusting (accelerating) vacuolar

glucose uptake according to cellular demands. This demon-

stration that VIK (a member of the MAP3K family) phospho-

rylates and regulates a transport protein changes our view

regarding the well-known role of MAP3K, i.e. phosphoryla-

tion of upstream kinases.

The demonstrated physical and functional interaction

with the kinase VIK suggests that AtTMT1 is affiliated to

Regulation of vacuolar glucose transport 897

ª 2011 The AuthorsThe Plant Journal ª 2011 Blackwell Publishing Ltd, The Plant Journal, (2011), 68, 890–900

the small but increasing group of Arabidopsis transporters

that are regulated by phosphorylation. This group so far

comprises only plasma membrane-located proteins, and

includes the root potassium channel AKT1 (Geiger et al.,

2009a), the guard cell anion channels SLAC1 and SLAH3

(Geiger et al., 2009b, 2011), the sodium transporter SOS1

(Quintero et al., 2011) and the P-type ATPase (Fuglsang

et al., 2007). Phosphorylation-dependent regulation of an

MFS-type sugar transporter has never been demonstrated

previously (Chang et al., 2004). As several phosphorylated

carriers were identified in the tonoplast, we assume that not

only AtTMT1 but also other AtTMTs and probably other

vacuolar membrane proteins are functionally modified via

phosphorylation.

EXPERIMENTAL PROCEDURES

Plant material and growth conditions

Arabidopsis plants were cultivated on soil in a growth chamber at20�C and 125 lmol quanta m)2 sec)1 under a 10 h light/14 h darkcycle. Plants were watered at regular intervals. Analyses of leafmaterial were performed on plants that had grown for 35 days (leafstage 3.7; Boyes et al., 2001). Cultivation in liquid culture mediumwas performed as described previously (Scheible et al., 2004;Kirchberger et al., 2008).

cDNA clones, Northern blot analysis and quantitative

RT-PCR

Quantitative RT-PCR using oligonucleotides vik_s (5¢-ATGGCTCCTGAAGTATTCAAGC-3¢) and vik_as (5¢-TCTTGAGAATGTCCAGAAACGACG-3¢) was performed to evaluate the expressionpattern of VIK. Quantification of mRNA was performed as describedpreviously (Wingenter et al., 2010) using expression of EF-1a(At1g07930) as reference. Northern blot analysis was performed asdescribed by Linke et al. (2002).

Identification of vik T-DNA mutants

The Arabidopsis T-DNA insertion lines vik1 (SALK_1330372) andvik2 (SALK_002267) were provided by the Nottingham ArabidopsisStock Centre (http://arabidopsis.info). These mutants were analysedvia PCR for the absence of functional vik transcript using primersko1_s (5¢-GAAGGTGTCGCTGAGATTGAG-3¢) and ko1_as (5¢-GA-ATTGATGACTTTTTCCTCCG-3¢) for vik1, and ko2_s (5¢-CAAATCCGCTGCTCATAAATC-3¢) and ko2_as (5¢-ACCATTACCATCTCCTGAGGG-3¢) for vik2, and for presence of the T-DNA insertion using theT-DNA-specific primer LB335 (designed as recommended by theNottingham Arabidopsis Stock Centre) and the respective vik-specific primer.

Quantification of respiratory use of [14C]-glucose and

non-aqueous fractionation

To analyse the [14C]-glucose-driven respiratory activity, we pre-pared seedlings 30 min after the onset of illumination. Materialfrom Wt and vik mutants grown for 12 days in liquid culture med-ium was transferred into 2 ml reaction tubes filled with 0.1 ml of100 lM [14C]-glucose at a concentration of 0.4 lCi per 100 ll. At theinside at the top of this tube, a small 0.5 ml reaction vessel con-taining 50 ll of 1 N KOH was fixed with grease to allow 14CO2

absorption. The seedlings were allowed to float on the solution, andincubation was continued for 6 h in the dark. The reaction was

stopped by adding 100 ll of 2 N HCl with a syringe through theclosed lid. The hole in the lid was sealed with grease, and, aftersubsequent incubation for 10 h, the released radioactively labelledCO2 was quantified in a scintillation counter (Hurth et al., 2005).Non-aqueous fractionation of plant material was performed asdescribed previously (Wingenter et al., 2010).

Uptake of [14C]-glucose into isolated mesophyll vacuoles

Isolation of Arabidopsis mesophyll vacuoles and quantification of[14C]-glucose uptake was performed as described previously (Em-merlich et al., 2003; Wormit et al., 2006).

