Download SUC1 and SUC2: two sucrose transporters from Arabidopsis

The Plant Journal (1994) 6(1), 67-77
SUC1 and SUC2: two sucrose transporters from
Arabidopsis thaliana; expression and characterization
in baker's yeast and identification of the histidinetagged protein
Norbert Sauer* and JLirgen Stolz
Lehrstuhl f~r Zellbiologie und Pflanzenphysiologie,
Universit~t Regensburg, 93040 Regensburg, Germany
Summary
An important, most likely essential step for the long
distance transport of sucrose in higher plants is the
energy-dependent, uncoupler-sensiUve loading into
phloem cells via a sucrose-H+ symporter. This paper
describes functional expression in Saccharomyces
cerevisiae of two cDNAs encoding energy-dependent
sucrose transporters from the plasma membrane of
Arabidopsis thaliane, SUCl and SUC2. Yeast cells
transformed with vectors allowing expression of
either SUC1 or SUC2 under the control of the promoter of the yeast plasma membrane ATPase gene
(PMA1) transport sucrose, and to a lesser extent also
maltose, across their plasma membranes in an
energy-dependent manner. The Ku-values for sucrose
transport are 0.50 mM and 0.77 mM, respectively, and
transport by both proteins is strongly Inhibited by
uncouplers such as carbonyl cyanide m-chlorophenylhydrazone (CCCP) and dinitrophenol (DNP), or
SH-group inhibitors. The VMAXbut not the Ka-values
of sucrose transport depend on the energy status of
transgenic yeast cells. The two proteins exhibit different patterns of pH dependence with SUC1 being much
more active at neutral and slightly acidic pH values
than SUC2. The proteins share 78% identical amino
acids, their apparent molecular weights are 54.9 kDa
and 54.5 kDA, respectively, and both proteins contain
12 putative transmembrane helices. A modified SUCIHis6 cDNA encoding a histidine tag at the SUC1
C-terminus was also expressed in S. cerevisiae. The
tagged protein is fully active and is shown to migrate
at an apparent molecular weight of 45 kDa on 10%
SDS--polyacrylamide gels.
Received 14 January 1994; revised 18 March 1994; accepted 30 March
1994.
*For correspondence (fax + 49 941 943 3352).
Introduction
The primary products of photosynthetic CO2 fixation are
exported from the chloroplast into the cytosol by the
triosephosphate translocator (FI0gge et aL, 1989). There
they serve for the synthesis of sucrose, the compound
which is used by most higher plants for long distance
transport of assimilated carbon. This transport occurs in
the sieve elements of the phloem and is necessary for the
carbon supply of photosynthetically inactive, often nongreen parts of plants such as roots, fruits, flowers, and
very young leaves. Phloem loading of sucrose has been
shown to be catalyzed by active, energy-dependent
transporters (Dale, 1987; Delrot and Bonnemain, 1981;
Komor et al., 1977;) which are presumably located in
the plasmalemma of the companion cells and/or the
plasmalemma of the actual transporting sieve elements.
Data obtained with transgenic plants expressing yeast
invertase in the leaf apoplast (von Schaewen et aL, 1990;
Sonnewald et aL, 1991) support this model of apoplastic
loading. High levels of external invertase hydrolyze
sucrose unloaded into the apoplast by the mesophyll cells,
thus causing reduced phloem loading, poor root growth,
and a concomitant accumulation of carbohydrates in the
leaf mesophyll.
Unloading of phloem in the sink tissues seems to occur
symplastically (Turgeon, 1989) or apoplastically (Wright
and Oparka, 1989). In the latter case unloaded sucrose is
hydrolyzed by cell wall-bound invertases and the resulting
monosaccharides are transported into the sink tissues by
specific transporters. Direct evidence for apoplastic
unloading has been obtained from recent work on a corn
mutant lacking cell wall invertase (Miller and Chourey,
1992). The mutation severely affected starch accumulation in kernels unable to hydrolyze unloaded sucrose.
During the last years genes or cDNAs for several transporters have been cloned, which are believed to be
involved in the various loading or unloading steps mentioned above. The STP1 gene from Arabidopsis thaliana
(Sauer et aL, 1990b) and the MST1 gene from Nicotiana
tabacum (Sauer and Stadler, 1993) encode highly similar
monosaccharide-H+ symporters. The latter one is
expressed almost exclusively in tobacco sink tissues and
data from Arabidopsis show that large gene families
encoding putative monosaccharide transporters are found
in higher plants (Sauer and Tanner, 1993). The function of
6?
68
Norbert Sauer and J(Jrgen Stolz
these transporters was confirmed by functional expression in Schizosaccharomyces pombe or Saccharomyces
cerevisiae (Sauer et aL, 1990b; Sauer and Stadler, 1993).
Riesmeier et aL (1992) reported the complementation
cloning in bakers yeast of the pS21 sucrose transporter
from spinach, an energy-dependent, active transporter of
the plasmalemma. The pS21 protein has a molecular
mass of 55 kDa and 12 putative transmembrane helices,
which is almost identical to what has been published for
the monosaccharide-H ÷ symporters from higher plants
(Sauer et aL, 1990b; Sauer and Stadler, 1993). Lemoine
et aL (1989) published an apparent molecular mass of
42 kDa for a sucrose transport protein from sugar beet
which again is identical to apparent molecular masses
reported for higher plant monosaccharide transporters
(Sauer and Stadler, 1993). Sequence comparison, however, does not reflect a high similarity between plant
mono- and disaccharide transporters.
