1984
Research Article
Identification of an animal sucrose transporter
Heiko Meyer, Olga Vitavska and Helmut Wieczorek*
Department of Biology and Chemistry, University of Osnabrück, 49069 Osnabrück, Germany
*Author for correspondence ([email protected])
Accepted 7 February 2011
Journal of Cell Science 124, 1984-1991
© 2011. Published by The Company of Biologists Ltd
doi:10.1242/jcs.082024
Journal of Cell Science
Summary
According to a classic tenet, sugar transport across animal membranes is restricted to monosaccharides. Here, we present the first report
of an animal sucrose transporter, SCRT, which we detected in Drosophila melanogaster at each developmental stage. We localized the
protein in apical membranes of the late embryonic hindgut as well as in vesicular membranes of ovarian follicle cells. The fact that
knockdown of SCRT expression results in significantly increased lethality demonstrates an essential function for the protein.
Experiments with Saccharomyces cerevisiae as a heterologous expression system revealed that sucrose is a transported substrate.
Because the knockout of SLC45A2, a highly similar protein belonging to the mammalian solute carrier family 45 (SLC45) causes
oculocutaneous albinism and because the vesicular structures in which SCRT is located appear to contain melanin, we propose that
these organelles are melanosome-like structures and that the transporter is necessary for balancing the osmotic equilibrium during the
polymerization process of melanin by the import of a compatible osmolyte. In the hindgut epithelial cells, sucrose might also serve as
a compatible osmolyte, but we cannot exclude the possibility that transport of this disaccharide also serves nutritional adequacy.
Key words: Drosophila melanogaster, Sucrose transporter, Sugar transport
Introduction
Transmembrane sugar transport in prokaryotes as well as in fungi
and plants is attained by a variety of transport proteins specific for
mono-, di- and oligosaccharides (Maiden et al., 1987; Sauer and
Stolz, 1994; Szkutnicka et al., 1989). By contrast, sucrose transport
has never been reported for animal cells. In lieu thereof, two major
families of monosaccharide transporters that are essential for
glucose uptake have been extensively studied, sodium-glucose
linked transporters (SGLT) and glucose transporters (GLUT). The
SGLT family represents Na+ symporters (Wright et al., 1994),
whereas the members of the GLUT family are facilitative
transporters (Thorens, 1996). One feature common to SGLT, GLUT
and other animal sugar transporters is the fact that sucrose has
never been reported to be a transported substrate.
In previous studies of active K+ transport across caterpillar
intestinal epithelium, we have learned that the disaccharide sucrose
has to be present in the bathing medium to sustain ion transport
activity (Meyer et al., 2004). Because this observation was
inconsistent with the role of sucrose as a simple extracellular
osmolyte, we hypothesized that sucrose has some function in
transepithelial transport or is itself a transported substrate. Although
proteins mediating the diffusion or active transport of sucrose are
unknown in animal cells, the genomes of insects and vertebrates
do include open reading frames that code for proteins similar to
plant sucrose uptake transporters (SUTs). In addition, a disaccharide
transporter with high similarity to these animal proteins and also
to plant SUTs (supplementary material Fig. S1), was identified in
the fission yeast Schizosaccharomyces pombe. Apparently, this
protein (SUT1) mainly transports maltose and, with reduced affinity,
also sucrose (Reinders and Ward, 2001). In contrast to other
disaccharide transporters from fungi, e.g. the maltose permeases
AGT1 or MAL61, the transporters with homology to plant SUTs
contain a highly conserved sequence motif (RxGRR) between the
second and the third transmembrane helix. Because this motif
occurs in every known sucrose transporter at exactly the same
position, it is considered to be a sucrose transporter signature,
presumably useful to identify new members belonging to this
protein family (Lemoine, 2000). In addition to sharing the RxGRR
motif (supplementary material Fig. S1, emphasized in blue), the
homologous protein from Drosophila melanogaster analysed
in our present study (SCRT; CG4484, FBgn0035968,
http://flybase.org) appears to have 12 membrane-spanning domains
(supplementary material Fig. S1, emphasized in black), an attribute
that is also shared with sucrose transporters. Another structural
aspect appears to be the long cytosolic, -helical N-terminal region
(supplementary material Fig. S1, emphasized in green), which is
present in SCRT, but also in all low-affinity plant sucrose
transporters with apparent Km values equal to or higher than 10
mM (Schulze et al., 2000). In summary, the predicted SCRT
structure is typical for a protein belonging to the major facilitator
superfamily (MFS) (Marger and Saier, 1993) and reveals
characteristic similarities to SUTs.
In the present study we determined, on the basis of genomic
information
from
D.
melanogaster
(FBgn0035968,
http://flybase.org), the stage- and tissue-dependent occurrence of
SCRT. In addition, we analysed the effects of RNAi-mediated scrt
knock-down in transgenic flies and expressed the protein in
Saccharomyces cerevisiae to glimpse at its function. We here
present evidence that SCRT is a transporter of sucrose. It is the first
sucrose transporter to be demonstrated in any animal.