Escherichia coli-based recombinant synthesis

of the AtTMT1 loop peptide and VIK

The AtTMT1 loop coding sequence was amplified and modified(insertion of XhoI sites and stop codon) using primers loop-sense(5¢-GTGTTTTATTTGCTCGAGTCTCCTCGTTGGC-3¢) and loop-anti-sense (5¢-CAACCTCGAGTTAACGCTTAACACCAGGTTC-3¢). PCRproducts were inserted into an EcoRV-linearized pBluescript plas-mid (Agilent Technologies, http://www.genomics.agilent.com), ex-cised using XhoI and cloned into an XhoI-linearized pET16b plasmid(EMD Chemicals, http://www.emdchemicals.com). The vik codingsequence was amplified and modified (insertion of XhoI sites) usingprimers vik-sense (5¢-CTCGAGATGAGCTCCGATTCACCGG-3¢) andvik-antisense (5¢-CTCGAGTAATGAAGTGAATAAGCCCC-3¢).

Site-directed mutagenesis of the aspartate cluster

in the AtTMT1 loop

Aspartates in the aspartate cluster of the AtTMT1 loop werereplaced by asparagine residues. To do this, the loop-encodingsequence was modified by site-directed mutagenesis using aQuikchange site-directed mutagenesis kit (Agilent, http://www.agilent.com), and the first base in each triplet encoding aspartate,namely Asp354 (GAC), Asp356 (GAT), Asp357 (GAC), Asp361 (GAT),Asp362 (GAT), Asp366 (GAT), Asp367 (GAT), Asp368 (GAT), Asp369(GAC), Asp371 (GAC) and Asp373 (GAT) was substituted by anadenine (A).

Recombinant protein synthesis

For recombinant protein synthesis, all pET16b-based expressionplasmids were transformed into E. coli (Rosetta2, DE3 pLysS; http://www.emdchemicals.com). Overnight cultures were inoculated inTerrific Broth medium (Duchefa, http://www.duchefa.com). At anOD600 of 0.5–0.6, T7 RNA polymerase expression was induced byaddition of 1 mM IPTG. Cells were harvested by centrifugation for 10min at 5 000 g (4�C) 1 h post-induction. Cells expressing the AtTMT1loop were resuspended in buffer containing 1 mM EDTA, 15%glycerol, 10 mM Tris, pH 7.0, and VIK-containing cells were resus-pended in IMAC binding buffer (300 mM NaCl, 20 mM imidazole,50 mM NaPi, pH 8.0). Resuspended cells were frozen in liquidnitrogen. To induce autolysis, cells were thawed at 37�C, and 1 mM

phenylmethylsulfonyl fluoride and 1 lg/ml of DNase were added.Then the cell lysate was centrifuged for 15 min at 20 000 g (4�C).

IMAC purification of VIK

The supernatant of VIK-expressing bacterial cell lysate was incu-bated with Ni-Sepharose 6-FF (GE Healthcare, http://www.gehealthcare.com) for 20 min on a stirrer (47 rpm/min), andSepharose beads were loaded onto an empty chromatographycolumn containing a frit. To remove non-specifically bound pro-teins, the column was flushed with binding buffer (300 mM NaCl,20 mM imidazole, 50 mM NaPi, pH 8.0) and washing buffer (300 mM

898 Karina Wingenter et al.

ª 2011 The AuthorsThe Plant Journal ª 2011 Blackwell Publishing Ltd, The Plant Journal, (2011), 68, 890–900

NaCl, 60 mM imidazole, 50 mM NaPi, pH 8.0). VIK was eluted usingelution buffer (300 mM NaCl, 500 mM imidazole, 50 mM NaPi, pH8.0).

IMAC purification of the AtTMT1 loop protein,

denaturation and refolding

After centrifugation of the lysate of cells expressing the AtTMT1loop, sediments were washed three times for 20 min, using ‘inclu-sion body’ washing buffer (300 mM NaCl, 1 mM EDTA, 0.1% TritonX-100, 30 mM Tris, pH 8.0). The final sediment was resuspended inbinding buffer (see above) supplemented with 6 M urea, and cen-trifuged. The supernatant was incubated with Ni-Sepharose 6-FF for20 min, and beads were subsequently applied to an empty chro-matography column containing a frit. To remove weakly boundproteins, the column was flushed with binding buffer and washingbuffer, both supplemented with 6 M urea. Finally, the denaturedAtTMT1 loop protein was eluted using elution buffer plus 6 M urea.Refolding of the loop protein was induced by chromatographic ex-change of the urea-containing elution buffer with PBS (100 mM

Na2HPO4, 140 mM NaCl, 2.7 mM KCl, 1.6 mM KH2PO4).

Affinity chromatography and identification of AtTMT1

loop-interacting proteins

The refolded AtTMT1 loop peptide was transferred from PBS tobinding buffer by use of a PD10 column (GE Healthcare, http://www.gehealthcare.com) and subsequently coupled to Ni-Sepha-rose 6-FF. Leaves of 5-week-old Arabidopsis plants were ground inbinding buffer, and the resulting extract was filtered and centri-fuged. The supernatant was flushed over a chromatography columncontaining Sepharose to which the peptide had been coupled. Thecolumn was then flushed successively with binding and washingbuffer. Finally, the AtTMT1 loop and interacting proteins wereeluted with elution buffer, and proteins were precipitated by addingfour volumes of acetone followed by centrifugation. The sedimentwas resuspended in SDS–PAGE loading medium, and proteins wereseparated by chromatography. After Coomassie staining, the wholelane was excised and sliced into small horizontal pieces.

