Conserved Glu-47 and Lys-50 residues are critical for UDP-N-acetylglucosamine/UMP antiport activity of the mouse Golgi-associated transporter Slc35a3

Expression and transport activity of the mouse SLC35A3 assessed in yeast using a whole-cell fluorescence approach

Mouse Slc35a3 (mSLC35A3), a protein consisting of 326 amino acids, shares between 95 and 96% sequence identity with four mammalian SLC35A3 variants, which previously have been experimentally characterized as Golgi-localized UDP-GlcNAc transporters (31, 37, 45, 46). To confirm the substrate specificity of mSlc35a3, Kl3 cells, a mutant strain of the yeast Kluveromyces lactis lacking a functional UDP-GlcNAc transporter, were transformed with either the empty vector (plasmid pE4) or pE4mSLC35A3. Subsequently, the cells were incubated with GSII lectin conjugated to FITC (GSII–FITC), which specifically binds GlcNAc-containing glycoconjugates. Only the suspension of Kl3 cells transformed with pE4mSLC35A3 showed fluorescence levels similar to that of WT Kl8 cells, carrying the functional UDP-GlcNAc transporter, indicating that they recovered the ability to bind the lectin GSII–FITC (Fig. 1A, dark gray bars). In this in vivo system, UDP-GlcNAc transport activity, mediated by mSLC35A3, is indirectly evaluated through the restoration of GlcNAc-containing glycoconjugates at the surface of Kl3 cells. This implicates at least three steps as follows: the restoration of the import of UDP-GlcNAc into the lumen of the Golgi apparatus through expression of an active transporter; the synthesis of GlcNAc glycoconjugates by specific lumenal transferases; and an appropriate membrane cell-sorting process of these products (47).

One aim of this work was to generate a collection of mutants and conveniently test their expression while avoiding time-consuming methods. To achieve this, the sequence of the green fluorescent protein (GFP) was fused to mSlc35a3. As shown in Fig. 1C, perinuclear fluorescence in Kl3 cells transformed with mSLC35A3–GFP can be visualized by confocal microscopy, consistent with the expression of mSlc35a3–GFP in the secretory pathway. Subsequent spectrofluorimetric measurements of the cell suspensions showed an increase in GFP fluorescence compared with cells transformed with the empty vector (pE4) or pE4mslc35a3 (Fig. 1A, white bars). Heating the cell suspensions for 1 min at 90 °C reduced the fluorescence signal of mSlc35a3–GFP-expressing cells to background levels of the control cells transformed with the empty vector. This is most likely due to the irreversible denaturation of the GFP fusion protein (Fig. 1, A, gray bars, and C).

The results presented in Fig. 1A indicate that fusing GFP to mSlc35a3 did not significantly alter its transport capacity (Fig. 1B) as revealed by the binding of the lectin. Next, the V180F mutation that had been shown to impair transport activity of the bovine Slc35a3 transporter (37) was introduced into the mouse SLC35A3 sequence, and subsequently the Slc35a3 mutant was expressed as GFP fusion in K13 cells. Although the expression level of the mutated variant was higher than the WT protein (Fig. 1A, white bars), cells expressing the V180F variant bound less than 25% of the GSII–FITC lectin compared with cells expressing mSlc35a3–GFP (Fig. 1B). This indicates a substantial decrease in UDP-GlcNAc transport activity. Taken together, our results suggest that this approach, based on heterologous expression of GFP-tagged proteins combined with GSII–FITC lectin recognition of GlcNAc glycoconjugates, can successfully be employed to evaluate the expression and activity of mSLC35A3.