Results
Expression of SCRT
The scrt gene (synonym CG4484) is located on the third
chromosome at position 67A3. Sequence analysis predicts a single
transcript of 2951 nucleotides resulting in a protein consisting of
599 amino acids (FBgn0035968, http://flybase.org). Initial
experimental evidence for this transcript can be inferred from
expressed sequence tags: among others, the clones LP06168,
CK02046 and EK216041 (FBgn0035968, http://flybase.org)
Journal of Cell Science
Animal disaccharide transporter
especially indicate transcription of the predicted isoform. Using
specific primers, we could confirm this prediction. By cloning and
sequencing the respective amplicon, we verified that the isolated
cDNA corresponds to the predicted transcript. To further ascertain
the issue, we analysed the stage dependence of transcript
occurrence. Northern blots with total RNA isolated from embryonic,
larval, pupal and adult Drosophila confirmed the presence of scrt
mRNA in all developmental stages. Although embryonic and pupal
stages exhibited moderate expression levels, strong expression was
evident in adult flies. Third instar larvae showed a weaker
expression than other stages tested (Fig. 1A). To ascertain whether
these differences in transcript abundance are also valid regarding
protein expression, we raised polyclonal antibodies against the
central cytoplasmic loop of Drosophila SCRT (supplementary
material Fig. S1, emphasized in red). The monospecificity of the
antibodies was confirmed by western blots. In embryonic, larval
and adult D. melanogaster protein preparations the antibodies
detected a single band, which corresponded well with the predicted
SCRT size of 66 kDa (Fig. 1B). The observation that no signal was
visible in protein preparations isolated from RNAi-mediated scrt
knockdown flies (actin-Gal4 ⫻ UAS-scrt-RNAi) provides strong
evidence for the monospecificity of the antibodies and the validity
of protein detection. In pupal protein preparations, the molecular
mass of the detected band was slightly increased. However, because
the pupal scrt transcript detected by northern blot (Fig. 1A) had
apparently the same size as the transcripts from the remaining
stages, which minimizes the possibility of alternative splicing, we
consider the larger protein as a post-translationally modified version
of SCRT. Indeed, sequence analysis revealed the presence of two
N-glycosylation sites and a high number of putative
phosphorylation sites in the protein (Motif Scan, http://myhits.isb-
Fig. 1. Stage-dependent expression of scrt transcript and SCRT protein.
(A)Expression of scrt analysed by northern blot. scrt mRNA is present in
embryonic, larval, pupal and adult Drosophila total RNA preparations as
shown by hybridization of an antisense riboprobe (arrow). The bottom panel
shows part of the radiant red stained gel with ribosomal RNA (rRNA) visible
to demonstrate the loading of comparable RNA amounts (15g per lane).
(B)Expression of SCRT analysed by western blot. SCRT is present in
embryonic, larval, pupal and adult Drosophila protein preparations (arrow).
The size of the detected protein corresponds to the predicted molecular mass
of SCRT (66 kDa). The slightly increased molecular mass of the larval protein
is presumably related to post-translational modifications. In adults expressing
scrt-specific hpRNA (adult RNAi), the amount of SCRT is reduced below
detection limits.
1985
sib.ch/cgi-bin/motif_scan), which renders glycosylation or
phosphorylation to be reasonable modifications.
To detect the protein in situ, we applied the antibodies against a
number of tissues in different developmental stages. Of these
tissues tested for SCRT expression, the most distinct signals were
apparent in the late embryonic hindgut and in ovarian follicle cells
(Figs 2, 3). Embryonic whole mount preparations showed a strong
immunostaining covering the complete hindgut (Fig. 2B–D).
Significantly, this staining is only observed in the final embryonic
stage (stage 17) prior to hatching and first meal, indicating that
Drosophila SCRT, like other proteins of metabolic relevance
(Arbeitman et al., 2002), is already expressed in the late embryo
to serve its purpose immediately in the first instar larval stage. The
observation that the relative expression of SCRT in embryos is
significantly higher compared to third instar larvae (Fig. 1) further
supports the assumption that SCRT activity is required in very
early larval stages already. To further localize Drosophila SCRT in
the hindgut, we also investigated cross-sections of the complete
embryo and found clear signals only at the very apical side of the
hindgut epithelial cells, whereas the rest of the tissues remained
unstained (Fig. 2E–J). Control experiments without primary
antibodies did not yield any signal (Fig. 2A). The location of
Drosophila SCRT at that exclusive site along the gastrointestinal
tract suggests a role in substrate uptake across the apical membrane.