All sucrose transporter activities studied so far seem to
have several features in common: their KM-values are in
the millimolar range, their pH optima are in the range of
pH 5-6, they are sensitive to SH group inhibitors, and they
are highly sensitive to uncouplers such as carbonyl
cyanide m-chlorophenylhydrazone (Buckhout, 1989;
Bush, 1990; Delrot, 1989; Riesmeier et a/., 1992).
It was not known whether higher plants possess only
one single gene for a sucrose transporter or whether two
or more transporters are simultaneously active to insure
optimal phloem loading, one of the central steps in carbohydrate partitioning. Furthermore it was not understood
whether loading of sucrose from the apoplastic space and
unloading of sucrose into the apoplastic space are catalyzed by one and the same transporter, e.g. regulated by
apoplastic pH, or whether different sucrose transporters
are needed for these two transmembrane steps.
In this paper we describe the identification of cDNA
clones encoding two sucrose transporters from
A. thaliana, SUC1 and SUC2, and their functional expression and characterization in S. cerevisiae. We also report
on the construction of an active, histidine-tagged SUC1
sucrose transporter and the identification of the tagged
protein on SDS-polyacrylamide gels.
Results
Isolation and characterization of SUC1 and SUC2
cDNA c/ones
Screening of an Arabidopsis cDNA library with radiolabeled DNA of the spinach sucrose transporter pS21
(Riesmeier et aL, 1992) resulted in 16 positive clones
(Z2001-2016). Some of the clones were lost during purification of the plaques, some had only very short inserts.
Only six clones were subcloned into the EcoRI site of
pUC19 and sequenced. Two of these clones did not show
any similarity to the spinach sucrose transporter. The
remaining four clones represented cDNAs which were
homologous to the pS21 sequence; however, the
sequences of clones pTF2011 and pTF2014 were clearly
different from the sequences of pTF2013 and pTF2015,
suggesting the existence of at least two putative sucrose
transporters in A. thaliana, pTF2011 was the only fulllength cDNA. To obtain a full-length clone for the second
group of cDNAs the library was rescreened with a 120 bp
EcoRI-Psll fragment from the 5'-end of pTF2013. Out of
many positive clones pTF2035 was the longest one and
full length. The gene-encoding mRNAs cloned in pTF2011
and pTF2014 was named SUC1, the gene for mRNAs
cloned in pTF2013, pTF2015, and pTF2035 was named
SUC2. Sequences of the full-length cDNAs are given in
Figure 1.
The longest open reading frame for SUCt is 1539 bp
long encoding a protein of 513 amino acid residues and a
calculated molecular weight of 54.9 kDa; the longest open
reading frame for SUC2 is 1536 bp long encoding a
protein of 512 amino acid residues and a calculated
molecular weight of 54.5 kDa. DNA sequences upstream
of the start ATGs of SUCI and SUC2are rich in A and T,
do not contain other ATG codons, and in the case of SUCI
possess a TAA stop codon within the SUC1 translation
frame. This all confirms the start ATGs given in Figure 1.
The sequence of SUCI ends with 20 A, the sequence
of SUC2 with 4 A, which could be the start of a longer
poly(A) tail.
The SUC1 and SUC2 proteins share a high number of
identical amino acids both with each other and with the
spinach pS21 sucrose transporter (Figure 2). There is a
strong structural similarity between sucrose transporters
and monosaccharide transporters since both groups of
membrane proteins have 12 putative transmembrane
helices (Sauer and Stadler, 1993; Sauer eta/., 1990b).
This structural similarity, however, does not continue on
the amino acid level. As shown in Figure 2, the three
sucrose transporters share between 65 and 77%
identical amino acids, the MST1 monosaccharide transporter from tobacco and the STP1 monosaccharide
transporter from Arabidopsis share 79%. A comparison
between mono- and disaccharide transporters gives only
about 20% identical amino acids, which is close to background (Figure 2). Nevertheless, there are a few regions
which are conserved between mono- and disaccharide
transporters, such as the short cytoplasmic loops between
the putative transmembrane helices 2 and 3 (loop 2/3) and
the cytoplasmic loops between the helices 8 and 9 (loop
8/9) of the different transporters (Table 1). These loops
always contain a short conserved sequence starting with a
small, neutral amino acid followed by two positively
charged amino acids (consensus sequence: AGRR).This
Arabidopsis
s u c r o s e transporters
69
Figure 1. Sequences and restriction maps of the EcoRI inserts of pTF2011 and pTF2035 encoding the A. tha/iana sucrose transporters SUC1 (a) and SUC2
(b), respectively.
Asparagine residues marked with an asterisk are part of a consensus sequence for possible N-glycosylation (N-X-S or N-X-T). Sequences also contain the
EcoRI linkers used for construction of the library (lower case letters) and the restriction sites for BamHI, EcoRI, EcoRV, Hindlll, Pstl and Scal. Boxed
sequences give the putative transmembrane helices.
70
Norbert Sauer and JEirgen Stolz
95 Identity
SUC1 SUC'2 pS21 STP1 MST1
SUC1
100
SUC2 85.5
77.6 ~64.4 21.9
100
65.9
20.0
18.7 20.3
pS21
79.9 81.8 1001 19.2
19.6
STPl
53.2 "45.9 47.2
100
79.5]
88.6
1001
MST1 52.6
49.8 46.6
Figure 2. Percentageof similarand identicalaminoacid residuesshared
by differenttransportersfor mono-and disaccharides.
Sequencecomparisonswere performedwith the programBESTFITof the
UWGCG DNA-analysissoftware(gapweight,3.0;gap lengthweight,0.1).
