View metadata, citation and similar papers at core.ac.uk brought to you by CORE

provided by Elsevier - Publisher Connector

FEBS Letters 586 (2012) 4082–4087

journal homepage: www.FEBSLetters.org

UDP-N-acetylglucosamine transporter and UDP-galactose transporter form heterologous complexes in the Golgi membrane ⇑ Dorota Maszczak-Seneczko a, Paulina Sosicka a, Michał Majkowski b, Teresa Olczak a, Mariusz Olczak a,

a Laboratory of Biochemistry, Faculty of Biotechnology, University of Wroclaw, Wroclaw, Poland b Laboratory of Cytobiochemistry, Faculty of Biotechnology, University of Wroclaw, Wroclaw, Poland

article info abstract

Article history: UDP-galactose transporter (UGT; SLC35A2) and UDP-N-acetylglucosamine transporter (NGT; Received 6 August 2012 SLC35A3) are evolutionarily related. We hypothesize that their role in glycosylation may be coupled Revised 2 October 2012 through heterologous complex formation. Coimmunoprecipitation analysis and FLIM–FRET mea- Accepted 8 October 2012 surements performed on living cells showed that NGT and UGT form complexes when overexpressed Available online 23 October 2012 in MDCK-RCAr cells. We also postulate that the interaction of NGT and UGT may explain the dual Edited by Judit Ovádi localization of UGT2. For the first time we demonstrated in vivo homodimerization of the NGT nucle- otide sugar transporter. In conclusion, we suggest that NGT and UGT function in glycosylation is combined via their mutual interaction. Keywords: UDP-N-acetylglucosamine transporter UDP-galactose transporter Structured summary of interactions: Heterologous complex NGT physically interacts with UGT2 by anti tag coimmunoprecipitation (View Interaction: 1, 2) Golgi apparatus NGT physically interacts with UGT1 by anti tag coimmunoprecipitation (View interaction) FLIM–FRET UGT2 physically interacts with NGT by fluorescent resonance energy transfer (View interaction) Coimmunoprecipitation NGT physically interacts with NGT by fluorescent resonance energy transfer (View interaction) UGT1 physically interacts with UGT2 by anti tag coimmunoprecipitation (View interaction) UGT1 physically interacts with NGT by fluorescent resonance energy transfer (View interaction)

Ó 2012 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

1. Introduction identified in the mutant cells cause inhibition of UGT production, resulting in glycoconjugates significantly defective in galactosyla- Cellular glycoconjugates play a variety of fundamental roles in tion and sialylation [3,8]. Two splice variants of UDP-Gal trans- the growth and development of eukaryotes, as well as in the porter (UGT1 and UGT2) have been identified in human tissues, host–pathogen interactions. The glycan moiety is synthesized the CHO and MDCK cell lines [9–11]. and modified by glycosyltransferases acting in the lumen of the Compared with mammalian UGT, knowledge regarding mam- endoplasmic reticulum (ER) and Golgi apparatus. The substrates malian UDP-N-acetylglucosamine transporter (NGT) is limited. required by glycosyltransferases are sugars activated by the addi- Among known UDP-GlcNAc transporters, SLC35A3 is assumed to tion of a mono- or diphosphate (UDP, GDP, or CMP). play a main role in glycosylation [12,13], while the function of They are transported into the ER or Golgi apparatus by SLC35D2 [14] and SLC35B4 [15] multispecific transporters appears sugar transporters (NSTs), which are hydrophobic with a to be less important. Compared with SLC35A3, which is ubiqui- molecular weight of 30–45 kDa [1,2]. Most predictions determine tously expressed, SLC35D2 and SLC35B4 are less common and tis- in these multitransmembrane proteins an even number of spans, sue-specific. Immunofluorescent microscopic analysis of NGT which results in the N and C termini facing the cytosol. (SLC35A3) overexpressed in CHO [13] and MDCK-RCAr [16] cells One of the best characterized NSTs is the UDP-galactose trans- demonstrated its localization in the Golgi apparatus. porter (UGT; SLC35A2). Detailed characterization of UGT was pos- NGT and UGT, two nucleotide sugar transporters involved in sible after mutant cell lines, such as MDCK-RCAr [3,4], CHO-Lec8 essential glycosylation processes, are evolutionarily related [2,17– [5,6], and Had-1 [7], had been generated. Non-sense mutations 19]. In addition, overexpression of NGT in UGT-defective cells partly restores galactosylation [17]. Therefore, we suspected that their role in glycosylation of macromolecules may be coupled and both ⇑ Corresponding author. Address: Faculty of Biotechnology, University of Wro- transporters may partially replace function played by its partner. claw, Tamka 2 St., 50-137 Wroclaw, Poland. Fax: +48 71 3752608. E-mail address: [email protected] (M. Olczak). In an attempt to gain an insight into this hypothesis, we analyzed