In ovarian follicle cells of adult flies, small vesicle-like structures
(1–2 m in diameter) were stained (Fig. 3A), whereas control
sections without primary antibodies did not exhibit any pattern
(not shown). In Drosophila and other insects, follicle cells produce
the chorion (eggshell) including the dorsal appendages of
developing embryos. During the formation of these structures,
many cell constituents, including proteins, are co-secreted by
follicle cells and can be found in the chorion after oviposition
(Waring, 2000). To ascertain whether SCRT is also secreted, we
immunostained the embryonic chorion and detected Drosophila
SCRT in vesicular structures of the dorsal appendages, which
looked very similar to the signal we had observed in the follicle
cells (Fig. 3D,G). Interestingly, the same vesicular structures were
labelled by a monoclonal antibody against melanin (Fig. 3H),
suggesting that these structures represent melanin-containing
organelles. However, in follicle cells the anti-melanin antibody did
not detect considerable amounts of melanin (Fig. 3B), indicating
that at this developmental stage melanin synthesis was not yet
occurring. Although the anti-melanin signal was restricted to the
dorsal appendages, an anti-SCRT signal was also observed as a
cell-shaped staining covering the whole chorion. Because the same
pattern was also detected by other antibodies used as controls
(anti-aldolase, Fig. 3E; anti-tubulin, not shown), this signal might
be unspecific. Stains without primary antibodies also resulted in a
weak cell-shaped staining, corroborating an unspecific signal,
whereas dorsal appendages remained completely unstained (Fig.
3F).
SCRT expression is vital for Drosophila
To ascertain the in vivo relevance of SCRT expression, we generated
RNAi lines utilizing the UAS–Gal4 system and analysed the effects
of scrt downregulation. High knockdown efficiency was confirmed
by northern blots (Fig. 4B). With both Gal4 driver lines being
balanced over CyO, and the UAS line being homozygous for the
scrt-RNAi construct, we could easily distinguish flies with expression
of scrt-specific hairpin RNA (hpRNA, normal wings) from flies
lacking the actin–Gal4 driver and thus also hpRNA expression (curly
Journal of Cell Science
1986
Journal of Cell Science 124 (12)
Fig. 2. Immunodetection of SCRT in the embryonic
hindgut. In late Drosophila embryos (stage 17) anti-SCRT
antibodies detect protein expression in the apical
membranes of hindgut epithelial cells. Expression seems to
be uniformly distributed across the whole tissue: (A)
control, (B) brightfield, (C) fluorescence, (D) merge.
Embryonic cross-sections show a clear signal at the apical
side of the hindgut: (E,H) brightfield, (F,I) fluorescence,
(G,J) merge.
wings). If the ubiquitous expression of scrt-specific hpRNA has no
effect on the viability of the animals, a ratio between normal and
curly wings of 1:1 would be anticipated. However, as depicted in
Fig. 4A, flies with curly wings occurred approximately twice as
often as flies with normal wings (67:33% on average), indicating a
vital role for SCRT in D. melanogaster. Half of the knockdown flies
died before reaching the adult stage. This result was valid for two
different Gal4 driver lines (BL4414, BL25374) and was irrespective
of the crossing type (male UAS–scrt-RNAi crossed to virgin female
actin–Gal4 and vice versa). The fact that, despite strong
downregulation of scrt, some animals reached the adult stage whereas
others did not is presumably based on the individual differences in
RNAi penetration. Although almost negligible, the residual amount
of scrt mRNA in the surviving RNAi flies (Fig. 4B) was apparently
sufficient for the animals to reach adult stage. Except for the increased
lethality however, no additional phenotype was apparent.
Fig. 3. Immunodetection of SCRT and melanin in follicle
cells and in the embryonic chorion. (A)In ovary
preparations, the follicle cells show a strong SCRT staining
based on vesicular structures of about 1–2m in diameter.
The nuclei of follicle cells are visible as black circles due to
the absence of staining (yellow arrows). (B)The application
of an anti-melanin antibody results only in a weak
background staining. (C)Merge of A and B. (D)In the
embryonic chorion, an unspecific cell-shaped staining
(white arrow,) as well as a very specific staining of the
dorsal appendages (green arrow) is apparent. This staining
is unique to SCRT antibodies, whereas the cell-shaped
staining is also detected by control antibodies (anti-aldolase,
E). (F)Controls without primary antibodies show a weak
cell-shaped staining but no staining of the dorsal
appendages. (G)Staining in the appendages is based on
vesicular structures of 1–2m in diameter (green arrows).
(H)The same structures are labelled by an antibody against
melanin (red arrows). (I)Merge of G and H.
Journal of Cell Science
Animal disaccharide transporter
Fig. 4. Effect of scrt RNAi on viability and gene expression.
Downregulation of scrt mRNA was realized by crossing scrt-RNAi flies (stock
105439, Vienna Drosophila RNAi Center) to two different actin–Gal4 driver
lines (BL4414 or BL25374, Bloomington Drosophila Stock Center). 1, 乆
BL4414 ⫻ 么 105439; 2, 乆 105439 ⫻ 么 BL4414; 3, 乆 BL25374 ⫻ 么 105439
and 4, 乆 105439 ⫻ 么 BL25374. The surviving G1 flies were collected,
divided into the control group (CyO, curly wings) and scrt-RNAi-expressing
group (Act5cGal4, normal wings) and counted. (A)Survival rate of flies
expressing scrt-specific hpRNA (Act5cGal4) compared with control flies
lacking the Gal4 driver element (CyO). Flies expressing scrt-specific hpRNA
show significantly increased lethality compared with control flies. In all
crossings, flies with curly wings occur about twice as often as flies with
normal wings. In individual experiments, total numbers of 650 (1), 786 (2),
696 (3) and 715 (4) flies were counted. Bars show means ± s.d.
