The solute carrier 45 family (SLC45) was defined in the course of the Human Genome Project and consists of four members, A1–A4, which show only 20–30% identity of amino acid sequences among each other. All these members exhibit an identity of ∼20% to plant H+/sucrose cotransporters. Recently, we expressed members of the murine SLC45 family in yeast cells and demonstrated that they are, like their plant counterparts, H+/sucrose cotransporters. In contrast with the plant proteins, SLC45 transporters recognise also the monosaccharides glucose and fructose as physiological substrates and seem to be involved in alternative sugar supply as well as in osmoregulation of several mammalian tissues. In the present study, we provide novel insights into the regulation of SLC45 transporters. By screening for interaction partners, we found a 14-3-3 protein as a promising candidate for control of transport activity. Indeed, co-expression of the gamma isoform of murine 14-3-3 protein in yeast and Xenopus oocytes led to a significant decrease in transport rates of the murine SLC45 transporters as well as of the plant H+/sucrose transporter Sut1.

Introduction

The mammalian solute carrier family 45 (SLC45) belongs to the major facilitator superfamily and consists of the four members A1 to A4. It emerged with the completion of the Human Genome Project in 2003 as a family of putative sugar transporters, which astonishingly bear a resemblance to plant sucrose transporters. However, the concept that animal proteins transport a disaccharide such as sucrose would challenge the well-accepted textbook dogma that mammalian sugar transport across membranes occurs only via transport of monosaccharides. Traditionally, two families of sugar transporters are found in animals, the GLUT and the SGLT proteins. GLUT proteins, also known as SLC2, are members of the major facilitative transporter superfamily, whereas SGLT proteins, also known as SLC5, are Na+/sugar cotransporters. Our recent investigation of the properties of murine SLC45 proteins in transformed yeast cells challenged this existing concept by showing that they are indeed able to transport the disaccharide sucrose, but also the hexoses glucose and fructose [1]. A further difference to the established sugar transporters arises from its mode of transport, an H+/symport, which is also likely the reason why these transporters have escaped the attention in scientific studies so far.

For many years, most of the knowledge regarding the SLC45 family was based on results derived from the Human Genome Project and from mRNA expression studies, while physiological functions have not been studied extensively. SLC45A1 has been shown to be a H+/glucose cotransporter which may be involved in the uptake of glucose into neuronal tissues during hypercapnia [2]. SLC45A2 seems to play a significant but not yet clearly defined role in the regulation of melanogenesis [36]. SLC45A3 was shown by us to be an H+-coupled sucrose transporter in the mammalian kidney, where sucrose evidently functions as a novel osmolyte in urine [7]. Most recently, we demonstrated that SLC45A4 is an H+-coupled sugar transporter in the plasma membrane of mammalian spermatozoa, proposed to be involved in their maturation in the epididymis [8]. Although it also transports sucrose across the plasma membrane of sperm cells, its physiological substrates may be fructose or glucose, which occur in the male reproductive tract at rather high concentrations.

Our recent insights into physiological functions of SLC45 transporters raised the question of how they are regulated. To answer this question, we addressed our study to the search for interaction partners of SLC45 transporters, because the interaction between proteins often helps to elucidate mechanisms of their regulation. We focused our study on the largest cytoplasmic part of transporters, their central loop between the sixth and seventh transmembrane domain. The crucial reason for this choice was data suggesting that the large central loop of related sugar transporters is not needed for transport, but may have instead a regulatory function [911]. Thus, mammalian H+/sugar transporters of the SLC45 family, mammalian facilitative transporters of the SLC2 family, plant H+/sucrose transporters and the Escherichia coli lactose permease belong to the major facilitator superfamily and are similar in structural features including 12 transmembrane-spanning domains, a cytosolic orientation of N- and C-termini, and a large cytosolic central loop between the sixth and seventh transmembrane domain. Interestingly, regulatory functions of the central loop are generally mediated by direct interaction with certain proteins. For example, sugar transport is allosterically regulated by binding of the intracellular regulatory protein, enzyme IIAglc, to the large central cytoplasmic loop of lactose permease in E. coli [1214]. Mammalian facilitators of the SLC2 family (GLUTs) are believed to be affected by several glucose transporter binding proteins (GTBPs) which may be an enzyme or nonenzyme, an adaptor or docking protein [10]. Thus, Liu et al. [15] have identified a 70 kDa cytosolic protein (GTBP70) in rat adipocytes that binds to the central loop of GLUT1, GLUT2 and GLUT4 transporters.

In a first approach, we coupled the recombinant central loops of murine SLC45A2, A3 and A4 transporters to beads, incubated them with cytosolic extracts from mouse brain or kidney and determined the nature of associated proteins by mass spectrometry. Among several detected putative interaction partners, 14-3-3 proteins appeared to be promising candidates for the regulation of SLC45 transporters as they mediate protein–protein interactions and regulate diverse metabolic and signalling pathways, including glycolysis [16].

The 14-3-3 proteins comprise a family of small acidic proteins with a molecular mass of ∼30 kDa [17]. They are highly conserved and abundantly expressed in all eukaryotes. Thus, seven members of the 14-3-3 family (beta, epsilon, gamma, eta, tau, zeta and sigma) have been found in mammals [18,19] while only two isoforms occur in yeast Saccharomyces cerevisiae, Bmh1 and Bmh2, [20]. The largest number of 14-3-3 members was found in plants, e.g. Arabidopsis thaliana, which expresses thirteen different isoforms [21]. The 14-3-3 proteins function as homo- or hetero-dimers through direct binding to client proteins, consequently modulating their properties. In this way, 14-3-3 proteins have been shown to regulate numerous cellular processes including cell cycle, protein trafficking, metabolism, cell proliferation and apoptosis [19,22,23].

In this study, we verified the detected interaction of SLC45 family members with the 14-3-3-γ protein by yeast two-hybrid analysis as well as by Far-western blot and pull-down assays with purified proteins. Furthermore, we examined the effect of 14-3-3-γ on the activity of SLC45 sugar transporters and, in addition, of the related plant H+/sucrose transporter Sut1 by uptake experiments using radioactively labelled sucrose. Finally, we analysed interactions by immunocytochemistry and western blot. In summary, we provide here novel insights into the 14-3-3 dependent regulation of H+/sucrose transporters from the mammalian SLC45 family, showing that this type of regulation may also be relevant for plant H+/sucrose transporters.