Values obtained from comparisonsof disaccharidetransportersonly or
monosaccharidetransportersonly are boxed (large box and small box,
respectively).
Table 1. Comparison of conserved sequences in the first and
second half of several transporters
Putative position
in the topological
model
Protein
Amino acid sequence and
position in the primary
sequence
Loop 2/3
SUCI:
SUC2:
pS21:
STPI:
MSTI:
96
95
101
106
106
K
R
R
K
K
Loop 8/9
SUCI:
SUC2:
pS21:
STPI:
MSTI:
356
352
362
344
342
Loop 6/7
SUCI:
SUC2:
pS21:
STPI:
MSTI:
C-terminus
SUCI:
SUC2:
pS21:
STPI:
MSTI:
F
F
F
F
L
G
G
G
G
G
RR
RR
RR
RL
RL
101
100
106
111
111
Wl G
Wl G
G L A
R WG
K L G
R KL
R KL
R MV
RRF
R RF
361
357
367
349
347
249
248
254
220
218
Q
P
Q
L
L
WS
WT
I T
V L
F L
P
P
I
P
P
PP
EP
DE
DT
ET
254
253
259
225
223
496
495
509
472
470
L
L
L
I
F
P
P
P
F
F
P
P
P
P
P
PP
PP
PP
ET
TT
501
500
514
477
475
$
S
S
L
L
R
R
R
R
R
sequence at this position has been discussed previously
as being typical for a superfamily of transporters (Griffith et
aL, 1992; Maiden et aL, 1987). Following transmembrane
helices 6 and 12 (loop 6/7 and C-terminus) another pair
of conserved sequences has been identified in monosaccharide transporters. At the same positions a pair of
conserved sequences is also found in the Arabidopsis
sucrose transporters; these sequences, however, are
different from those found in STP1 and MST1 (Table 1).
For various monosaccharide transporters it has been
postulated that their present structure with 12 transmembrane segments might have evolved, by gene duplication
and fusion of an ancestral transporter gene which only had
six transmembrane helices (Griffith et aL, 1992; Maiden et
aL, 1987; Sauer and Tanner, 1993). This is confirmed by
several striking sequence conservations between the first
and the second half of these monosaccharide transporters. The data presented in Table 1 show that such
conserved regions are also found in SUC1, SUC2, and to
a lesser extent also in pS21 sucrose transporters.
There is one consensus sequence for N-glycosylation
found in SUC1 (asparagine 155) and two consensus
sequences are found in SUC2 (asparagine residues 154
and 402; Figure 1). SUC2-Asn402 is located in a putative
extracellular loop and might therefore be a candidate for
N-glycosylation in the endoplasmic reticulum. SUC1Asn155 and SUC2-Asn154 are located at identical
positions within longer highly conserved regions of the
two proteins and the consensus sequence is also
conserved in the spinach pS21 sucrose transporter. The
location of this N - N - T consensus sequence within a
putative transmembrane helix, however, suggests that
this asparagine is not glycosylated in the mature protein.
The calculated isoelectric points of the SUC1 and
SUC2 sucrose transporters are 9.25 and 9.55, respectively. These values are in the same range as those
determined for the spinach pS21 sucrose transporter and
the STP1 and MST1 monosaccharide transporters (8.81,
9.49 and 9.26, respectively).
Heterologous expression of SUC 1 and SUC2 in baker's
yeast
For functional investigations SUC1 and SUC2 had to be
expressed in yeast cells which had been shown to be
excellent expression systems for plant sugar transporters
before (Riesmeier et aL, 1992; Sauer and Stadler, 1993;
Sauer et aL, 1989, 1990a, 1990b). Baker's yeast grows
very efficiently on sucrose as sole carbon source due to
secreted invertase which hydrolyzes extracellular
sucrose, and the resulting monosaccharides are taken up
by a large set of various transporters (Bisson et aL, 1993).
The uptake of sucrose by specific transporters has
also been reported (Santos et aL, 1982). Both extracellular sucrose hydrolysis by invertase and direct transport of sucrose are strongly repressed during growth in the
presence of high concentrations of D-glucose. Thus,
glucose-grown wild-type cells of S. cerevisiae should be
an excellent system for the characterization of plant
sucrose transporters.
Arabidopsis sucrose transporters
Transformation of S. cerevisiae was performed
using the newly constructed NEV-E Escherichia coil~
S. cerevisiae shuttle vector which has a promoter/terminator box of the S. cerevisiae plasma membrane H*-ATPase
PMAI gene containing a unique EcoRI cloning site
(Figure 3). This vector allows direct insertion of SUC1 and
SUC2 cDNAs. The PMA 1 promoter was chosen because
it is very active in glucose-grown yeast cells, and the H÷ATPase is one of the most prominent proteins of the
plasma membrane.
Figure 4 shows that sucrose uptake into S. cerevisiae
control cells grown on 2% D-glucose is negligible. Cells
expressing either SUC1 or SUC2, however, transport
14C-labeled sucrose at high rates indicating that these two
proteins are indeed transporters for the disaccharide
sucrose. To exclude the unlikely possibility that this transport is due to secreted invertase activity and subsequent
uptake of labeled monosaccharides, transport experiments were repeated in the presence of a large excess of
unlabeled D-glucose (10 mM) added to the uptake test
1 rain prior to the labeled sucrose (Figure 4). The results
clearly show that an excess of D-glucose does not have an
inhibiting effect but rather a stimulating effect on the determined uptake rates by transgenic yeast cells. Thus, cell
wall invertase activity can be excluded.