Topology analysis of mSlc35a3

To identify amino acid residues relevant for transport activity, we first analyzed the primary amino acid sequence of mSlc35a3, using the topology predictors TOPCONS, MEMSAT-SVM, HMMTOP, and TMHMM. As summarized in Table S1, 8–10 transmembrane helices (TMHs) were predicted for mSLC35A3. The data obtained from the different prediction programs varied the most on TMHs 3 and 4, whereas TMHs 1–2 and 5–10 were predicted by all methods used, with slight extension differences (Fig. S1). Similar results were obtained for other well-characterized UDP-GlcNAc transporters from different species, including Homo sapiens and Caenorhabditis elegans as well as for different members of subfamily A, e.g. mouse Slc35a1, the only NST for which the topology has been experimentally determined (32). Based on this information, a 10-transmembrane helical model was proposed as an initial working hypothesis for mSlc35a3 (Fig. 2). Given the even number of TMHs and the positive inside rule (48), we conclude that both the N and C termini would be oriented to the cytoplasmic side. A general analysis of the amino acid distribution in the model showed an accumulation of charged and polar residues in luminal and cytoplasmic loops, whereas within the transmembrane helices hydrophobic residues prevailed. A detailed assessment of the transmembrane helices in the model revealed that five TMHs (1, 3, 4, 6, and 9) were enriched in amino acids with polar characteristics, whereas only three charged residues were identified, namely Glu-47 and Lys-50 located in TMH2 and Lys-120 in TMH4 (Table 1).

Cys-scanning mutagenesis of transmembrane helix 2

As shown in Fig. 2, the mSlc35a3 amino acid sequence contains three cysteine residues: Cys-54 in TMH2, Cys-64 in the loop between TMH2 and TMH3, and Cys-184 in TMH6. To generate a collection of cysteine-unique mutants, we first produced a cysteine-less variant (CL). Therefore, mSlc35a3 variant containing substitutions corresponding to C54S/C64S/C184S was generated and fused to GFP. The expression level of CL-GFP in Kl3 cells was similar to that of WT–GFP (Fig. S2). After incubation with GSII–FITC, cells bound close to 80% of the lectin compared with cells expressing the WT protein (Fig. 3). As assessed by phenotypic complementation of Kl3 cells, all cysteine replacements were well-tolerated by the protein, indicating that they are not essential for transport activity and concluding that the CL mutant is functionally active in vivo.

Based on the bioinformatic results obtained for the mSlc35a3 sequence (Fig. 2 and Table 1), further experimental analysis focused on the putative TMH2. Notably, TMH2, which was predicted by all membrane topology programs used in this study, is highly hydrophobic and contains two charged amino acids embedded in the membrane. Charged and polar residues in transmembrane domains of transport proteins are usually involved in the binding of hydrophilic substrates, domain interactions, or conformational changes as part of the transport mechanism (49). Consequently, the corresponding codon of each amino acid between leucine 38 to lysine 60 was substituted by a cysteine codon using CL as a backbone, and a collection of 23 cysteine-unique mutants fused to GFP was generated. As shown in Fig. S2, all Kl3 transformants exhibited GFP fluorescence higher than cells transformed with the empty vector, which decreased to mock background levels after heat treatment. Incubation with GSII–FITC showed that most of the cells expressing cysteine-unique mutants bound the lectin, with fluorescence intensity values higher than 40% of the WT, consistent with a restoration of the UDP-GlcNAc import into the lumen of the Golgi apparatus (Fig. 3). The only exceptions were mutants A42C, V44C, E47C, and K50C, which revealed a specific binding of GSII–FITC lower than 20% that of WT cells, indicative of a severe impairment of transport activity.

Phenotypic complementation of Kl3 cells requires the synthesis and incorporation of an active form of mSlc35a3 in membranes of the secretory pathway (ER/Golgi apparatus). To exclude incorrect membrane insertion or organelle localization as a cause for the inability of these mutants to transport UDP-GlcNAc, two approaches were undertaken. First, localization of the C-terminally GFP-tagged mutants was evaluated by confocal microscopy. Kl3 cells transformed with a plasmid carrying different mutants and CL were grown to an OD₆₀₀ = 2, and the cellular localization of the GFP fluorescence signal was analyzed. Similar to CL-GFP–expressing cells, perinuclear fluorescence was observed for all mutants tested, a pattern consistent with expression in the secretory pathway (Fig. 4A). Second, membranes were isolated by ultracentrifugation, and proteins abundant in these membrane fractions were separated by SDS-PAGE and analyzed by in-gel fluorescence. As shown in Fig. 4B, a predominant band for all mutants with mobility close to the 60-kDa marker was observed. This is consistent with the calculated molecular mass of 55 kDa corresponding to the fusion protein mSlc35a3–GFP (Fig. 4). In agreement with the microscopy data, but not with whole-cell fluorescence results (Fig. S2), the intensity of the main band of mutant 44 was slightly lower compared with the others. Taken together, these results strongly suggest that all variants were well-integrated into the membranes of the secretory pathway, but in an inactive form.