Materials and methods

Yeast two-hybrid analyses

The yeast two-hybrid screening was performed according to the Matchmaker Gold Yeast Two-Hybrid System (Clontech) using the Mate & Plate Library — Universal Mouse (normalised). The regions of the central loop between the sixth and the seventh transmembrane domains of SLC45 transporters served as ‘baits'. They were chosen after prediction of protein topology by the algorithms SOSUI [24] and TMHMM [25] and comprise the following amino acid sequences: SLC45A2-loop 240APLRDAATDPPSQQDPQGSSLSASGMHEYGSIEKVKNGGADTEQPVQEWKNKKPSGQSQRTMSMKSLLRALVNMPSHY317; SLC45A3-loop 218EEAVLGPPEPAEGLLVSAVSRRCCPCHVGLAFRNLGTLFPRLQQLCCRMPRTLR272 and SLC45A4-loop 256EQYSPQQDRGPEDPTLPGTSVQPGAPAPASRLSSLGGGMQDGSPPFPDEVQSEHELSLDYLDVDIVRSKSDSVLHMADATLDMEPQLLFLHDIEPSIFQDASYPSTPQSTSQELLRAKLPRLSTFLRESTKEDDTLLDNHLNEAKVPNGRGSPPINSLSRSKVDLKPSVTSGSMRRRRHMFHRQASSTFSYYGKIGSHCYRYRRANAVVLIKPSRSMSDLYDLQQRQRSRHRNQSGATASSGDTESEEGETETTVRLLWLSMLKMPK523. The sequences encoding the loops were cloned into the pGBKT7 vector in frame with the sequence for the Gal4 DNA-binding domain and a c-Myc epitope tag using the NcoI and SmaI restriction sites. The Y2HGold Yeast Strain containing the bait proteins was mated with the Y187 Yeast Strain, containing the normalised mouse cDNA library. Diploids were selected on DDO/X plates, and blue colonies, showing activation of the reporter gene MEL1, were patched out on higher stringency QDO/X plates. In addition to MEL1, these plates were also selected for activation of the two reporter genes HIS3 and ADE2. Those detected interactors, which activated the Gal4-responsive reporters in the absence of the bait protein as well as all nuclear proteins were excluded from further analysis as false positives.

After 14-3-3-γ had been found by screening as a candidate for interaction, ‘pray'-plasmid pGADT7 containing the 14-3-3-γ sequence was amplified in E. coli, purified and used for co-transformation with pGBKT7 containing one of the SLC45-loops. Yeast cells carrying both plasmids were plated onto a QDO/X (40 µg/ml X-α-Gal in Dropout/-Ade/-His/-Leu/-Trp, 0,67% yeast nitrogen base, 2% glucose) as well as onto a DDO (Dropout/-Leu/-Trp, 0,67% yeast nitrogen base, 2% glucose) medium and incubated at 30°C for 3 days.

Cloning and purification of recombinant proteins

For both pull-down assays (with mouse tissues as well as with purified 14-3-3-γ) and for Far-western blot analysis, the same regions of the central loop of SLC45 transporters used in yeast two-hybrid experiments (see above) were expressed as a fusion to maltose binding-protein (MBP). For this, the loop sequences were cloned into pMAL-c2 by XbaI and HindIII restriction sites in frame to the N-terminal MBP-tag. After expression in E. coli BL21(DE3)pLysS, recombinant proteins were purified by affinity chromatography using amylose according to the manufacturer's instructions.

Murine 14-3-3-γ (AF058799.1) was cloned and expressed as a His-tagged protein by amplifying the coding region of the sequence and ligation into the pET-29b vector (Novagen) using NdeI and XhoI restriction sites. After purification by Nickel-NTA chromatography (Qiagen) according to the manufactur's protocol, 14-3-3-γ was dialysed into 20 mM Tris–HCl, 0.2 M NaCl, pH 7.5, and used for pull-down assays or for Far-western blots.

Pull-down assays with tissue extracts

For tissue extracts, mouse brains or kidneys were homogenised in cold 20 mM Tris pH 7.4, 150 mM NaCl, 1 mM EGTA, 1 mM EDTA, 1 mM DTT and 5 mM Pefabloc using an Ultra-Turrax (IKA). After centrifugation at 100 000×g and 4°C for 1 h, supernatants were used for assays. For each sample of pull-down, ∼0.1 ml fresh amylose beads were saturated with one of the three purified MBP-fusion proteins (A2-, A3-, A4-loops) or with MBP alone, and incubated with 1 ml of tissue extract containing 3 mg of total protein in 20 mM Tris pH 7.4 and 0.5 mM NaCl for 4 h at 4°C and mild rotation. After stringent washing steps with incubation buffer and with the same buffer containing 0.1% Triton X-100, proteins were eluted by incubation of beads with 10 mM maltose for 10 min and collected by centrifugation at 200×g and 4°C for 1 min. Eluted proteins were precipitated overnight at −20°C with 5 volumes of acetone, separated by a centrifugation at 16 000×g for 12 min and dried overnight. After digestion with 2.5 μg/ml trypsin (Roche, sequencing grade) in 5% acetonitrile/50 mM NH4HCO3 at 30°C overnight, proteins were purified using the Supel-Tips C18 pipette tips and analysed by Electro Spray Ionisation (Amazon Speed ETD, Bruker) following after HPLC (Ultimate 3000, Dionex).

After exclusion of proteins found also in negative controls with MBP alone, we combined the data from both tissues and analysed them using QIAGEN's Ingenuity Pathway Analysis (IPA, QIAGEN Redwood City, www.ingenuity.com).

Pull-down assays with purified 14-3-3-γ

For pull-down experiments, purified 14-3-3-γ-His was bound to fresh Nickel-NTA agarose and incubated overnight at 4°C with purified MBP, MBP-A2-loop, MBP-A3-loop or MBP-A4-loop fusion proteins in binding buffer. After stringent washing with buffer containing 20 mM Tris–HCl, pH 7.9, 30 mM imidazole, 0.5 M NaCl and 0.1% Triton X-100, proteins were eluted with buffer containing 250 mM imidazole in 20 mM Tris–HCl, pH 7.9 and 0.5 M NaCl, and analysed by Western blot. For this, eluates were loaded on a gel and transferred on a nitrocellulose membrane after SDS–PAGE. After 1 h blocking with 5% (w/v) skim milk powder in TNNT buffer [20 mM Tris–HCl pH 7.5, 0.5 M NaCl, 0.02% [w/v] NaN3 and 0.05% [v/v] Tween 20], the membrane was probed with a monoclonal antibody against MBP (New England Biolabs), and finally with an anti-mouse antibody conjugated to alkaline phosphatase (Sigma). Treatment of membrane with primary as well as with secondary antibodies was performed in 2.5% milk/TNNT for 1 h at room temperature.

Far-western blot

For the Far-western blot analysis of the central loops, 1.5 or 3 µg of purified 14-3-3-γ were transferred onto a nitrocellulose membrane after SDS–PAGE. The membrane was cut into pieces and was blocked with 5% (w/v) skim milk powder in TNNT buffer for 1 h. The membrane pieces were separately incubated with 0.5 µM of A2-, A3- or A4-loop in the presence of 2.5% milk in TNNT buffer for 1 h. MBP without any loops was used as a negative control of interaction. After three washing steps with TNNT buffer for 10 min each, the membrane pieces were probed with a monoclonal antibody against MBP, and finally with an anti-mouse antibody conjugated to alkaline phosphatase. Treatment of membranes with primary as well as with secondary antibodies was performed in 2.5% milk/TNNT for 1 h at room temperature.