In order to study the stimulating effect of D-glucose on
sucrose transport apparent VM~X and KM-Values for
sucrose uptake by the SUC1 and SUC2 transporters were
compared in transformed yeast cells, both in the presence
and absence of D-glucose. The KM-values determined are
in the range of 0.45-0.77 mM and are in good agreement
71
with values published for sucrose transporters from other
organisms (Table 2; Buckhout, 1989; Delrot and
Bonnemain, 1981 ; Komor, 1982; Riesmeier et a/., 1992).
The effects of D-glucose on the apparent KM-values, however, are neglegible and cannot explain the increased
uptake rates. In contrast the apparent VMAXof sucrose
transport is increased three- and six-fold for SUC1 and
SUC2, respectively.
Taken together all these results suggest that uptake of
sucrose by SUC1 and SUC2 is energy dependent and that
in cells grown on D-glucose the machinery for sucrose
metabolism is not sufficiently active to allow maximal
energization of the yeast plasma membrane; addition of
the optimal carbon source D-glucose energizes the
plasma membrane much better resulting in increased
transport rates for sucrose with little or no influence on the
KM-Values. Similar conclusions were drawn by Bush
(1990) from experiments performed with plasma membrane vesicles from sugar beet. In this case changes of
the membrane potential Au/influenced only the VMAXand
not the KM.
.~ 1o
,o
S~tJCl
/"
9
7
|,
4
EcoRI
1
O-
1
2
4
6
8
10
o
o 2
4
6
8 !o
time [mini
Rgure 4. Uptake of sucrose into transgenic S. cerevisiaecells.
Uptake of sucrose into S. cerevisiae cells transformed with NEV2011R
(SUC1) or NEV2035R (SUC2) in the presence (e) or absence (O) of 10
mM o-glucose. (11) Shows sucrose uptake into control cells transformed
with NEV2011F or NEV2035F (SUC1 or SUC2 in antisense orientation).
The sucrose concentration was 1 mM in all experiments, the pH was 5.5.
Table 2. KMand VMAXvalues of the SUC1 and SUC2proteins for
sucrose transport at pH 5.5
No glucose
10 mM glucose
KM
VM~X*
450 I~M
45.4
500 I~M
153.8
KM
VMAX*
*llmol h-1 x g fresh weight.
530 pM
12.0
770 ~M
76.9
SUC 1
Figure 3. The E. coNS. cerevisiaeshuttle vector NEV-E.
The E. coNS. cerevisiae shuttle vector NEV-E was used for expression of
SUCt and SUC2 proteins in baker's yeast under the control of the S. cerevisiae PMA1 promoter. In the NEV-N and NEV-X vectors the unique EcoFII
cloning site is replaced by a Nott or Xhol site, respectively.
SUC2
72
Norbert Sauer and JOrgen Stolz
Substrate specificity, and sensitivity to inhibitors and
pH dependence
To determine the substrate specificities of the SUC1 and
SUC2 proteins transport of 14C-labeled sucrose was
studied in the presence of various other di- and trisaccharides that might be potential substrates for a
sucrose transporter. Only maltose inhibits the transport of
sucrose, whereas neither lactose nor the trisaccharide
raffinose have any influence on the transport rates
(Table 3). The glucose derivates cc-phenylglucoside and
~-phenylglucoside both inhibit transport of sucrose by
SUC1 and SUC2 which is in clear contrast to what has
been published by Hecht et aL (1992). The authors
showed a competitive inhibition of sucrose transport by
~-phenylglucoside but not by ~-phenylglucoside and
discussed a central role for the ¢z-glycosidic linkage in
substrate recognition by the sucrose symporter.
Protonophores such as dinitrophenol or carbonyl
cyanide m-chlorophenyl-hydrazone (CCCP) inhibit sucrose transport catalyzed by the SUC1 and SUC2 proteins
(Table 4) confirming the energy dependence of the two
transporters. Both transporters are also sensitive to SH
group inhibitors such as N-ethylmaleimide (NEM) or
p-chloro-mercuriphenylsulphonic acid (PCMPS) which
has already been described by various authors (Table 4;
Delrot and Bonnemain, 1981 ; Williams et aL, 1992).
Whereas the substrate specificities and the sensitivities to certain inhibitors seem to be the same for the SUC1
Table 3. Inhibition of SUC1 and SUC2 sucrose transport by other
sugars
and SUC2 proteins, their activities at different pH values
clearly differ (Figure 5). SUC1 is equally active at pH 5 or 6
and the relative activity decreases to only about 50%
when the pH is increased to 7. SUC2 is much more active
at acidic pH values with a loss of 80% of the relative activity when the pH is shifted from 5 to 6. At pH 7 SUC2 is
almost completely inactive. Both types of pH dependence
have already been described for other sucrose transporters, but always just for one specific transporter from
one specific plant: a protein studied in plasma membrane
vesicles from sugar beet (Bush, 1990) has a pH dependence which is very similar to what is found for SUC1 ; the
pH dependence of the pS21 sucrose transporter from
spinach (Riesmeier et aL, 1992) is very similar to that of
SUC2.