Analysis of involvement of Glu-47 and Lys-50 residues in mSLC35A3-mediated transport through conservative substitution analysis

To study the importance of residues Ala-42, Val-44, Glu-47, and Lys-50 for the function of mSlc35a3, conservative replacements were made using the WT sequence as backbone. Kl3 cells transformed with the mutant alleles A42V, A42G, V44A, V44I, or V44L fused to GFP showed fluorescence levels similar to those observed for well-expressed proteins (white bars, Fig. S3) and values of GSII–FITC binding between 50 and 140% compared with Kl3 cells expressing the WT transporter (Fig. 5). The reduced binding of GSII–FITC in Kl3 cells expressing A42G (50%), but not in A42V (90%), suggests the importance of a lateral group, absent in the small and hydrophilic amino acid glycine. In contrast, cells expressing all Val-44 mutants exhibited an increased abundance of glycoconjugates, suggesting that the changes of V44A, V44I, or V44L improve the function of mSlc35a3 and increase the concentration of luminal UDP-GlcNAc. These results indicate that conservative substitutions of Ala-42 or Val-44 are well-tolerated as revealed by the in vivo transport activity. However, our data further suggest that cysteine replacements in these positions are incompatible with the function of mSlc35a3.

To assess the role of glutamic acid 47 for mSLC35A3 activity, mutations to alanine (E47A), aspartic acid (E47D), or glutamine (E47Q) were generated, and Kl3 cells were transformed with the corresponding variants. As shown in Fig. S3, expression of the mutant E47A was not detectable, whereas E47D and E47Q were expressed at similar levels compared with the WT protein. The in vivo activity was assessed by incubation of Kl3 transformants with GSII–FITC lectin. Cells expressing the E47D mutant bound 47% of the lectin compared with control cells transformed with the WT protein. By contrast, the substitution E47Q completely abolished the biosynthesis of GlcNAc glycoconjugates and showed no binding. The significance of lysine 50 was examined by substituting Lys-50 with alanine (K50A), histidine (K50H), or arginine (K50R). Kl3 cells were transformed with the corresponding mutant variants, and expression was monitored by whole-cell GFP fluorescence. As is shown in Fig. S3, no expression was detectable for K50H, whereas the K50A and K50R variants were expressed at similar levels to those of the WT protein. Upon incubation of Kl3 cells expressing the K50R mutant with GSII–FITC, low levels of GlcNAc were detected, suggesting severely impaired transport activity.

To explore a possible interaction between both charged amino acids, double mutants were generated and expressed in Kl3 cells (Fig. S3). To elucidate whether a charge misbalance in the individual mutations (E47C or K50C) caused the impairment in transport activity, both charges were neutralized (E47A/K50A). To analyze whether their specific positions in TMH2 are of particular relevance, both residues were interchanged (E47K/K50E). Finally, a mutant containing substitutions of the residues that lead to simultaneous changes in length but conservation of the original charge (E47D/K50R) was generated. As shown in Fig. 5, neutralization of the charge or interchange of the position disrupts the production of GlcNAc glycoconjugates on the surface of Kl3 cells transformed with the corresponding plasmids. Kl3 cells expressing the E47D/K50R mutant bound less than 20% of the lectin compared with cells expressing the WT protein, suggesting an additive effect of the corresponding individual mutations.