(B)Subsequently to counting, total RNA was extracted from flies of each
group, subjected to northern blot and probed with a scrt antisense riboprobe.
Strong downregulation of scrt mRNA is apparent in the RNAi groups
(Act5cGal4) compared to the control groups (CyO). The bottom panel shows
part of the radiant red stained gel with ribosomal RNA (rRNA) visible to
demonstrate the loading of comparable RNA amounts (15g/lane).
SCRT is a functional sucrose transporter
To assess a functional role of Drosophila SCRT, we expressed the
protein in yeast cells, which are a commonly used system for the
characterization of plant sucrose transporters (Riesmeier et al.,
1992; Sauer and Stolz, 1994). Complementation assays showed
that the expression of Drosophila SCRT in the yeast strain SuSy7
(Riesmeier et al., 1992) enabled the transformed cells to grow on
minimal media containing 2% sucrose as the sole carbon source
at a pH of 4, but not at neutral or slightly alkaline pH (Fig. 5).
One key feature of the SuSy7 strain is its inability to grow on
sucrose due to a deletion of its invertase gene. In yeast, invertase
is a secreted enzyme that mediates the extracellular hydrolysis of
sucrose to glucose and fructose. Thus, the lack of invertase
prevents the extracellular hydrolysis of sucrose and thereby the
subsequent uptake of the respective monosaccharides. A second
key feature of SuSy7 is the insertion of the sucrose synthase gene
from potato, enabling the strain to metabolize intracellular
sucrose. Due to these genetic modifications, SuSy7 is eminently
suited to identify sucrose transporters by complementation assays.
The fact that expression of Drosophila SCRT enabled this strain
to grow normally on a sucrose-containing minimal medium
provides strong evidence that SCRT is able to transport sucrose.
As a positive control, StSUT1 which is a well-characterized
sucrose transporter from Solanum tuberosum (Riesmeier et al.,
1993), was analogously expressed and led to growth
characteristics comparable to those observed with Drosophila
SCRT. By contrast, the transformation of empty expression
1987
Fig. 5. Complementation of sucrose uptake by SCRT expression. The
functionality of SCRT is shown by complementation of a yeast strain
incapable of sucrose uptake (SuSy7). Under acidic conditions (pH 4), the
expression of SCRT under the control of the PMA1 promoter enables this
strain to grow normally compared with the very slow growth rate of cells
transformed with the empty expression vector as a control. At neutral pH
values (pH 7), the growth rate of the cells expressing SCRT is largely reduced,
and diminishes completely under alkaline conditions (pH 8.5). As a positive
control, StSUT1 was expressed in the same strain and showed growth
characteristics similar to SCRT. Transformed cells were grown for 5 days at
29°C on minimal media.
vectors as a negative control resulted in negligible background
growth of the cells.
To confirm the results obtained by the complementation tests,
we expressed SCRT in the yeast strain RS453, which is a commonly
used strain for the expression and characterization of
monosaccharide (Sauer and Stadler, 1993) and disaccharide
transporters (Sauer and Stolz, 1994). We made use of the fact that
RS453 cells grown on glucose-containing media strongly repress
the uptake of sucrose as well as extracellular hydrolysis of sucrose
by invertase (Sauer and Stolz, 1994). Accordingly, glucose-grown
yeast cells are naturally well suited for the heterologous expression
and characterization of sucrose transporters. Northern blots ensured
that the SCRT expression construct was transcribed by the
transformed yeast cells. As expected, an antisense probe detected
a clear band of anticipated size, whereas the application of a sense
probe did not elicit any signal (not shown). The transformed strain
was subsequently used for uptake measurements with [14C]sucrose
as a substrate. As shown in Fig. 6, the expression of Drosophila
SCRT under the control of the strong PMA1 promoter (Sauer and
Stolz, 1994) enabled the yeast cells to accumulate sucrose in a
time-dependent manner, being linear up to at least 2 minutes.
Control experiments showed that the kinetics of sucrose uptake
were considerably blunted in yeast cells transformed with the
empty expression vector. Clearly, SCRT is a transporter of sucrose.
To ascertain whether the pH dependence observed in the
complementation tests reflected pH-dependent sucrose transport,
we performed uptake experiments at different pH values. Sucrose
1988
Journal of Cell Science 124 (12)
Journal of Cell Science
Fig. 6. Time dependence of sucrose uptake by yeast cells (RS453)
expressing SCRT. Compared to yeast cells transformed with empty
expression vectors (open squares), cells expressing SCRT (closed squares)
show a significant increase in time-dependent uptake of [14C]sucrose. Uptake
was measured in the presence of 100 mM sucrose at pH 4. Data points
correspond to the means ± s.d. of seven independent preparations. FW, fresh
weight.
transport was most efficient under acidic conditions, whereas
neutral or slightly alkaline pH abolished transport completely (Fig.