For the Far-western blot with 14-3-3-γ, 1.5 or 3 µg of purified loops or MPB were loaded onto a SDS-gel and transferred onto a nitrocellulose membrane. After blocking in 5% milk/TNNT, the membrane was incubated with 0.5 µM 14-3-3-γ in the presence of 2.5% milk in TNNT buffer for 1 h. Bound 14-3-3-γ was detected using a monoclonal antibody against His-tag (Qiagen) and visualised with alkaline phosphatase conjugated anti-mouse antibodies as above.

Transport assays in yeast

For sucrose uptake experiments, full length coding sequences of mouse SLC45A1 (NM_173774.3), SLC45A2 (NM_053077.3), SLC45A3 (NM_145977.2), and SLC45A4 (NM_001033219.3) as well as of potato Sut1 (NM_001318624.1) were cloned into pDR196 shuttle-vector as described by Bartolke et al. [1]. The coding sequence for murine 14-3-3-γ was amplified as a His-tagged version from pET29-His-14-3-3-γ plasmid and cloned into the YEp351 yeast/E. coli shuttle-vector using HindIII and SphI restriction sites. YEp351 is a standard yeast multicopy vector carrying a 2 µm replication origin and a URA3 selection marker [26].

The yeast strain HOD55-4B [MATα ura3-FS leu2-3,112 his3-11,15 kanMX-KlPGK1p::SUC2int mal0] was described previously [1]. To obtain a bmh1 deletion lacking the 14-3-3 protein and producing only the internal form of invertase, strain HOD242-1A (MATa ura3-FS leu2-3,112 his3-11,15 kanMX-KlPGK1p-SUC2int bmh1::SpHIS3 mal0) was constructed. For this purpose, a PCR-based one-step gene replacement method was employed, using the oligonucleotide pair (5′-AAGTGAGAAGAAAAAGCAAGTTAAAGATAAACTAAAGATAAAACTTCGTACGCTGCAGGTCGAC-3′ and 5′- TTTCTTTTTTTTAGTAATTTCTCTTTAGATTTATCAGAATACTTAGCATAGGCCACTAGTGGATCTG-3′) and plasmid pUG27 [27] as a template. The PCR product was introduced into the diploid strain DHD5 [28] and clones carrying the heterozygous bmh1::SpHIS3 deletion were selected on synthetic medium lacking histidine. Correct gene replacement was confirmed by PCR using flanking oligonucleotides. One correct clone was sporulated and subjected to tetrad analysis yielding the haploid segregant HOD240-1D (MATa ura3-52 leu2-3,112 his3-11,15 bmh1::SpHIS3 MAL3 SUC2 GAL). A backcross of this strain with HOD55-4B followed by sporulation and tetrad analysis yielded the desired segregant HOD242-1A.

For transport assays, yeast cultures were grown in minimal YNB medium supplemented with auxotrophic requirements and 2% (w/v) sucrose or glucose (both from Sigma in a purity ≥99.5%) to late exponential growth phase. Cells were collected by centrifugation, washed first with water, then with uptake buffer (55 mM sodium phosphate, pH 5.7), and finally resuspended in 750 µl of the same buffer to an OD600 of 30. Cells were kept on ice until shortly before the assays. After pre-incubation at 30°C for 3 min sugar uptake was initiated by adding 14C-sucrose (final concentration of 10 mM, specific radioactivity of 2.5 μCi/ml, purchased from Hartmann Analytic, Germany). Reactions were performed for 3 min at 30°C and stopped by 5-fold dilution of the sample with ice-cold water. Cells of each sample were washed with further 10 ml ice-cold water and collected by vacuum filtration on a 0.45 µm cellulose acetate filter (Whatman). Accumulated 14C-sucrose was measured by scintillation counting (Beckman LS 6500).

Experimental procedures in Xenopus laevis oocytes

Murine 14-3-3-γ coding sequence was amplified as a His-tagged version from pET29-His-14-3-3-γ plasmid, cloned into pGEMHE vector and used for synthesis of cRNA. The sequence of potato sucrose transporter Sut1 was first fused to a Green Fluorescence Protein (GFP) by cloning into pEGFP-N1 (Clontech). This Sut1-GFP construct was then amplified by PCR, cloned into the pGEMHE vector and used for synthesis of cRNA. cRNAs were synthesised from NheI-linearized plasmids using the mMessage mMachine-T7 in vitro transcription kit (Ambion) according to the manufacturer's protocol. cRNA concentration and purity were verified by measuring the UV absorbance using the Implen NanoPhotometer® (Implen, Germany).

Oocytes were obtained from Ecocyte Bioscience (Germany) and injected with 23 ng cRNA of Sut1 together with or without 23 ng of 14-3-3 cRNA. The corresponding volume of RNase free water was used for mock injection of oocytes. Upon injection, the oocytes were incubated 3 days at 18°C in Barth's buffer (88 mM NaCl, 2 mM KCl, 0.82 mM MgSO4, 0.66 mM NaNO3, 0.77 mM CaCl2, 5 mM HEPES/NaOH, pH 7.6) containing 12 mg/l gentamycin with daily medium changes. Three days after injection, oocytes were used for uptake experiments with [14C]sucrose, for Western blot and for immunocytochemistry.

For uptake experiments, oocytes were washed in oocyte-uptake-buffer (5 mM MES, 115 mM NaCl, 2 mM KCl, 2 mM CaCl2, 1 mM MgCl2, pH 5.6) and pre-incubated for 5 min in a buffer containing 50 µM CCCP/0.01% DMSO or in a buffer containing only 0.01% DMSO. Uptake was started by adding [14C]sucrose at a final concentration of 10 mM and performed for 1 h at 20°C. Reactions were stopped by 20-fold dilution of the sample with ice-cold phosphate-buffered saline (PBS; 10 mM sodium phosphate buffer and 150 mM NaCl, pH 7.4). After washing steps with ice-cold PBS, single oocytes were lysed in 0.1% sodium dodecyl sulfate and analyzed by scintillation counting. The CCCP inhibitable part of sucrose uptake was determined in each single experiment by subtraction of uptake values in the presence of CCCP from uptake values measured without CCCP. Statistical analysis of transport experiments was performed by two-tailed Student's t-test.

For immunocytochemistry, oocytes were fixed in 4% formaldehyde/PBS overnight at 4°C, washed three times with PBS for 20 min each, and incubated in Roti-Block (Carl-Roth, Germany) for 1 h at room temperature. After an overnight incubation at 4°C with an antibody against GFP (Abcam) at a dilution of 1:1000 in Roti-Block, oocytes were washed with Roti-Block (three times for 10 min) and probed with an anti Rabbit-Alexa 488 antibody (Abcam; dilution of 1:200) in Roti-Block for 1 h at room temperature. Then oocytes were washed for 5 min twice with PBS/0.05% Tween 20 and once with PBS only, were embedded by tissue-freezing medium (Leica Microsystems) and frozen in melting iso-pentane chilled in liquid nitrogen. Sections of 6 μm were cut on a cryostat (Leica Microsystems) at a chamber temperature of −20°C, collected on SuperfrostPlus microscope slides (Menzel-Gläser, Germany), covered with Vectashield (Vector Laboratories), and analysed with an Olympus IX70 fluorescence microscope using Metamorph (Molecular Devices, version 6.2) software.