Glu-47 and Lys-50 are critical for UDP-GlcNAc/UMP exchange activity of mSlc35a3

Phenotypic complementation of Kl3 cells was used as an efficient way to screen and identify relevant residues. However, limited information could be obtained about the specific properties of the mSlc35a3 variants. To study the UDP-GlcNAc transport properties of mSLC35A3 and the effect of the mutations E47D, E47Q, K50R, and K50H in detail, the WT protein as well as the variants were heterologously expressed in Saccharomyces cerevisiae. Membrane proteins were reconstituted into liposomes, and the corresponding proteoliposome preparations, preloaded with UMP, were incubated with UDP-GlcNAc, and nucleotide sugar uptake was evaluated by MS. UDP-GlcNAc uptake was saturable in a time- and concentration-dependent manner (Fig. 6, A and B). Determinations of the kinetic parameters of UDP-GlcNAc transport activity mediated by the WT mSLC35A3 revealed a K_m of 22 μm and V_max of 894 nm s⁻¹ (Table 2). In contrast, proteoliposomes containing the corresponding mutant variants E47D, E47Q, K50R, or K50H showed UDP-GlcNAc accumulation over time with variable levels between 1 and 10% compared with the WT (Fig. 6B). Velocities were dramatically affected in Glu-47 and Lys-50 substitutions with values close to 10 and 1% of WT mSlc35a3. The apparent affinities of the variants for UDP-GlcNAc showed a decrease for all mutants except for E47D (Table 2).

EXXK motif is specific to subfamily A members and conserved among species

A multiple alignment generated from the putative TMH2 amino acid sequences of mSlc35a3 and other members of the mouse SLC35 family revealed that the EXXK motif containing Glu-47 and Lys-50 is present only in subfamily A (Fig. 7), indicating uniqueness of the motif and possibly providing distinct structural/functional properties. In addition, the motif is found in characterized NSTs across a variety of species ranging from unicellular organisms like Entamoeba histolytica (50) and yeast (51) to worms (4), plants (52), and mammals (29, 45). This suggests an early occurrence of this subfamily in eukaryotes and an adaptation to transport diverse substrates during evolution.

Interactions between TMH2/TMH7 and TMH2/TMH8 were predicted by coevolution studies of amino acid residues

Evolutionary constraints on the function and structure of proteins are reflected in conserved interactions between pairs or groups of amino acids (53). To evaluate possible interactions involving residues of TMH2 in the context of the whole protein, the mSlc35a3 sequence was analyzed using the algorithm EVfold_membrane. This algorithm extracts coevolution patterns from multiple sequence alignments using an entropy maximization approach (54). The scoring of evolutionarily constrained pairs, many reflecting interactions between residues close in space, suggests that residues from TMH2 interact with residues of TMH7 and TMH8. As shown in the contact map (Fig. S4) from 330 evolutionary couplings, specifically, the five pairs Val-44/Leu-221, Ile-51/Phe-214, Ile-51/Ser-217, Phe-48/Ser-217, and Ile-55/Ile-210 are predicted to interact between TMH2 and TMH7, and the two pairs Ala-53/Ala-253 and Leu-49/Val-248 are predicted to interact between TMH2 and TMH8. Our results revealed no coevolution pairs between TMH7 and TMH8 suggesting a localization on opposite sides of TMH2. For the highly conserved residues Glu-47 and Lys-50, no interaction with residues of other TMHs were predicted.

Finally, the 3D structure of the protein was computed using EVfold_membrane, based only on the predicted residue contacts and restrictions imposed by topology (54). The resulting model, shown in Fig. S5, contains 10 TMHs with an arrangement resembling the structure of members of the drug/metabolite transporter (DMT) superfamily that have been crystallized so far (44, 55, 56).

Homology model of mSlc35a3

A homology model of mSlc35a3 was generated using the Phyre2 Protein Fold Recognition Server (57). The model was built on the basis of the structure of Vrg4 (PDB code 5OGE), and the quality of the model was tested using the structure-validated web server Molprobity (58). A comparison of the homology model (Fig. 8) with the model obtained using coevolutionary information (Fig. S5) shows that they are highly similar. The resulting models contain 10 TMHs folded in two with a central cavity limited by TMH2–4 and TMH6–9, whereas TMH5 and TMH10 outside this core structure appear as a zipper. Residues Glu-47 and Lys-50 seem to be oriented to the same side of TMH2 facing the central cavity of the protein (Fig. 8B).

Materials

Common chemicals used in this work were of analytical grade and were obtained from Sigma. Yeast extract and peptone were obtained from Britannia (Argentina).

Bioinformatic analysis

The primary amino acid sequences of the SLC35 family proteins were analyzed using the following topology predictors: TOPCONS (61), MEMSAT-SVM (62), HMMTOP (63), and TMHMM (64). Sequence alignments were generated using Clustal Omega (65).