7). For reasons of comparison, we did the same experiments with
yeast cells expressing the plant sucrose transporter StSUT1 and
observed an almost identical stimulation of sucrose uptake with
decreasing pH [not shown here, but already reported for, e.g.
AtSUC1 and AtSUC2 (Sauer and Stolz, 1994)]. Experiments
regarding the concentration dependence of SCRT under conditions
of initial velocity revealed that half maximal [14C]sucrose uptake
requires concentrations in the range of 15 mM (Fig. 8). Thus, the
affinity of Drosophila SCRT for sucrose was in line with that of
low affinity sucrose transporters from plants, e.g. proteins belonging
Fig. 8. Concentration dependence of sucrose uptake by yeast cells (RS453)
expressing SCRT. Sucrose uptake rate measured after 1 minute of transport
(initial velocity) increases with elevated sucrose concentrations. Values are
presented with background transport (empty vector) subtracted. The
hyperbolic curve was fitted using Origin 7 (Originlab) and the apparent Km
was 16±5 mM. Uptake was measured at pH 4 and the data points correspond
to the means ± s.d. of three independent preparations. FW, fresh weight.
to the SUT4 or the SUT2/SUC3 subfamilies, which show similar
apparent Km values (Schulze et al., 2000; Weise et al., 2000). To
obtain initial data on the substrate specificity of SCRT, sucrose
uptake was competed with other mono- and disaccharides that
might be potential substrates for the transporter. The addition of
non-labelled sucrose itself led, due to the lower specific
radioactivity, to a 70% decrease of detected radioactivity (Table 1).
Among the other sugars, the most efficient competitor of sucrose
uptake was trehalose. It is noteworthy that the addition of glucose
augmented sucrose transport instead of diminishing it. The latter
result was also reported for different sucrose transporters from
plants, e.g. SUC1 and SUC2 from Arabidopsis thaliana (Sauer and
Stolz, 1994). As a possible reason for this effect, the authors
proposed that the increased transport activity is based on a more
efficient yeast plasma membrane energization caused by the
increased metabolism of the optimal carbon source glucose. An
almost complete inhibition of transport was caused by the addition
of the protonophore carbonyl cyanide m-chlorophenylhydrazone
(CCCP), which is consistent with a proton-coupled sucrose uptake
mechanism (Table 1).
Table 1. Transport properties of SCRT in yeast cells
Sugar added
None
Sucrose
Trehalose
Maltose
Lactose
Galactose
Glucose
CCCP
Fig. 7. pH dependence of sucrose uptake by yeast cells (RS453) expressing
SCRT. Sucrose uptake after 10 minutes of transport was measured at different
pH values. Transport activity is high under acidic conditions whereas neutral
or slightly alkaline pH abolishes the uptake of radiolabelled substrate. Data are
presented with background transport (empty vector) subtracted and correspond
to the means + s.d. of three independent assays. FW, fresh weight.
Activity (%)
100
31±16
56±16
75±11
86±10
73±4
124±1
7±3
Putative substrates were added at fivefold excess (final concentration 300
mM) and CCCP was added to a final concentration of 50 M, 2 minutes
before the assay was started by the addition of radiolabelled sucrose. Values
are presented with background transport (empty vector) subtracted and
represent relative uptake rates after 10 minutes of transport at pH 4 compared
with [14C]sucrose uptake without additional sugars or inhibitors. Values
correspond to the means ± s.d. of at least three independent assays.
Journal of Cell Science
Animal disaccharide transporter
Fig. 9. Rooted ML tree (lnL–15818.5) of human proteins belonging to the
SLC45 family, SCRT from D. melanogaster, and similar proteins from
fungi and plants as outgroups. The tree shows the relationship of the proteins
to each other. Bootstrap values are given at the branches. The scale bar
indicates substitutions per position. SpSUT1, Schizosaccharomyces pombe glucoside transporter (NP_594387.1); NfSUT, Neosartorya fischeri putative
sucrose transporter (XP_001266016.1); AfSUT, Aspergillus fumigatus sucrose
transporter (XP_754012.1); PmSUT, Penicillium marneffei putative sucrose
transporter (XP_002147999.1); StSUT1, Solanum tuberosum sucrose
transporter (CAA48915.1); VvSUT2, Vitis vinifera sucrose transporter
(AAL32020.1); AtSUC3, Arabidopsis thaliana sucrose transporter
(NP_178389.1); LeSUT4, Lycopersicon esculentum sucrose transporter
(AAG09270.1). NCBI sequence accession numbers
(http://www.ncbi.nlm.nih.gov/sites/entrez) are given in brackets.