For the Western blot, 10–12 oocytes were homogenised in 150 µl of 20 mM Tris–HCl, pH 8.0 containing a protease inhibitor (Roche Diagnostics) and centrifuged 15 min at 1000×g and 4°C in order to remove cell debris and egg yolk [29]. The resulting supernatant was collected, boiled with Laemmli buffer and served as crude extract. After SDS–PAGE, proteins were transferred onto a nitrocellulose membrane and probed with an antibody against GFP as well as an antibody against His-tag. For this purpose, the membrane was first blocked with 5% skim milk powder in TNNT buffer for 1 h and then incubated with a mix of primary antibodies: anti His-tag (Qiagen) and anti GFP (Abcam). Primary antibodies were visualised with secondary alkaline phosphatase conjugated antibodies: anti-mouse and anti-rabbit (both from Sigma). Treatment of the membrane with primary as well as with secondary antibodies was performed in 2.5% milk/TNNT for 1 h at room temperature.

Quantification of Sut1 signal at oocyte plasma membrane and in immunoblot were determined by the fluorescence intensity detection method according to ref. [30]. For that the signal intensities within 150 boxes (5 × 5 pixel/box; 50 boxes/oocyte) placed on the plasma membrane of three single oocytes expressing Sut1 without 14-3-3 were analysed and compared with that of three oocytes expressing Sut1 together with 14-3-3. Quantification of Sut1 signal in immunoblot was performed by measurement of signal intensities within seven 20 × 2 pixel boxes placed on each protein band. Statistical analysis was performed by two-tailed Student's t-test.

Results

Screening for interaction partners of SLC45 sugar transporters

To find putative interaction partners of SLC45A2, SLC45A3 and SLC45A4, we purified their recombinant big intracellular central loops between the sixth and seventh transmembrane helices as the MBP fusion proteins, coupled them to beads, incubated them with cytosolic extracts from mouse brain or kidney and determined the nature of associated proteins by mass spectrometry. Although the identification scores for most of the found proteins were less than 50 and did not appear to represent reliable results, we performed a further analysis of data in order to get any ideas about regulation of the transporters. For this reason, we excluded proteins found also in negative controls with MBP alone, combined the data from both tissues and analysed them using QIAGEN's Ingenuity Pathway Analysis (IPA, QIAGEN Redwood City, www.ingenuity.com). In this way, we got a first indication for the involvement of 14-3-3-mediated signalling pathway for transporters of the SLC45 family (Table 1).

Table 1
Top canonical pathways
A2-loop Remodeling of epithelial adherens junctions 
Gap junction signaling 
Gluconeogenesis I 
Sertoli cell — Sertoli cell junction signaling 
14-3-3-mediated signaling 
Germ cell — Sertoli cell junction signaling 
A3-loop Remodeling of epithelial adherens junctions 
14-3-3-mediated signaling 
Epithelial adherens junction signaling 
Germ cell — Sertoli cell junction signaling 
Gap junction signaling 
A4-loop Sertoli cell — Sertoli cell junction signaling 
A2-loop Remodeling of epithelial adherens junctions 
Gap junction signaling 
Gluconeogenesis I 
Sertoli cell — Sertoli cell junction signaling 
14-3-3-mediated signaling 
Germ cell — Sertoli cell junction signaling 
A3-loop Remodeling of epithelial adherens junctions 
14-3-3-mediated signaling 
Epithelial adherens junction signaling 
Germ cell — Sertoli cell junction signaling 
Gap junction signaling 
A4-loop Sertoli cell — Sertoli cell junction signaling 

Since pull-down assay identify, in general, not only direct but also mediated interactors, and since the overall scores of proteins detected by mass spectrometry were rather low, we complemented this screen with a second approach, the yeast two-hybrid analysis. For this assay, the central A2, A3 and A4 loops were used as bait in the Y2HGold Yeast Strain which was mated with the Y187 Yeast Strain, containing the normalised mouse cDNA library. The number of screened clones for the A2 loop was calculated to be 1.9 × 107 and the mating efficiency was 27.8%. Out of this screen, 35 positive clones were able to grow on the high stringency selective plates. After the elimination of false positives, two genuine positive proteins were found: guanylate kinase and G-protein coupled receptor-associated sorting protein 2. The number of screened clones for the A3 loop was 1.5 × 107 with a mating efficiency of 14.9%. After excluding false positives, 39 genuine positive clones were left. A surprisingly high number of RNA- and DNA-binding proteins were identified which led us to doubt the efficacy of the screen. There were, however, two candidates supporting a putative physiological role for SLC45A3 in myelination: the ganglioside-induced differentiation-associated protein 1-like 1 and the major prion protein precursor. For the A4 loop, 107 clones could be screened with a mating efficiency of 5.5%, and 27 genuine clones were identified after excluding false positives and duplicates. For instance, five of them are involved in the ubiquitin proteasome pathway, three proteins are involved in spermatogenesis and further protein is also highly expressed in testis and in proliferating cells. Noticeably, three protein modulators were identified that are involved in diverse signalling pathways, including carbohydrate metabolism. One of them was the gamma isoform of 14-3-3 proteins (14-3-3-γ).

Results of both screens [for detailed information, see (31)], the pull-downs from tissue extracts followed by mass spectrometry as well as the yeast two-hybrid assays, detected proteins involved in carbohydrate metabolism, protein turnover and degradation, and signalling, and suggested putative regulatory roles for the central cytosolic loops of the SLC45 sugar transporters. Mass spectrometry indicated the 14-3-3 signalling pathway as a putative regulatory pathway for the transporters and the yeast two-hybrid assay found an interaction of 14-3-3-γ with the A4 loop. Since 14-3-3 proteins are involved in the regulation of glycolysis, and because both the A3 and the A4 loops contain a putative 14-3-3 binding site, as suggested by an ELM motif search, 14-3-3-γ seemed a good candidate for the regulation of the SLC45 transporters and was chosen for further analysis.

Interaction of SLC45 transporters with 14-3-3-γ protein

The 14-3-3-γ protein, detected as putative interaction partner of SLC45A4, seems us to be a very interesting candidate for regulation of the SLC45 transporter family in general. Co-transformation of murine 14-3-3-γ with constructs encoding the murine A2-, A3- and A4-loops into the yeast strain Y2HGold indeed showed an interaction not only with the loop of A4 initially employed, but also with the loop of A3 (Figure 1). The A2-loop did not display interaction under these conditions.

Interaction of 14-3-3-γ with A3-loop and A4-loop.

Figure 1.
Interaction of 14-3-3-γ with A3-loop and A4-loop.