Tissue specificity of the expression of SUC1 and SUC2
Northern blot analyses were performed to see in which tissues mRNAs of SUCI and SUC2can be detected (Figure
6). Total RNA was isolated from different tissues of
A. thaliana plants, separated on agarose gels, and
hybridized to 32p-labeled sequences from the 3'-ends of
SUCI and SUC2 cDNA clones pTF2024 and pTF2013,
respectively. The highest levels of SUCI and SUC2
mRNA expression were found in the leaves of Arab#
dopsis plants, with no significant difference between
100
% activity
Sugar added (mM)
None
Maltose (2)
Maltose (10)
Lactose (10)
Raffinose (10)
c~-phenylglucoside (1)
~-phenylglucoside (1)
SUC1
SUC2
100
76
33
95
106
18
28
100
74
34
108
93
20
28
Table 4. Sensitivity of SUC1 and SUC2 to inhibitors
% activity
Inhibitor added (mM)
None
N-ethylmaleimide (100)
PCMPS (100)
Dinitrophenol (50)
CCCP (50)
0
SUC1
SUC2
100
100
41
44
53
5
61
49
57
4
5
6
7
pH
Figure 5. pH dependance of sucrose transport in transgenic yeast cells
expressing either SUC1 (O) or SUC2 (0).
Measurements were performed at 1 mM sucrose in 25 mM NaPO4 buffer
pH 5, 6 or 7; all experiments were performed in the presence of 10 mM
D-glucose. Experiments were repeated twice giving identical results.
Arabidopsis sucrose transporters
young and old rosette leaves. A similar level of SUCl
mRNA was seen in roots, a tissue showing hardly any
expression of SUC2. Reduced levels of both SUCl and
SUC2 mRNAs were found in flowers; hardly any signal
could be detected in RNAs isolated from stems.
73
The tagged SUC1-His6 protein could be identified on
10% SDS-polyacrylamide gels when dodecyl-~-D-maltoside (DDM) extracts from a total membrane fraction of
SUC1 -His6 expressing yeast cells were allowed to bind to
a Ni+-NTA matrix, washed with increasing concentrations
Identification of the histidine-tagged protein by
SDS-PAGE
To obtain biochemical information on one of the two SUC
proteins the cDNA clone pTF2011 (SUC1) was modified in
such a way that the resulting protein was elongated by five
additional histidine residues at the C-terminus. Since the
last amino acid residue in the wild-type SUC1 protein is
histidine (Figure 1), the modified SUC1 protein ends with
six histidine residues in a row (SUC1-His6). This so-called
histidine tag (Hochuli et aL, 1988) has been used by other
authors for the purification of recombinant membrane proteins using the specific binding of the histidine tag to Ni 2÷nitrilotriacetic acid (NTA) columns (Loddenkotter et aL,
1993; Waeber etaL, 1993; ). After introduction of the additional sequences into the SUCl open reading frame the
resulting construct was ligated into the NEV-E vector
to allow functional tests of the recombinant protein in
S. cerevisiae. Figure 7(a) shows sucrose transport by
S. cerevisiae cells expressing SUC1 -His6-mRNA either in
sense or in antisense orientation. It is obvious that both
activity and energy dependence (= glucose stimulation) of
the tagged protein (mRNA in sense orientation) are similar
to that of the wild-type SUC1 protein (Figure 4). There is
no detectable sucrose transport in cells expressing SUC1His6 mRNA in antisense orientation nor does glucose
stimulate the transport of sucrose into control cells
(Figure 7a).
Figure 6. Northern blot analyses of tissue-specific expression of SUC1
and SUC2 mRNAs.
Twenty micrograms of total RNA isolated from different tissues were
loaded per lane: yL, young rosette leaves; mL,mature rosette leaves; St,
stems; Ro, roots; FI, flowers. The size of the detected mRNA was 2 kbp for
both genes. Transcript lengths were determined by comparison with
ribosomal RNAs.
(a)
6
-
SUC1-His6
3
0
0
2
4
6
8
10
time [min]
Figure 7. Transport activity of the SUC1 sucrose transporter tagged with
five additional C-terminal histidine residues (SUC1-His6) and identification
of the tagged protein by purification on Ni*-NTA agarose.
(a) Uptake of sucrose at an external pH of 5.5 in the presence (O) or
absence (©) of 10 mM D-glucose by yeast cells expressing the SUC1-His6
protein (NSY2011H6R), and uptake by control cells in the absence of
D-glucose (n); addition of 10 mM D-glucose to control cells has no effect on
sucrose uptake (data not shown).
(b) Silver stained SDS-polyacrylamide gel showing the purification of
SUC1-His6 from dodecylmaltoside solubilized membrane proteins of
NSY2011H6 with Ni*-NTA agarose: flow through (lane 1) and eluates
obtained with buffer E (lane 2), 10 mM (lane 3), 20 mM (lane 4), 30 mM
(lane 5) and 100 mM imidazole (lane 6). Lane 7 shows the 100 mM
imidazole eluate obtained from dodecylmaltoside solubilized total
membranes of control cells (JSY301 = RS453 transformed with the NEV-E
shuttle vector) which were treated exactly the same way. Ten microliters of
each fraction were separated on the SDS gel.
74
Norbert Sauer and JOrgen Stolz
of imidazole, and finally eluted from the column with
100 mM imidazole (Figure 7b). The SUC1-His6 protein
corresponds to the prominent band at 45 kDa in lane 6 of
the protein gel in Figure 7b. The protein is eluted to some
extent already during the 20 mM and 30 mM imidazole
washes (lanes 4 and 5) but most of the protein is eluted
during the 100 mM imidazole washing step. In DDM
extracts of identically treated control cells transformed
with the NEV-E expression vector alone this band is
missing (lane 7).