Discussion
We have provided the first experimental evidence for a sucrose
transporter in animal cells. However, as depicted in Table 1, the
major insect blood sugar trehalose (Wyatt and Kale, 1957) has a
certain potential to compete with sucrose for transport, indicating
that this disaccharide might also be a substrate for SCRT, yet with
a reduced potency. The Drosophila SCRT resembles sucrose
transporters in plants, including a crucial region that is considered
to be a sucrose transporter signature. Its localization in the apical
membrane of hindgut epithelial cells suggests a role in
transmembrane and/or transepithelial sucrose transport. An
intestinal sucrose transporter would appear particularly
advantageous for animals such as Drosophila that rely primarily
on a fruit diet, which contains sucrose in concentrations of up to
0.2 mol/l (Beruter, 2004; Etxeberria and Gonzalez, 2005). This
high abundance is a strong argument for the assumption that at
least in the hindgut tissue sucrose represents the main substrate of
SCRT. Furthermore, if the blood sugar trehalose was the transported
disaccharide in the hindgut, we would assume the transporter to be
located in the basolateral instead of the apical membrane of
epithelial cells. The pH-dependent growth of transformed yeast
cells at pH 4 but not at neutral or slightly alkaline pH (Fig. 5),
together with the result that uptake of radiolabelled sucrose requires
acidic conditions (Fig. 7), suggests that Drosophila SCRT might
be, like the plant sucrose transporters, an H+/sucrose symporter.
Additional evidence for proton-coupled transport arises from our
finding that the uncoupler CCCP abolishes sucrose transport almost
1989
completely (Table 1). Indeed, the pH in the insect hindgut is
slightly acidic (Harrison, 2001), thus supporting our speculation.
The transport of sucrose or other disaccharides such as the
insect blood sugar trehalose into melanin-containing organelles
might also be an H+ co-transport because in mammals, mature
melanin-synthesizing melanosomes are, in contrast to premature
melanosomes, not thought to be acidic (Ancans et al., 2001; Tabata
et al., 2008). Nevertheless, the function of Drosophila SCRT in the
melanosomal membrane is less clear. We suggest that it plays a
role in volume control during the polymerization of tyrosine at the
beginning of melanin synthesis. Indeed, the putative sucrose
transporter membrane-associated transporter protein (MATP),
which was identified in the Japanese killifish and exhibits high
similarities to SCRT and also to plant sucrose transporters
(supplementary material Fig. S1), has been proposed to provide
osmotic balance in melanosomes during melanin biosynthesis
(Fukamachi et al., 2001). Similar postulations were made for
human MATP (SLC45A2), in which distinct point mutations,
presumably inactivating the protein, result in a novel form of
oculocutaneous albinism, OCA4 (Newton et al., 2001).
Furthermore, MATP-mutant mice exhibit small, crenated
melanosomes compared with the ovoid or round melanosomes in
the wild type, which was considered an indication for an
osmoregulatory dysfunction (Lehman et al., 2000). Because in
vivo substrates of the vertebrate melanosomal transporters are
entirely unknown, our data on the highly similar SCRT might be
particularly advantageous in understanding the function of sucrose
transporters in melanin synthesis. Although there is so far no
experimental proof of the necessity of disaccharides in melanosomal
volume regulation, the potential to act not only as compatible
osmolytes but also as stabilizers of protein structures and enzymatic
activities has been shown extensively for sucrose (Cioni et al.,
2005; Meyer et al., 2004; Ou et al., 2001; Wang et al., 1995) and
for trehalose (Felix et al., 1999; Kaushik and Bhat, 2003; Zancan
and Sola-Penna, 2005). Thus, in addition to the proposed
osmoregulatory functions, sucrose or trehalose might be crucial for
protein functionality inside the melanosomal microcompartment.
Besides SLC45A2, which displays the highest degree of amino
acid identity with SCRT (supplementary material Fig. S1), the
remaining three members of the human SLC45 family also show
remarkable similarities to Drosophila SCRT. In a phylogenetic
analysis, SCRT is sister to a clade comprising SLC45A1, SLC45A2
and SLC45A4. SLC45A3 is sister to this clade (Fig. 9). In contrast
to SLC45A1 and SLC45A4 that have not yet been assigned to any
physiological process, SLC45A3 has been reported to be involved
in prostate cancer (Helgeson et al., 2008; Tomlins et al., 2007).
Because up to now no physiological substrate for this putative
transporter has been identified, its involvement in the disease is
rather elusive.
Our observation that, except for increased lethality, scrt
knockdown flies do not show any apparent phenotype is presumably
due to the residual expression of scrt mRNA in the surviving
RNAi flies (Fig. 4B). Apparently, the remaining expression of
SCRT in these flies is not only sufficient for the individuals to
develop to adults, but might also be adequate to conceal additional
phenotypes like albinism, which could be anticipated to occur
according to the observations reported on similar vertebrate proteins
(MATPs). In future experiments, the use of tissue- or stage-specific
driver lines might be beneficial in identifying additional RNAimediated phenotypes. However, the fact that ubiquitous
downregulation of SCRT induces lethality is quite an interesting
1990
Journal of Cell Science 124 (12)
result on its own because it demonstrates that the physiological
functions fulfilled by SCRT are absolutely essential.