An interaction analysis in yeast showed an activation of the Gal4 responsive reporter genes and growth on a selective QDO/X plate of 14-3-3-γ co-transformed with A3-loop and A4-loop, but not with the A2-loop. 14-3-3-γ did not auto-activate the reporter genes when co-transformed with the empty pGBKT7 vector. Another negative control, pGBKT7-Lam co-transformed with pGADT7-T into Y2HGold showed no growth, whereas the positive control, a co-transformation of pGBKT7-53 with pGADT7-T grew well. Growth on a DDO plate confirmed the successful transformation into the Y2HGold yeast strain.

Figure 1.
Interaction of 14-3-3-γ with A3-loop and A4-loop.

An interaction analysis in yeast showed an activation of the Gal4 responsive reporter genes and growth on a selective QDO/X plate of 14-3-3-γ co-transformed with A3-loop and A4-loop, but not with the A2-loop. 14-3-3-γ did not auto-activate the reporter genes when co-transformed with the empty pGBKT7 vector. Another negative control, pGBKT7-Lam co-transformed with pGADT7-T into Y2HGold showed no growth, whereas the positive control, a co-transformation of pGBKT7-53 with pGADT7-T grew well. Growth on a DDO plate confirmed the successful transformation into the Y2HGold yeast strain.

To substantiate the observed interactions, pull-down assays with recombinant SLC45-loops and 14-3-3-γ were performed. For this purpose, murine 14-3-3-γ was expressed as a His-tag fusion, while SLC45-loops were obtained as fusions to MBP. Purified 14-3-3-γ was coupled to the Ni2+-NTA beads, incubated with purified SLC45-loops and eluted from the beads after several washing steps. Figure 2 shows that the A3 and A4 loops but not the loop of A2, were co-eluted with 14-3-3-γ.

Pull-down assay of 14-3-3-γ with SLC45-loops.

Figure 2.
Pull-down assay of 14-3-3-γ with SLC45-loops.

Purified and bead-coupled 14-3-3-γ was incubated with purified MBP or MBP-fusion of A2-, A3- and A4-loops and eluted from the beads after washing off unbound compounds. The eluates were transferred onto a nitrocellulose membrane after SDS–PAGE and analysed by Western blot. Each lane contained 20 µl eluate. (A) Ponceau staining of the membrane shows the band of 14-3-3-γ (∼30 kDa). Weak stainings in the last two lanes already indicate the presence of the A3-loop and the A4-loop. (B) Immunological detection of the same membrane with an antibody against MBP shows the presence of A3-loop (∼50 kDa) and A4-loop (∼80 kDa) in the eluates. *Band of unknown origin.

Figure 2.
Pull-down assay of 14-3-3-γ with SLC45-loops.

Purified and bead-coupled 14-3-3-γ was incubated with purified MBP or MBP-fusion of A2-, A3- and A4-loops and eluted from the beads after washing off unbound compounds. The eluates were transferred onto a nitrocellulose membrane after SDS–PAGE and analysed by Western blot. Each lane contained 20 µl eluate. (A) Ponceau staining of the membrane shows the band of 14-3-3-γ (∼30 kDa). Weak stainings in the last two lanes already indicate the presence of the A3-loop and the A4-loop. (B) Immunological detection of the same membrane with an antibody against MBP shows the presence of A3-loop (∼50 kDa) and A4-loop (∼80 kDa) in the eluates. *Band of unknown origin.

To exclude the possibility that the interaction of 14-3-3-γ with the A3- and A4-loops was mediated by another protein, for instance co-purified from bacterial cells, we confirmed the interactions by Far-western blots. The 14-3-3-γ protein was blotted onto a membrane after SDS–PAGE and separately incubated with purified SLC45 loops. The bound loops were detected with an antibody against MBP. In this case the interaction of 14-3-3 with A3 or A4 was clearly confirmed (Figure 3A,B). In contrast, when the loops were transferred onto a membrane, incubated with native 14-3-3-γ and bound 14-3-3-γ was probed via an anti His-tag antibody, only an interaction with A4-loop was observed (Figure 3C,D).

Interaction of 14-3-3-γ with SLC45-loops in Far-western blot.

Figure 3.
Interaction of 14-3-3-γ with SLC45-loops in Far-western blot.

(A) Purified recombinant 14-3-3-γ was transferred onto a nitrocellulose membrane after SDS–PAGE and stained by Ponceau. (B) The membrane from A was cut into four pieces and used for Far-western blot with purified MBP-fusion proteins: MBP alone, A2-loop, A3-loop or A4-loop. Bound proteins were detected with anti-MBP antibody. (C) Purified MBP alone, MBP-fusion proteins with A2-, A3- or A4-loop, respectively, were transferred onto a nitrocellulose membrane after SDS–PAGE and stained by Ponceau. (D) The membrane from C was incubated with purified His-tag-14-3-3-γ and bound 14-3-3-γ was detected with anti His-tag antibody.

Figure 3.
Interaction of 14-3-3-γ with SLC45-loops in Far-western blot.

(A) Purified recombinant 14-3-3-γ was transferred onto a nitrocellulose membrane after SDS–PAGE and stained by Ponceau. (B) The membrane from A was cut into four pieces and used for Far-western blot with purified MBP-fusion proteins: MBP alone, A2-loop, A3-loop or A4-loop. Bound proteins were detected with anti-MBP antibody. (C) Purified MBP alone, MBP-fusion proteins with A2-, A3- or A4-loop, respectively, were transferred onto a nitrocellulose membrane after SDS–PAGE and stained by Ponceau. (D) The membrane from C was incubated with purified His-tag-14-3-3-γ and bound 14-3-3-γ was detected with anti His-tag antibody.

The Far-western blots strongly suggested a direct interaction between A4-loop and 14-3-3-γ. For the lack of interaction with the A3-loop at least two explanations are possible: Either the first detected interaction was indeed mediated by another protein (co-purified with A3-loop), thus indicating a false positive result, or the 14-3-3-γ required the A3-loop in its native conformation and cannot interact in its SDS denatured form.

Effect of 14-3-3-γ protein on activity of SLC45 transporters

Since 14-3-3 proteins exert regulatory functions on many different proteins, we wondered, whether the binding of 14-3-3-γ can affect the activity of SLC45 transporters. Because of the observed direct interaction of 14-3-3-γ with A4-loop, we decided to start further investigations with SLC45A4. Thus, we overexpressed a mouse 14-3-3-γ construct in the yeast strain HOD55-4B, also producing mouse SLC45A4. The recipient yeast strain is deficient for disaccharide transporters and does not produce extracellular invertase, as previously shown in our [14C]sucrose uptake assays studying the SLC45 transporters [1]. As shown in the two left-hand columns of Figure 4, the modified yeast strain can transport sucrose, a feature not affected by co-expressing of the 14-3-3-γ construct. Because both yeast orthologues of 14-3-3 proteins, Bmh1 and Bmh2, remained intact in the HOD55-4B strain with a possible interference in the assays, we next deleted the BMH1 gene, since deletion of both genes is synthetically lethal [20]. Indeed, co-expression of mouse 14-3-3-γ in the Δbmh1 yeast strain HOD242-1A caused a strong decrease in uptake rate of SLC45A4 transporter (Figure 4, right-hand columns). Interestingly, the comparison of transport rates of SLC45A4 without mouse 14-3-3-γ (Figure. 4, both black columns) showed that sucrose uptake in the wild-type strain with intact BMH1 and BMH2 genes was significantly lower than in the Δbmh1 strain. This indicates that both proteins, mouse 14-3-3-γ and yeast Bmh1, negatively regulate transport activity of SLC45A4.