Molecular biology

Unless otherwise stated, standard molecular biology protocols were applied as described in Ref. 66. The Mus musculus slc35a3 gene (mSLC35A3) was PCR-amplified from cDNA (clone ID 5150304, Open Biosystem) using DNA polymerase (Bioline, USA) and primers XhoI–Mmf 5′-GCGGCGCTCGAGATGTCTGCCAACCTAAAATAT-3′ and Mm-EcoRI 5′-CGCCGCGAATTCCTATGCTTTAGTGGGATTTCC-3′. The resulting PCR product was digested with XhoI and EcoRI and subsequently ligated into the pE4 vector (47) to obtain the plasmid pE4-mslc35a3 for expression in the K. lactis mutant 3 (Kl3). To generate a GFP fusion (mslc35a3-GFP) in pE4, the mslc35a3 sequence was introduced into the vector p426Gal-GFP (67) by homologous recombination and subsequently subcloned into the pE4 vector.

In the first step, the PCR product was obtained using the following primers (EcoRI): mouse RECf (5′-TCGACGGATTCTAGAACTAGTGGATCCCCCGAATTCATGTCTGCCAACCTAAAATAT-3′) and mouse RECr (5′-AAATTGACCTTGAAAATATAAATTTTCCCCTGCTTTAGTGGGATTTCCTGC-3′). The plasmid p426Gal-GFP was digested with SmaI and transformed into S. cerevisiae FGY217 (kindly provided by Prof. Ljungdahl). The plasmid p426Galmslc35a3–GFP, purified from S. cerevisiae, was digested with EcoRI to release the insert EcoRI–mslc35a3–GFP–EcoRI and subsequently ligated into the vector pE4. The correct orientation was confirmed by colony PCR using appropriate primers. Plasmid p426Gal–GFP was obtained essentially as described previously (67). In brief, the GFP sequence was PCR-amplified from pUG35 plasmid (kindly provided by Dr. Johannes Hegemann), using the following primers: TEVGFPfor (5′-GAAAATTTATATTTTCAAGGTCAATTTTCTAAAGGTGAAGAATTATTCAC-3′) and GFPhisrev (5′-TTAATGATGATGATGATGGTGGTGGTGTTTGTACAATTCATCCATACC-3′). The corresponding PCR fragment was used as template in the second round of PCR using the following primers: RecTEVfor (5′-TCTAGAACTAGTGGATCCCCCCCCGGGGAAAATTTATATTTTCAAGGTC-3′) and Rechisrev (5′-GATAAGCTTGATATCGAATTCCTGCAGTTAATGATGATGATGGTGGTGG-3′) to add recombination sequences. Finally, the PCR product was introduced into the pRS426GAL1 plasmid (kindly provided by Dr. James Konopka) digested with SmaI by homologous recombination into the FGY217 yeast strain.

To obtain the CL mutant, the following protocol was used. First, two fragments were obtained by PCR. Fragment A with primers (EcoRI) mouseRECf/C54S/C64Sr and fragment B with primers C184Sf/mouseRECr (Table S2) using mslc35a3 cDNA as template were used. After purification, both fragments were used in PCR to obtain the full-length fragment with the corresponding serine codons replacing the cysteine codons. The cysteine-unique mutants were generated using the primers listed in Table S2 (L38C to K60C) and employing the megaprimer method (68) in two rounds of PCR. In the first round, a fragment (megaprimer) containing the mutation was obtained in combination with primer (EcoRI)mouseRECf. In the second round, the megaprimer was used in combination with the primer mouseRECr to obtain the full-length version. In both rounds, the CL sequence obtained was used as a template. Finally, single- and double-point mutations of WT-mslc35a3 were obtained essentially as described for a unique cysteine mutant using the WT cDNA as template and the corresponding primers (E47Dr, E47Qr, E47Ar, E47Cr, K50Rr, E47K/K50Er, E47D/K50Rr, V44Ar, V44Lr, and V44Ir). The identity of the cloned fragments was confirmed by DNA sequencing.