The existence of a sucrose transporter in D. melanogaster extends
our current understanding of sugar transport in animals. Protein
expression in the Drosophila hindgut emphasizes this in particular
by indicating a direct involvement of SCRT in sucrose uptake in
the intestinal tract. Future research will clarify how extensively the
common conception that sugar transport across animal membranes
is restricted to monosaccharides is violated.
Materials and Methods
Journal of Cell Science
Strains and growth conditions
D. melanogaster Oregon R strain was grown on a medium containing 1.35% agar,
3.75% baker’s yeast, 6.4% sucrose and 16.5% maize grit. Compounds were mixed,
boiled briefly and transferred to breeding containers. Breeding was conducted at
22°C. To knock down scrt mRNA expression in the complete animal, flies from the
scrt-specific RNAi stock 105439 (Vienna Drosophila RNAi Center, VDRC,
http://www.vdrc.at/) were either crossed to the driver line BL4414 (Act5C-Gal4/CyO)
or to the line BL25374 (Act5C-Gal4/CyO, Bloomington Drosophila Stock Center,
http://flystocks.bio.indiana.edu/) and reared at 29°C, 12:12 photoperiod and 70%
relative humidity. In all cases, 5–8 virgin females were crossed to 10–15 males, with
the mated flies being transferred to new breeding containers every second day. The
genotype of the resulting G1 progeny was identified utilizing the CyO marker for
chromosome 2. Flies showing the curly wing phenotype were considered wild type,
whereas flies with normal wings expressed scrt-specific hpRNA. High knockdown
efficiency was confirmed by northern blots.
For cloning in Escherichia coli, strain DH5 (Hanahan, 1983) was used. In the
case of transport tests with [14C]sucrose as a substrate, SCRT was expressed in the
S. cerevisiae strain RS453 (Sauer and Stadler, 1993). For complementation
experiments, the yeast strain SuSy7 (Riesmeier et al., 1992), a kind gift from Norbert
Sauer, Friedrich-Alexander University, Erlangen-Nürnberg, Germany, was used as
an expression system.
Molecular cloning of full length scrt and the central loop
Full length scrt was obtained by RT-PCR using D. melanogaster embryonic cDNA
as a template and the primers 5⬘-ATGGTTGGAGTTACTGCCGAC-3⬘ (forward,
fw) and 5⬘-CTAGACATATAACACAAACAT-3⬘ (reverse, rv), respectively. The
cDNA was reverse transcribed from embryonic total RNA (1 g per reaction) using
the AMV First Strand cDNA Synthesis Kit for RT-PCR (Roche, Basel, Switzerland).
In order to ligate the resulting fragment into the NEV-E yeast expression vector
(Sauer and Stolz, 1994), EcoRI restriction sites were introduced at both ends into the
cDNA by appropriate primer design. To amplify the sequence coding for the central
cytoplasmic loop, the primers 5⬘-CGTGAAATTCCATTGCCCTTG-3⬘ (fw) and 5⬘GCGCATCGAGTAGGGCATGAT-3⬘ (rv) were used. Subsequently, the PCR-product
was ligated into the pET16b expression vector (Novagen, EMD Biosciences,
Madison, WI) employing NdeI and BamHI restriction sites, respectively. Expression
in E. coli Rosetta (DE3) cells (Novagen, Darmstadt, Germany) was done essentially
as described (Meyer et al., 2009) and the resulting soluble protein (fused to a Nterminal 10⫻His-tag) was purified via Ni-NTA Agarose (Qiagen, Hilden, Germany)
according to the manufacturer’s instructions. Templates for riboprobe synthesis were
generated with primers 5⬘-ATGGTTGGAGTTACTGCCGAC-3⬘ (fw) and 5⬘AAGATGTGAAATTCAAGGCTG-3⬘ (rv). All constructs were validated by
sequencing.
Phylogenetic analysis
We used a maximum likelihood (ML) analysis to reconstruct the phylogenetic
relationship of proteins belonging to the human SLC45 family and SCRT from D.
melanogaster (CG4484, http://flybase.org), and complemented the data with similar
proteins from fungi and plants as outgroups. The amino acid data set was aligned
with the aid of MUSCLE (Edgar, 2004). The most appropriate substitution model
for the ML analysis was LG+I+G+F as determined on the basis of the Akaike
information criterion using ProtTest (Abascal et al., 2005). ML analyses were
conducted with RAxML 2.7.6 (Stamatakis, 2006) using 100 replicate searches
starting from randomized maximum parsimony trees. Confidence values for the
edges of the best ML tree were determined on the basis of 100 bootstrap replicates.