Effect of 14-3-3 protein on sucrose uptake by SLC45A4.

Figure 4.
Effect of 14-3-3 protein on sucrose uptake by SLC45A4.

HOD55-4B and Δbmh1 yeast strains were co-transformed with pDR196-SLC45A4 together with mouse 14-3-3-γ in YEp351vector or with empty vector YEp351 only. [14C]sucrose uptake was performed for 3 min. Yeast cells transformed with both empty vectors, pDR196 and YEp351, served as negative controls and their uptake values were subtracted as background from all presented data. Data points correspond to the means ± s.d. of four independent experiments running in triplicates (*P < 0.05, ***P < 0.001).

Figure 4.
Effect of 14-3-3 protein on sucrose uptake by SLC45A4.

HOD55-4B and Δbmh1 yeast strains were co-transformed with pDR196-SLC45A4 together with mouse 14-3-3-γ in YEp351vector or with empty vector YEp351 only. [14C]sucrose uptake was performed for 3 min. Yeast cells transformed with both empty vectors, pDR196 and YEp351, served as negative controls and their uptake values were subtracted as background from all presented data. Data points correspond to the means ± s.d. of four independent experiments running in triplicates (*P < 0.05, ***P < 0.001).

We also expressed the other three members of the SLC45 family in the Δbmh1 yeast strain and checked the transport rates in the presence and absence of mouse 14-3-3-γ. Co-expression of 14-3-3-γ led to a significant reduction in sucrose uptake rate of SLC45A2 transporter, while the activity of SLC45A1 as well as that of SLC45A3 appeared to remain basically unaffected (Figure 5).

Mouse 14-3-3-γ reduces sucrose uptake rate of SLC45A2 and of SLC45A4.

Figure 5.
Mouse 14-3-3-γ reduces sucrose uptake rate of SLC45A2 and of SLC45A4.

Cells of Δbmh1 strain were co-transformed with pDR196-SLC45A1, pDR196-SLC45A2, pDR196-SLC45A3 or pDR196-SLC45A4, respectively, together with mouse 14-3-3-γ in YEp351vector or together with empty vector YEp351 only. Uptake rate was measured using [14C]sucrose and shown as a relative values, where the 100% values correspond to transport rate of each transporters without 14-3-3-γ. Yeast cells transformed with both empty vectors, pDR196 and YEp351, served as negative controls and their uptake values were subtracted as background from all presented data. Data points correspond to the means ± s.d. of three or four independent experiments running in triplicates (n.s.: not significant, *P < 0.05, ***P < 0.001).

Figure 5.
Mouse 14-3-3-γ reduces sucrose uptake rate of SLC45A2 and of SLC45A4.

Cells of Δbmh1 strain were co-transformed with pDR196-SLC45A1, pDR196-SLC45A2, pDR196-SLC45A3 or pDR196-SLC45A4, respectively, together with mouse 14-3-3-γ in YEp351vector or together with empty vector YEp351 only. Uptake rate was measured using [14C]sucrose and shown as a relative values, where the 100% values correspond to transport rate of each transporters without 14-3-3-γ. Yeast cells transformed with both empty vectors, pDR196 and YEp351, served as negative controls and their uptake values were subtracted as background from all presented data. Data points correspond to the means ± s.d. of three or four independent experiments running in triplicates (n.s.: not significant, *P < 0.05, ***P < 0.001).

Plant sucrose transporters and 14-3-3-γ protein

The members of the SLC45 family show only 20–30% identity of amino acid sequences among each other, and they all exhibit an identity with plant H+/sucrose cotransporters of ∼20% [6]. This similarity prompted us to check whether a 14-3-3 protein may also affect plant H+/sucrose cotransporters. For this purpose we cloned the gene encoding the potato H+/sucrose transporter Sut1 into pDR196 vector, co-expressed this transporter with or without mouse 14-3-3-γ protein in the Δbmh1 yeast strain, and analysed uptake rates using [14C]sucrose (Figure 6). In these assays, 14-3-3-γ had a similar effect on the plant Sut1 transporter as on mammalian SLC45A4 and SLC45A2, i.e. it significantly decreased the rate of sucrose uptake.

Mouse 14-3-3-γ reduces sucrose uptake rate by Sut1 in Δbmh1 strain.

Figure 6.
Mouse 14-3-3-γ reduces sucrose uptake rate by Sut1 in Δbmh1 strain.

Cells of Δbmh1 yeast strain were transformed with pDR196-Sut1 together with mouse 14-3-3-γ carried on the YEp351vector or together with empty vector YEp351 only, and used for uptake assays with [14C]sucrose. Yeast cells transformed with both empty vectors pDR196 and YEp351 served as a negative control and values were subtracted as background. Data points correspond to the means ± s.d. of three independent experiments running in triplicates (*P < 0.05).

Figure 6.
Mouse 14-3-3-γ reduces sucrose uptake rate by Sut1 in Δbmh1 strain.

Cells of Δbmh1 yeast strain were transformed with pDR196-Sut1 together with mouse 14-3-3-γ carried on the YEp351vector or together with empty vector YEp351 only, and used for uptake assays with [14C]sucrose. Yeast cells transformed with both empty vectors pDR196 and YEp351 served as a negative control and values were subtracted as background. Data points correspond to the means ± s.d. of three independent experiments running in triplicates (*P < 0.05).

To confirm the effect of 14-3-3 protein on the activity of H+/sucrose cotransporters in another experimental model, we tried to express the murine SLC45 proteins and plant Sut1 together with murine 14-3-3-γ in Xenopus oocytes. Unfortunately, only the plant Sut1 transporter was functionally produced in this system. Therefore, Xenopus oocytes were incubated with [14C]sucrose in the absence and in the presence of the specific protonophore carbonyl cyanide m-chlorophenylhydrazone (CCCP). CCCP destroys the proton gradient over the membrane and is widely used to study proton-coupled mechanisms of transport [32,33]. Thus, the portion of sucrose uptake that can be inhibited by CCCP reflects the transport mediated exclusively by an H+/sucrose cotransporter such as Sut1. Figure 7 shows that oocytes expressing Sut1 accumulated significantly more sucrose than oocytes expressing both Sut1 and 14-3-3-γ (black columns), while in the presence of CCCP oocytes of both groups took up sucrose in similar negligible amounts (white columns). Comparison of the calculated CCCP inhibitable part of sucrose uptakes using unpaired t-test confirmed the decrease of sucrose uptake in the presence of 14-3-3-γ with a value of P < 0.001.

14-3-3-γ decreased rate of sucrose uptake by Sut1 expressed in Xenopus oocytes.