Discussion
Full-length cDNAs encoding two sucrose transporters
from A. thaliana were cloned and compared with
sequences from other mono- and disaccharide transporters (see results). The data suggest that both types of
plant transporters, despite their low overall similarities
(Figure 2), may have evolved from a common ancestral
gene probably coding for a protein with only six transmembrane helices (Maiden et aL, 1987; Marger and Saier,
1993).
Purification of a histidine-tagged SUC1 protein (SUC1His6) from transgenic S. cerevisiae allowed for the first
time direct identification of a sucrose transporter on 10%
SDS-polyacrylamide gels. The apparent molecular weight
of 45 kDa is very close to the apparent molecular weight of
42 kDa suggested by Lemoine et aL (1989) from immunological studies. Unpublished work from our own group
on the glucose-H + symporter from Chlorella (Sauer and
Tanner, 1989) showed that the molecular weight on 10%
SDS gels was increased by 2 kDa upon the addition of
six C-terminal histidines suggesting that the apparent
molecular weight of the untagged SUC1 protein is about
43 kDa. This difference of 12 kDa between the molecular
weight calculated from the DNA sequence (55 kDa) and
the one determined from SDS gels (43 kDa) compares
well with the results from plant monosaccharide transporters such as the tobacco MST1 transporter where the
difference is 16 kDa (58 kDa calculated and 42 kDa on
SDS gels).
Both SUC1 and SUC2 are active, energy-dependent,
and probably H+ symporting sucrose transporters as can
be seen from the results obtained with transgenic yeast
(Figures 4 and 7a). Cells grown on p-glucose as sole carbon source repress the expression of both the transporter
and metabolic enzymes for sucrose. As a consequence,
sucrose transported into the cells by SUC1 or SUC2 is
metabolized only to a limited extent, not allowing optimal
energization of the plasma membranes. Addition of
D-glucose, which is taken up by glucose uniporters,
supplies the cells with enough energy for optimal sucrose
transport by a putative sucrose-H* symporter (Table 2).
This confirms data from many other laboratories postu-
lating that sucrose is transported together with protons
(Buckhout, 1989; Delrot and Bonnemain, 1981; Komor el
al., 1977; and others). Sensitivities of SUC1 and SUC2 to
SH group inhibitors and uncouplers are more or less
identical to what has been published for the spinach pS21
sucrose transporter and for other plant sucrose transporters studied in planta (Delrot, 1989, for review;
Riesmeier et al., 1992). The strong inhibitory effect of
uncouplers such as CCCP also points towards a sucroseH+ symport by SUC1 and SUC2.
Studies on the substrate specificities of the two
Arabidopsis transporters show that both proteins accept
sucrose and maltose but neither lactose nor the trisaccharide raffinose. Both a-phenylglucoside and ~-phenylglucoside inhibit the transport of sucrose very efficiently
at concentrations of 1 mM. With the exception of the
relatively strong inhibition by I]-phenylglucoside all other
specificity data agree with published data on other
sucrose transporters (Delrot, 1989).
Transport rates in the expression system described
using S. cerevisiae and the NEV-E shuttle vector with the
yeast PMA 1 promotor are in the same range as the rates
described for plant monosaccharide transporters
expressed in Schizosaccharomyces pombe (Sauer et
al., 1990a, 1990b) or in S. cerevisiae (Sauer and Stadler,
1993) reaching maximal rates of 80-150 I~mol h-~ x g
fresh weight (Table 2) and values of 50-100 iJmol h-1 × g
fresh weight during standard uptake conditions with 1 mM
sucrose under energized conditions. These values are
also comparable with sucrose transport rates described in
the literature which are between 10 and 500 I~mol h-~ x g
fresh weight (for review see Komor, 1982). The differences in transport rates published for the spinach pS21
sucrose transporter (about 2 p~mol h-~ x g fresh weight)
and the Arabidopsis SUC1 and SUC2 transporters do not
reflect different activities in the plant, since the pS21 transporter reaches similar rates when tested in the expression
system which was used for SUC1 and SUC2 (data not
shown).
It seems that the substrate specificities and inhibitor
sensitivities of the various sucrose transporters described
from different plants are all comparable regardless of
whether they were studied in vesicles, whole plant cells, or
in a heterologous system. There are, however, differences
in the pH dependences of the sucrose transporters
published for different organisms. The sucrose transporter
from sugar beet studied by Bush (1990) is fully active
between pH 5 and 6, and the activity decreases to about
40% when the pH is increased to 7. On the other hand the
activity of the pS21 sucrose transporter from spinach
(Riesmeier et aL, 1992) increases with decreasing pH
values. Both types of pH dependence are found in the
A. thalianasucrose transporters: SUC1 is fully active at pH
5 and 6 and transports with 50% activity at pH 7. The activ-
Arabidopsis sucrose transporters
ity of SUC2, however, which is very low at pH 7 increases
slowly at pH 6 and shows a further, drastic, increase at
pH 5. Measurements at lower pH values show an even further increase in activity for SUC2 but not for SUC1 (data
not shown). These differences might be important for
regulation of the activity of the two sucrose transporters in
the plasma membrane of Arabidopsis. According to the
theory of apoplastic loading and unloading, sucrose transport is necessary for the unloading of sucrose from the
mesophyll cells, for phloem loading and for phloem
unloading. It could be that loading and unloading of
phloem are catalyzed by one and the same transporter
regulated by factors such as the sucrose concentrations
on both sides of the membrane or the apoplastic pH. In
contrast two sucrose transporters, both expressed in the
phloem, might be regulated by these parameters, with one
transporter catalyzing primarily the loading, the other
transporter the unloading of phloem. Both possibilities
would of course imply that sucrose transporters such as
SUC1 and SUC2 can also catalyze the efflux of sucrose
out of cells under certain conditions. This has to be studied
with purified and reconstituted sucrose transporters.