Yeast strain, transformation, and growth media

For transformation of the K. lactis mutant 3 (Kl3, Mata, uraA, mnn2-2, arg2⁻ K⁺, pKD1⁺) a lithium acetate/PEG method was utilized (69). Cells were grown in complete YPD media (0.75% yeast extract, 1.13% peptone, 2.2% dextrose), and transformants were selected by their ability to grow at 28 °C in the absence of uracil (−Ura media) on plates containing 0.67% yeast nitrogen base with ammonium sulfate without amino acids (YNB), 0.08% complete supplemented medium minus Ura (Ura⁻), 2.0% dextrose, and 2.0% agar. WT K. lactis cells (Kl8), used as a positive control for the UDP-GlcNAc transporter complementation assays, were grown at 28 °C in YPD media.

Cell surface labeling of K. lactis with GSII–FITC

To assay the activity of the mouse Slc35a3 variants, Kl3 cells transformed with the corresponding pE4 vector (31) were grown overnight in 5 ml of synthetic uracil dropout media to an OD₆₀₀ = 1–2 as described earlier (31). Cells were collected by centrifugation and washed with Solution I (150 mm NaCl, 0.5 mm CaCl₂). Cell density was estimated at 600 nm (OD₆₀₀), and an aliquot, corresponding to 2 units of OD₆₀₀, was isolated by centrifugation and subsequently resuspended in 200 μl of solution I. GFP fluorescence (excitation, 485 nm/emission, 514 nm) was measured before and after incubation at 90 °C for 1 min to estimate the expression of the corresponding mSLC35A3–GFP variants (WT or mutants). After the 90 °C incubation step, the cells were harvested by centrifugation; the supernatant was discarded; and the pellet was incubated in the presence of 25 μl of GSII–FITC (EY Laboratories) labeling solution (3 parts of lectin + 1 part 150 mm NaCl, 4 mm CaCl₂) for 1 h at 25 °C. After this step, the cells were pelleted, washed with 1.5 ml of solution I, and resuspended in 200 μl of solution I before the GFP fluorescence was determined. Fluorescence measurements were normalized to the OD₆₀₀.

Confocal microscopy of yeast expressing mSlc35a3-GFP

Kl3 cells transformed with pE4 and pE4-MmSLC35A3–GFP (WT or mutants) were mounted on YSB buffer and imaged using an Olympus Fluoview FV1000 spectral laser-scanning confocal microscope with a ×60 oil immersion lens using 473 nm excitation. GFP fluorescence emission was collected between 500 and 550 nm.

Analysis of the membrane integration of the mSlc35a3–GFP fusions using in-gel fluorescence analysis

Total membrane fractions were prepared using glass beads to disrupt the cell pellets derived from 10-ml yeast cultures (OD₆₀₀ = 1–2) in YSB buffer (10% glycerol, 5 mm EDTA, 1 mm phenylmethylsulfonyl fluoride, 50 mm Tris-HCl, pH 7.6). The suspensions were centrifuged at 2,000 × g followed by centrifugation of the supernatants at 100,000 × g for 1 h. Resulting membrane pellets were resuspended in YSB buffer prior to protein separation by electrophoresis on 12% SDS-polyacrylamide gels (70). Gels were scanned using a Storm 840 molecular imager (Amersham Biosciences) to determine GFP fluorescence.

In vitro transport assay

UDP-N-acetyl-α-d-glucosamine (UDP-GlcNAc) was obtained from Sigma. The yeast strain INVScI (Thermo Fisher Scientific) was transformed using the S.c. EasyComp^TM transformation kit (Thermo Fisher Scientific). Yeast growth, cell disruption, membrane isolation, membrane protein reconstitution into liposomes, transport activity assays, and MS were performed as described previously in detail (12). Kinetic parameters were calculated by nonlinear regression using the Prism 7 application (GraphPad Software, La Jolla, CA).

Coevolution analysis

The coevolution analysis of residues pairs of MmSlc35a3 was performed using the EV coupling web interface (http://evfold.org/evfold-web/evfold.do)⁸ (54).

Homology modeling

The homology model of mSlc35a3 was generated using the Phyre2 Protein Fold Recognition Server (http://www.sbg.bio.ic.ac.uk/phyre2) (57). The model was built on the basis of the structure of Vrg4 (PDB code 5OGE). Model quality was tested using the structure-validate web server Molprobity (http//molprobity.biochem.duke edu)⁸ (58).