Figure 7.
14-3-3-γ decreased rate of sucrose uptake by Sut1 expressed in Xenopus oocytes.

Potato sucrose transporter Sut1 was fused to GFP and expressed in Xenopus oocytes alone or together with murine 14-3-3-γ protein fused with a His-tag. On the third day after cRNA injection oocytes were used for uptake experiments with 14C-sucrose. Uptake was performed for 1 h at 20°C in Oocyte-uptake-buffer (5 mM MES, 115 mM NaCl, 2 mM KCl, 2 mM CaCl2, 1 mM MgCl2, pH 5.6) in the presence of CCCP/DMSO or of DMSO only. After washing steps with cold PBS the [14C]-sucrose accumulation by singe oocyte was analysed. Data points correspond to the means ± s.d. of thirteen single oocytes analysed by three independent experiments (***P < 0.001).

Figure 7.
14-3-3-γ decreased rate of sucrose uptake by Sut1 expressed in Xenopus oocytes.

Potato sucrose transporter Sut1 was fused to GFP and expressed in Xenopus oocytes alone or together with murine 14-3-3-γ protein fused with a His-tag. On the third day after cRNA injection oocytes were used for uptake experiments with 14C-sucrose. Uptake was performed for 1 h at 20°C in Oocyte-uptake-buffer (5 mM MES, 115 mM NaCl, 2 mM KCl, 2 mM CaCl2, 1 mM MgCl2, pH 5.6) in the presence of CCCP/DMSO or of DMSO only. After washing steps with cold PBS the [14C]-sucrose accumulation by singe oocyte was analysed. Data points correspond to the means ± s.d. of thirteen single oocytes analysed by three independent experiments (***P < 0.001).

In a further approach, we visualised the signal of GFP fused to the Sut1 transporter in oocytes. In vivo fluorescence observed in a binocular revealed that the signal of Sut1-GFP was definitely stronger in oocytes expressing Sut1 alone than in oocytes additionally expressing 14-3-3-γ (Figure 8A). To find out whether this dissimilarity was caused by different densities of transporter in the plasma membrane, cryosections of oocytes were produced, probed with an antibody against GFP and visualised under a microscope. Indeed, signals in oocytes co-injected with 14-3-3-γ cRNA were hardly detectable at the plasma membrane, while controls without 14-3-3-γ cRNA showed a strong Sut1-GFP signal at their plasma membranes (Figure 8B). Analysis of signal intensities confirmed with a value of P < 0.001 an ∼3-fold reduction in fluorescence at the oocytes plasma membrane when 14-3-3 was co-expressed (Figure 8D, left).

Reduction in Sut1 expression in the presence of 14-3-3-γ.

Figure 8.
Reduction in Sut1 expression in the presence of 14-3-3-γ.

Potato sucrose transporter Sut1 was fused to GFP and expressed in Xenopus oocytes alone or together with murine 14-3-3-γ protein fused to a His-tag. (A) expression of Sut1-GFP was analysed in vivo by a fluorescence binocular. (B) Oocytes from A were fixed, probed in 6 µm cryosections with an antibody against GFP and analysed by fluorescence microscopy. Bar 25 µm. (C) 12, 6 or 3 µg of oocytes crude extract were transferred onto a nitrocellulose membrane after SDS–PAGE. The membrane was probed with both an anti His-tag and an anti GFP antibody. Primary antibodies were visualised with secondary alkaline phosphatase conjugated antibodies: anti-mouse and anti-rabbit. 1: oocytes injected with Sut1 cRNA, 2: oocytes injected with Sut1 cRNA as well as with cRNA of 14-3-3-γ. (D) quantification of Sut1 signal at the oocyte plasma membrane (left) and in immunoblot (right). Data points correspond to the means ± s.d. (***P < 0.001).

Figure 8.
Reduction in Sut1 expression in the presence of 14-3-3-γ.

Potato sucrose transporter Sut1 was fused to GFP and expressed in Xenopus oocytes alone or together with murine 14-3-3-γ protein fused to a His-tag. (A) expression of Sut1-GFP was analysed in vivo by a fluorescence binocular. (B) Oocytes from A were fixed, probed in 6 µm cryosections with an antibody against GFP and analysed by fluorescence microscopy. Bar 25 µm. (C) 12, 6 or 3 µg of oocytes crude extract were transferred onto a nitrocellulose membrane after SDS–PAGE. The membrane was probed with both an anti His-tag and an anti GFP antibody. Primary antibodies were visualised with secondary alkaline phosphatase conjugated antibodies: anti-mouse and anti-rabbit. 1: oocytes injected with Sut1 cRNA, 2: oocytes injected with Sut1 cRNA as well as with cRNA of 14-3-3-γ. (D) quantification of Sut1 signal at the oocyte plasma membrane (left) and in immunoblot (right). Data points correspond to the means ± s.d. (***P < 0.001).

Finally, we determined the total amount of Sut1 transporter in both groups of oocytes by Western blot. For that, 3, 6 and 12 µg of total protein from crude oocyte extracts were separated in a SDS gel, transferred onto a membrane and probed with an antibody against GFP as well as with an anti His-tag antibody detecting the mouse 14-3-3-γ-His-tag fusion protein (Figure 8C). Both proteins were expressed: Sut1-GFP (upper band) and 14-3-3-γ-His-tag (lower band). Quantification of band intensities revealed that the total amount of Sut1-GFP in oocytes co-expressing 14-3-3-γ was ∼2-fold smaller than in control oocytes (Figure 8D right).

Discussion

In the present study, we investigated the involvement of 14-3-3 proteins in regulation of proton-coupled sugar co-transporters of the SLC45 family. Although the precise mechanism and the underlying physiological purpose have been elucidated only in a small number of 14-3-3 interactions, three principal mechanisms of regulation by direct interaction with 14-3-3 proteins have been described so far [34,35,22]. As ‘clamping' we understand the mechanism when binding of the 14-3-3s leads to stabilisation of client proteins in their activated or deactivated conformation. For instance, binding of 14-3-3 dimers to the auto-inhibitory domain of the plant plasma membrane H+-ATPase prevents the domain movement and finally the inactivation of proton pumping. Anchoring of the nicotinic acetylcholine receptor to the microtubule cytoskeleton by 14-3-3 proteins is one of the examples for scaffolding as a further type of regulation. Thus, anchoring 14-3-3 target proteins in a specific cellular compartment may increase their accessibility and facilitate interaction with other proteins. Finally, 14-3-3 binding may also lead to physical masking of specific motifs in client proteins, and in this way to impeding or at least retarding canonical cell processes. For instance, 14-3-3 proteins have been shown to regulate the endoplasmic reticulum export of several client proteins by occlusion of their interaction site with the coat protein complex I, a key player in the biosynthetic delivery of membrane proteins. It is well established that 14-3-3 proteins bind to phosphorylated serine or threonine residues in many client proteins (for reviews, see [2123]). However, not all 14-3-3 protein interactions are phosphorylation-dependent [3643]. For instance, both human and Dictyostelium non-muscle myosin II have been shown to interact in vitro and in vivo with 14-3-3 directly and phosphorylation-independently [44]. Another example is 14-3-3-zeta from bovine brain which binds to the phosphorylated as well as the nonphosphorylated microtubule-associated protein tau [45].