Gel slices were washed twice with 50 ll of medium A (50 mM

ammonium bicarbonate) followed by 50 ll of medium B (50 mM

ammonium bicarbonate, 50% acetonitrile). For carbamidomethyla-tion, slices were incubated with 10 mM dithiothreitol in mediumA (30 min) and subsequently with 5 mM iodoacetamide in mediumA (30 min). Then the washing step using media A and B wasrepeated twice to remove excess reagents. The gel pieces weredried under vacuum and re-hydrated using 6 ll trypsin solution(12.5 ng ll)1 in medium A), and digested at 37�C.

The resulting peptides were eluted using 25 ll 5% formic acid andsubjected to nano-LC-MS/MS using a Dionex Ultimate 3000 nano-HPLC system coupled online to a QStar XL hybrid tandem massspectrometer (ABSciex GmbH, http://www.absciex.com/). MS/MSspectra were searched against the Arabidopsis InformationResource database (http://www.arabidopsis.org) using Mascot(version 2.2, http://www.matrixscience.com). Proteins with at leasttwo as significantly scored and manually verified peptide MS/MSspectra were considered.

In vitro phosphorylation assay of the AtTMT1 loop

The AtTMT1 loop peptide and VIK were transferred to kinase buffer(Hastie et al., 2006) using PD10 columns. The proteins were mixed,and the reaction was started by adding 0.2 mM [c-32P]-ATP (Hart-mann Analytic, http://www.hartmann-analytic.de), and allowed toproceed at 30�C. The reaction was terminated by addition ofSDS–PAGE loading medium and immediate heating to 99�C. After

separation via SDS–PAGE, the radioactive labelling was visualizedand quantified with a phosphor imager (Packard B431200, http://www.matrixscience.com).

Surface plasmon resonance (SPR) analysis

The SPR interaction studies were performed using a Biacore 2000(Biacore Life Sciences, http://www.biacore.com). Purified AtTMT1loop protein and the Asp cluster mutant were immobilized as theligand to the detection channel on a CM5 chip (Biacore Life Sci-ences, http://www.biacore.com) in BIA buffer (10 mM HEPES,150 mM NaCl, 3 mM EDTA, 0.005% v/v Polysorbat 20, pH 7.4).Purified recombinant VIK protein was perfused over the referencechannel and over the detection channel as the analyte. Bindingwas measured in real time using BSA as a control protein.

Bimolecular fluorescence complementation (BiFC) analysis

The coding sequences of VIK and MEKK1 (as a control) were fusedto the C-terminal half of YFP (vik::yfpCT and mekk1::yfpCT) and thecoding sequence of AtTMT1 was fused to the N-terminal half of YFP(tmt1::yfpNT). Using the USER technique (Nour-Eldin et al., 2006),constructs were cloned into pSAT-derived vectors (Geiger et al.,2009b, 2011) for transient expression under the control of a 35Spromoter. For documentation of BiFC within protoplasts and vacu-oles, images were taken using a confocal laser-scanning micro-scope (Leica TCS SP5; http://www.leica-microsystems.com).

ACKNOWLEDGEMENTS

Work in the laboratories of H.E.N., J.R., R.H. and D.G. was financiallysupported by the Deutsche Forschungsgemeinschaft (FOR1061 andGE2195/1-1). We thank Ilka Haferkamp (Plant Physiology, Universityof Kaiserslautern, Germany) for critical reading our manuscript.H.E.N. received additional support from the Federal State ofRhineland-Palatinate (Landesschwerpunkt Membrantransport,Research Initiative Membrane Biology).

SUPPORTING INFORMATION

Additional Supporting Information may be found in the onlineversion of this article:Figure S1. Schematic view of the structure of AtTMT-type carriers.Figure S2. Purification of recombinant AtTMT1 loop proteins andrecombinant VIK.Figure S3. IMAC-based affinity chromatography of putative AtTMT1loop-interacting soluble proteins from Arabidopsis leaves.Figure S4. Molecular characterization of vik T-DNA insertion lines.Figure S5. Fresh weight of wild-type and vik T-DNA plants grown inliquid culture.Table S1. Putative interaction partners of the recombinant AtTMT1loop protein identified by mass spectrometry.Please note: As a service to our authors and readers, this journalprovides supporting information supplied by the authors. Suchmaterials are peer-reviewed and may be re-organized for onlinedelivery, but are not copy-edited or typeset. Technical supportissues arising from supporting information (other than missingfiles) should be addressed to the authors.

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