Presently we do not even know in which cells SUCI and
SUC2 are expressed.
First information on the tissue specificities of SUC1 and
SUC2 expression was obtained from Northern blot
analyses (Figure 7), The highest levels for both sucrose
transporter mRNAs were found in rosette leaves of
Arabidopsis, supporting the thesis of phloem loading in
the green leaves by sucrose transporters. An interesting
difference was determined for the expression levels of
SUCI and SUC2 mRNAs in roots. Whereas the amount of
SUC1 mRNA in roots was similar to the levels found in
leaves only tiny amounts of SUC2 mRNA were detectable
in this tissue. This might be another indication for different
functions of the SUC1 and SUC2 proteins in Arabidopsis
plants.
Transgenic plants with downregulated expression
levels of SUC1 or SUC2 mRNAs and plants expressing
~-glucuronidase under the control of the SUC1 or SUC2
promoters will help to understand the function of these two
transporters and to illuminate the processes of carbohydrate partitioning in higher plants.
75
Isolation of SUC 1 and SUC2 cDNA clones
A cDNA library of A. thaliana strain Columbia wild-type in :;~gtl0
(Dr Andreas Bachmair; Max-Planck-lnstitut for Z0chtungsforschung, KOln, Germany) was screened with radioactively
labeled pS21 cDNA, encoding the spinach sucrose transporter
(generouslyprovided by Dr Wolf Frommer; IGF Berlin, Germany).
Plaques (100 000) were screened at a density of about 10 000
p.f.u, per 8 cm plate. Plaque hybridizationwas performed at 37°C
in 2 x SSC (1 x = 0.15 M NaCI, 0.015 M sodium citrate), 30% formamide, 1 x Denhardt's solution (Denhardt, 1966), 0.1% SDS,
and 100 Ilg m1-1 salmon sperm DNA. Hybridized nitrocellulose
filters were washed in 1 x SSC/0.1% SDS (w/v) for 30 min at
37°C, and in 0.1 x SSC/0.1% SDS for 30 min at 37°C, followed by
exposure to Kodak-X-OmatAR film.
Nucleic acid sequencing
cDNA clones were sequenced using the method of Sanger et aL
(1977). Exonuclease III deletions and subclones of restriction
fragments provided complete sequences of all clones. Sequence
comparisons were performed using the software of the Heidelberg Unix Sequence Analysis Resources (HUSAR).
Construction of yeast expression vectors NEV-X, NEV-E
and NEV-N
The yeast/E, coil shuttle vector YEp352 (Hill et aL, 1986) was linearized with EcoRI, treated with mung bean nuolease and religated to removethe EcoRI site from the polylinker. Subsequently
a 1.95 kb Hindlll fragment from the plasmid pRS1024, with promoter and terminator sequences of the S. cerevisiae plasma
membrane ATPase gene PMA1 (Serrano et aL, 1986) was
inserted into the unique Hindlll site of the polylinker, resulting in
the yeastJE, coil shuttle vector NEV-X. Plasmid pRS1024 is a
YEp351 (Hill et aL, 1986) derived plasmid with this Hindlll-fragment in the. polylinker; construction of this promoter/terminator
box has been described (Villalba et aL, 1992). The unique Xhol
cloning site which separates promoter and terminator sequences
in NEV-X was deleted by mung bean nuclease treatment of the
linearized plasmid and replaced by an EcoRI or a Notl site. The
resulting expression/shuttlevectors (NEV-E, NEV-N and NEV-X,
respectively) have a total length of roughly 7200 bp, carry yeast
URA3 and ampicillin-resistancegenes as selectable markers and
unique cloning sites for EcoRI, Nolt, or Xhol between the promoter and the terminator of the S. cerevisiae PMA 1 gene.
Expression of SUC 1 and SUC2 in baker's yeast
Experimentalprocedures
Yeast, bacteria/strains and plant material
For cloning in Escherichia coil we used the strain DH5e¢
(Hanahan, 1983). Expression in Saccharomyces cerevisiae was
performed with the strain RS453, which had already been used
for expressionof monosaccharidetransporters before (Sauer and
Stadler, 1993). Plants of Arabidopsis thaliana C24 were grown in
the greenhouse.
EcoRI inserts of plasmids pTF2011 and pTF2035 containing fulllength cDNA clones of SUC1 and SUC2, respectively,were isolated and ligated directly into the unique EcoRI restriction site of
NEV-E. The resulting plasmids NEV2011R and NEV2035R carry
their inserts in sense orientation; NEV2011F and NEV2035F
carry the inserts in the antisense orientation. Plasmids were
transformed into S. cerevisiae strain RS453 (Mata, ade2-1, trpl 1, can1-100, leu2-3,112, his3-1, ura3-52). Transformed yeast
cells were named NSY2011R, NSY2011F, NSY2035R and
NSY2035F, respectively.