The regulatory potential of 14-3-3 proteins seems to be enormous, since more than 200 proteins have been identified as their direct interaction partners [16,46]. For sugar transporters, however, only indirect effects of 14-3-3 proteins have been reported so far. For instance, 14-3-3 binding to the Rab GTPase-activating protein TBC1D4/AS160 is required for activation of trafficking of GLUT1 or GLUT4 containing vesicles from the cytosol to the plasma membrane [4749]. In addition, interaction with 14-3-3 proteins has been shown for GTBP70, which binds and regulates mammalian GLUT1, GLUT2 and GLUT4 transporters [15]. Furthermore, the amount of yeast glucose transporter Hxt6 at the plasma membrane is regulated by 14-3-3 via the adaptor protein Rod1 [50].

In this study, a novel direct interaction of 14-3-3 proteins with sugar transporters is shown. By yeast two-hybrid analysis as well as by pull-down assays we detected the interaction of 14-3-3-γ with two members of the SLC45 family, A3 and A4. Far-western blots confirmed a direct interaction only for the SLC45A4 member, while no interaction with A3 could be observed when 14-3-3-γ was incubated with SDS-treated SLC45A3. We assume that the interaction between 14-3-3-γ and A3 was mediated by a protein co-purified from E. coli cell extract, which is in line with the results from sucrose uptake experiments, where SLC45A3 appeared not to be affected by 14-3-3-γ co-expressed in yeast cells. In contrast, co-expression of murine 14-3-3-γ in yeast led to the reduction in transport activity of SLC45A4 as well as of SLC45A2, although the A2 member did not show any direct interaction with 14-3-3-γ in the other assays. This discrepancy can probably be explained by the fact that all interaction studies were performed only with the cytosolic central loop of the transporters, but not with the complete proteins as for the transport assays. Therefore, a putative 14-3-3 interaction site within another domain of the A2 transporter cannot be excluded. Indeed, 14-3-3 binding sites outside the loop cannot be excluded for the A4 transporter, too.

Another possible explanation would be that SLC45A3 interacts with the γ-isoform, but that a heterodimer with another mammalian 14-3-3 isoform is required for the regulation of transport activity. However, only yeast Bmh2 as a putative partner for dimerisation was present during uptake assays in yeast. While 14-3-3-sigma preferentially forms homodimers [51], the other mammalian isoforms can act as both homo- and heterodimers. The specific composition of dimers has been discussed as having functional implications in the differential action of 14-3-3 proteins, because all isoforms are ubiquitously expressed in mammalian tissues and are highly conserved. Thus, the amino acid sequence identity of the γ-isoform to other mouse isoforms ranges between 75 and 64%. The 14-3-3 proteins are highly conserved also evolutionary: mouse 14-3-3-γ shares an identity of more than 60% with both yeast isoforms as well as with plant 14-3-3 proteins. Due to their high similarity, 14-3-3 proteins seem to be well compatible among each other. Although disruption of both yeast genes BMH1 and BMH2 is lethal, indicating a critical role for the protein function, both genes have been successfully complemented by a 14-3-3 protein from Arabidopsis thaliana [20]. In our study, co-expression of mouse 14-3-3-γ also affected the plant H+/sucrose cotransporter Sut1, which shows a high similarity to SLC45 proteins, by decreasing its uptake activity in yeast cells as well as in Xenopus oocytes. Furthermore, yeast Bmh1 seems to have a similar influence on the mouse SLC45A4 transporter, since deletion of the BMH1 gene led to an increase in uptake activity (Figure 4). Thus, universality of 14-3-3 proteins and their functional compatibility appears to occur in all three phyla, animals, plants and fungi.

Due to the direct interaction of SLC45 transporters with 14-3-3-γ and due to the reduction in transport by co-expression of 14-3-3-γ, we first assumed that 14-3-3 binding leads to the occlusion of functional transporter sites needed for the interaction with their substrates, sugars or protons. In this case, only Sut1 properties themselves, but not its localisation at the plasma membrane of Xenopus oocytes would be affected. However, analysis of cryo-sections demonstrated a significant reduction in the Sut1 fluorescence signal at the plasma membrane of oocytes when Sut1 was co-expressed with 14-3-3-γ (Figure 8B,D left). Therefore, transporter trafficking seems to be negatively affected by 14-3-3-γ. Trafficking is a prevalent regulatory mechanism of transporters, because translocation from or to the membranes allows for fast and precise control of their activity, and therewith for a high flexibility under changeful conditions. Since regulation normally occurs on several levels and alteration of total protein amount is well-known phenomenon, we checked this point and found that also the total amount of Sut1 sugar transporter significantly decreased when 14-3-3-γ was co-expressed (Figure 8C,D, right). Whether a reduced protein synthesis or a boosted protein degradation is responsible for the decrease in Sut1 protein amount could not be elucidated in our present study. Interestingly, 14-3-3-γ has been shown to bind to the kidney urea transporter UT-A1, and to have a negative effect on its uptake activity by increasing its ubiquitination and degradation [52]. This scenario seems us to be plausible also for the Sut1 transporter: binding of 14-3-3-γ may lead to a conformational change of Sut1, thus making it more accessible to ubiquitination. In contrast, it appears to us rather unlikely that protein synthesis was affected, because the expression in oocytes was induced by injection of cRNA (capped and poly-adenylated), thus reducing the influence of 14-3-3-γ on protein synthesis merely to translational step.

Although regulatory mechanisms of transporters by 14-3-3 proteins have not yet been defined in detail, our study presents the first finding of regulation in the mammalian SLC45 family. Moreover, we showed here for the first time the effect of 14-3-3 proteins on Sut1, a plant H+/sucrose transporter.

Abbreviations

     
  • GFP

    green fluorescence protein

  •  
  • GTBPs

    glucose transporter binding-proteins

  •  
  • MBP

    maltose binding-protein

Author Contribution

Screening for interaction partners as well as experiments shown in Figures 13 were designed and analysed by R.B., H.W. and O.V., and were performed by R.B.. Experiments shown in Figures 46 were designed and analysed by K.T., H.W. and O. V., and were performed by K.T.. Experiments shown in Figures 7 and 8 were designed and performed by O.V., and analysed by O.V., K.T. and H.W.. J.J.H. created the yeast bmh1 deletion strain and advised on yeast metabolism. The paper was written by O.V. and H.W. O.V. and H.W. conceived and supervised the project.

Funding

This work was supported by the Deutsche Forschungsgemeinschaft [VI 673/2-1 to O.V., WI 698/7-1 to H. Wieczorek], by the Lichtenberg Ph.D. Program (to R.B.) and by the ‘Incentive Award of the Faculty of Biology/Chemistry’ of the University of Osnabrück (to O.V.).

Competing Interests

The Authors declare that there are no competing interests associated with the manuscript.

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