Expression in S. cerevisiae and transport assays
All plasmids were transformed into yeast strains via electroporation (1 pulse, 1.5 kV,
25 F, 200 ) with 1 g plasmid DNA per transformation approach. The resulting
strains were grown on minimal media containing 0.67% yeast nitrogen base (BD,
Franklin Lakes, NJ), 1% casaminoacids (BD), 2% D-glucose (Serva, Heidelberg,
Germany) and 25 mM sodium phosphate (pH 6.0). After reaching an optical density
of 1.5 at 600 nm, 20 ml of cells were harvested by centrifugation (5 minutes at 3.500
g), washed first with 20 ml H2O followed by 20 ml of incubation buffer (minimal
media as described above, with substitution of glucose by variable concentrations of
sucrose at different pH-values adjusted with 25 mM sodium phosphate), centrifuged
again and resuspended in 1 ml of incubation buffer. Cells were kept on ice until uptake
assays were started by the addition of radiolabelled substrate solution ([14C]sucrose
0.2 Ci/l; GE Healthcare, Munich, Germany). Assays with 1 mM, 5 mM and 25 mM
sucrose in the incubation buffer employed 0.4 Ci, whereas assays with 60 mM
sucrose contained 1 Ci and assays with 100 mM sucrose contained 2.5 Ci. In all
cases, aliquots (100 l) were taken and transferred into 1 ml of incubation buffer to
stop the uptake process. After filtration (cellulose acetate filters, 0.45 m, Schleicher
and Schuell, Germany), the filters containing yeast cells were transferred into
scintillation vials and the amount of accumulated [14C]sucrose was measured by
scintillation counting (Beckman LS 6500).
Complementation tests
For complementation, transformed SuSy7 strains were grown at 29°C on SD minimal
media containing 0.67% Yeast Nitrogen Base (BD), 0.002% tryptophan (Sigma), 2%
sucrose (analytical grade; Serva, Heidelberg, Germany) and 25 mM sodium phosphate
(variable pH values).
Immunohistochemistry
SCRT antisera were raised in guinea pigs (Charles River, Kisslegg, Germany) against
the expressed and Ni-NTA-purified central cytoplasmic loop of the protein (amino
acid positions 286–390; supplementary material Fig. S1). Antibody specificity was
confirmed by antigen blocking (50 l of sera were incubated with 40 g of purified
antigen in 1 ml PBS for 1 hour at room temperature) and the resulting sera were
controlled by western blot and tissue staining. For whole mount preparations, embryos
were dechorionized, devitellinized and fixed (Patel, 1994). If the embryonic chorion
was stained, incubation in Klorix:water (1:1) was omitted. Fixation was performed in
4% formaldehyde in PBS (137 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, 2 mM
KH2PO4, pH 7.4) for 25 minutes. Antibody application was carried out in 2% bovine
serum albumin, 0.1% SDS in PBS. Blocking was done in the same buffer for 1 hour
at room temperature. Primary antibodies (anti-SCRT, raised in this study, and antimelanin antibodies, a kind gift from Joshua Nosanchuk, Albert Einstein College of
Medicine, New York) and secondary antibodies (anti-guinea-pig Cy3-conjugate and
anti-mouse FITC-conjugate, both from Sigma) were applied overnight (4°C) at dilutions
of 1:1.000 and 1:200, respectively. Washing was done in PBS with 0.05% Tween 20.
Prior to VectaShield (Vector Laboratories, Burlingame, CA) coating, whole mount
stained embryos were incubated in glycerin for 48 hours. For cryo-sections (5 m),
stained specimen were subjected to an increasing sucrose gradient (10, 20 and 30% in
PBS) prior to embedding them in Tissue Freezing Medium (Jung, Germany). Freezing
was conducted in isopentane cooled in liquid nitrogen. Subsequent to sectioning
(Leica CM1900 cryomicrotome), frozen tissues were transferred to superfrost glass
slides (Microm International, Walldorf, Germany), fixed by short-term warming (10
seconds, 50°C), washed three times (PBS, 5 minutes each) and embedded in
VectaShield. Fluorescence was visualized using either a reverse microscope (Olympus
IX70, 100⫻, NA 1.35) or a confocal laser scanning microscope (LSM 5 Pascal, Zeiss,
40⫻, NA 1.3).
Western blot and northern blot analysis
SDS-PAGE and western blots were carried out essentially as described (Wieczorek et
al., 1991) with the variations that proteins (15 g per lane) were transferred to
nitrocellulose membranes instead of PVDF, and milk powder (5%) was used as
blocking reagent instead of gelatine. Purified SCRT antiserum was applied at a dilution
of 1:5,000 and visualized by anti-guinea pig alkaline phosphatase conjugated antibody
(1:10,000; Sigma).
Northern blots were generally conducted as described (Meyer et al., 2010) with total
RNA (15 g per lane) and a hybridization temperature of 65°C.
This work was supported in part by the DFG (Research training
group 612) and the Hans Mühlenhoff foundation. We thank Klaus W.
Beyenbach (Cornell University) for valuable comments on the
manuscript and Torsten Struck for giving support regarding the
phylogenetic analysis.
Supplementary material available online at
http://jcs.biologists.org/cgi/content/full/124/12/1984/DC1
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