76
Norbert Sauer and JOrgen Stolz
Construction of histidine-tagged SUC l
Transformation of S. cerevisiae
To allow simple identification and purification of the SUC1 protein
five additional histidine residues were added to the C-terminal
end of SUC1 resulting in a protein with a tail of six histidine
residues (his tag): PCR was performed on pTF2011, which carries a full-length cDNA clone of SUC1 in pUC19, using universal
sequencing primer and oligonucleotide SUC1/3 (5'- G "ETA GTG
AAT TCA GTG GTG GTG GTG GTG GTG GAA TCC TCC CAT
GGT CG -3'), which carries one EcoRI t'estriction site, one TCA
(antisense for a TGA stop codon), five times GTG (antisense for
CAC codon for histidine) and a sequence binding to the last 20
bases of the SUC1 open reading frame. Following extensive
restriction digest the resulting 1630 bp EcoRI fragment was
ligated directely into the unique EcoRI cloning site of the yeast/
E. coil shuttle vector NEV-E. Orientation was determined by
restriction digest and yeast cells (RS453) were transformed with
vectors containing the modified SUC1 cDNA in sense orientation
(NEV2011H6R) or in antisense orientation (NEV2011H6F). The
resulting yeast strains were named NSY2011H6R and
NSY2011H6F, respectively.
Yeast cells were transformed according to the protocol of Gietz el
aL (1992). URA* transformants were identified on 1.8% agar minimal plates (2% glucose, 0.67% yeast nitrogen base without
amino acids, and supplements).
Identification of histidine-tagged SUC 1 by SDS-PAGE
Cells expressing the histidine-tagged SUC1 protein were grown
in minimal medium to an ODs78 of 1.0. Cells (200 ml) were
harvested, washed once with distilled water, once with buffer K
(25mM Tris-HCI pH 7.5, 5mM EDTA), and resuspended in 100 I~1
of buffer K (on ice). In a small glass tube 1 g of glass beads (diameter 0.45 mm) was added to the cells, mixed with 3 I~l of
protease inhibitor mix (PIM: 0.1 M phenylmethylsulfonylfluodde
and 0.25 M p-amino-benzamidine in dimethyl sulfoxide) and
vortexed four times for 30 sec (put on ice between the individual
vortexing steps). Homogenized cells were centrifuged for 5 min at
1000 g and the supernatant again for 60 min at 50 000 g. The
membrane pellet was resuspended in 420 I~1of buffer 0 (100 mM
NaPO4 buffer pH 8.0, 50 mM NaCI) and 2 I~1of PIM, and solubilized for 5 min on ice with 60 ~1 of 20% dodecyl-~-D-maltoside
(DDM; Sigma) in buffer 0. The solubilized membranes were
mixed with 480 pJ of buffer 0 and centrifuged for 10 rain at 50 000
g and the supernatant was incubated for 1 h at 4°C with 200 td of
Ni2+-NTA-agarose in an Eppitube (Quiagen; washed twice with
buffer 0). The material was filled into a minicolumn and washed
with 2 ml of buffer E (100 mM NaPO4 buffer pH 8.0,250 mM NaCI,
0.05% DDM), 2 ml buffer E with 10 mM imidazole, 10 ml buffer E
with 20 mM imidazole, 3 ml buffer E with 30 mM imidazole, and
finally 1 ml buffer E with 100 mM imidazole. Ten microliters of
each wash were analyzed by SDS-polyacrylamide gel electrophoresis on 10% gels (Laemmli and King, 1970) and silver
stained (Heukeshoven and Dernick, 1988).
RNA isolation and Northern blots
Total RNA from different tissues of A. thaliana was isolated as
described earlier (Sauer et aL, 1990b). RNA was separated on
1% agarose gels containing formaldehyde and transferred to
nitrocellulose filters as described by Maniatis et al. (1982). Blots
were hybridized to gene-specific 32p-labeled Hincll fragments
from the 3'-ends of cDNA clones pTF2024 (331 bp; SUC1) or
pTF2013 (292 bp; SUC2). Both fragments covered the last 60 bp
of the respective coding sequences plus the entire 3'-untranslated sequences. Conditions for prehybridization, hybridization,
and washes were as described (Sauer et al., 1990b).
Transport tests
For transport tests S. cerevisiae strains NSY2011R/F,
NSY2035R/F and NSY2011H6R were grown in minimal medium
to an OD57s of 0.5-1,5. For each transport test 20 OD of cells
were harvested, washed twice with 25 mM NaPO4 buffer pH 5,5,
and resuspended in I ml of the same buffer, Cells were shaken in
a rotary shaker at 30°C and tests were started upon addition of
10 p.I of 10 mM ~4C-sucrose (7400 Bq from UJ4C-sucrose:
23.0 GBq mmol-1; Amersham), For certain experiments unlabeled o-glucose (10 raM) was added 1 min prior to the addition of
labeled sucrose, Samples were withdrawn at given intervals, filtered on nitrocellulose filters (0.8 I~m pore size) and washed with
an excess of cold water, Incorporation of radioactivity was determined by scintillation counting. The analogs a-phenylglucopyranoside and ~-phenylglucopyranoside were generously provided
by Dr T,J, Buckhout (University of Kaiserslautern, Germany), For
experiments on the pH dependence of sucrose transport cells
were treated the same way but washed and resuspended in
25 mM NaPO4 buffer pH 5, 6 or 7. All transport experiments were
performed at least twice.
Acknowledgments
We want to thank Dr Widmar Tanner for his continuous interest in
this work and for critically reading the manuscript. We also want
to thank Miss Birgit Huber for skillful technical assistance and Dr
Wolf Frommer (IGF Berlin, Germany) for the cDNA of the spinach
sucrose transporter. This work was supported by the Deutsche
Forschungsgemeinschaft (SFB 43/C5) and a grant to NS from the
Bundesministerium fLir Forschung und Technik (BEO210310331A).
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EMBL Data Library accession numbers X75365 (SUC1) and X75382 (SUC2).