0014-5793/$36.00 Ó 2012 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.febslet.2012.10.016 D. Maszczak-Seneczko et al. / FEBS Letters 586 (2012) 4082–4087 4083 the ability of NGT and UGT to form heterologous complexes in the Plan-Neofluar (NA 1.3) objective (Zeiss). The bandpass Golgi membrane. 500 ± 20 nm emission filter (Semrock, Lake Forest, IL, USA) was used and suitable controls to exclude the excitation cross-talk arti- 2. Materials and methods facts were performed. Laser power was adjusted to give an average photon rate of 104–105 photons to avoid the pile-up effect and sig- 2.1. Construction of expression plasmids, cell maintenance and nificant photobleaching. About 500 photons per pixel were ac- transfection quired and the photon statistics were further improved by 2 Â 2 pixel binning. A robust fitting-free fast-FLIM approach was used MDCK-RCAr mutant cells were grown and transfected with to reconstruct FLIM images in which every pixel was colored expression plasmid(s) (Supplementary Table 1) as described previ- according to the photon’s average arrival time. ously [17,20]. Stable transfectants expressing NGT and UGT1/UGT2 combinations were selected in complete media containing 600 lg/ 2.4. Analysis of FLIM–FRET experiments ml G-418 sulfate (InvivoGen, Toulouse, France). For the control pull-down experiment, MDCK-RCAr mutant cells and MDCK-RCAr Two florescence decay models for FLIM–FRET data analysis cells stably expressing either B4-FLAG (human UDP-xylose/N-ace- were applied. Validity of decay model was determined by weighted tylglucosamine transporter; SLC35B4) or UGT2-FLAG were residuals, which for a good fit fluctuate randomly around zero with 2 transiently transfected with HA-HsNGT expression plasmid (Sup- no obvious trends and by v value, which is close to 1 for a good fit. plementary Table 1) and analyzed 4 days after transfection. First, FLIM–FRET data were tail-fitted to single exponential decay. Such model described properly decays obtained from control cells, 2.2. Cell lysis, coimmunoprecipitation and Western blotting however, was insufficient when applied to decays obtained from cells expressing both eGFP-NGT and its mRFP-tagged putative Cells (1 Â 109) were washed in PBS and lysed using lysis buffer interacting partners. In this instance data were tail-fitted to bi- (50 mM Tris–HCl; pH 8.0 with 1% NP-40) supplemented with pro- exponential decay model: I(t)=A1exp(Àt/s1)+A2exp(Àt/s2), where tease inhibitor cocktail and 1 mM EDTA. Lysates were centrifuged s stands for fluorescence lifetime and A for amplitude of individual at 14000Âg for 15 min. Supernatants were pre-incubated over- contribution. The applied model allows resolving both fluorescence night at 4 °C with anti-HA rabbit polyclonal antibody (1.5 lg; lifetime and fractional contribution of interacting and non- Abcam, Cambridge, UK) and then incubated with Protein A-Sephar- interacting donor molecules. Fitting to a single-exponential decay oseÒ 4B (25 ll; Invitrogen, Camarillo, CA, USA) for 4 h at 4 °C. model besides its invalidity may also lead to underestimation of Samples were centrifuged at 2000Âg for 5 min, pellets washed five FRET efficiency as in such case it is assumed that all donors interact times with lysis buffer, suspended in poly- with one or more acceptor(s). To calculate an average lifetime acrylamide (SDS–PAGE) sample buffer, and and fractional amplitudes, respective formulae were used: sav = incubated for 30 min at 65 °C. Proteins were separated in 10% (A1s1)+(A2s2)/A1 + A2 and Ai = Ai/Ai + Ai+1. SDS–PAGE gels and transferred onto nitrocellulose membranes (Whatman, Dassel, Germany). After blocking, incubation with 3. Results and discussion anti-c-myc (0.1 lg/ml; Abcam) or anti-FLAG (1 ng/ml; Sigma– Aldrich) antibody, and subsequently with horseradish peroxi- 3.1. Coimmunoprecipitation demonstrates complex formation dase-coupled goat anti-chicken IgY (0.2 lg/ml; Abcam) or between NGT and UGT anti-mouse IgG (1:10000; Promega, Madison, WI, USA) antibody was carried out. Immunoreactive proteins were visualized with Several reports demonstrated that NSTs function in the form of an enhanced chemiluminescence system (PerkinElmer, Whaltam, homodimers [21–23] or higher homooligomers [24], and UGT MA, USA). Proteins subjected to coimmunoprecipitation were also forms heterologous complexes with ceramide galactosyltransfer- separated by SDS–PAGE and stained with colloidal Coomassie ase [25]. Recently we showed that UGT splice variants undergo Brilliant Blue (PAGEBLUE Protein Solution; Fermentas, partial relocalization when coexpressed, suggesting their mutual Vilnius, Lituania). interaction [16]. Moreover, we demonstrated that overexpression of NGT may partially restore galactosylation of N-glycans in 2.3. Confocal and fluorescence lifetime imaging microscopies of living MDCK-RCAr and CHO-Lec8 mutant cells defective in UGT activity cells [17]. Residual galactosylation detected in the mutant cells [11,26–28], at least in part, suggests that other NSTs may support MDCK-RCAr mutant cells were transiently transfected with this process in the absence of UGT. Taking into account similarity expression construct(s). After 48 h cells were seeded onto the 40- of NGT and UGT (Supplementary Fig. 1) [2,17–19] we assumed that mm cover glasses (Thermo Scientific, Rockford, IL, USA) placed in these two NSTs may form heterologous complexes in the Golgi 60-mm culture dishes and allowed to grow for additional 24 h. membrane. The standard growth medium was replaced with the phenol-free To confirm our hypothesis, we first employed coimmunoprecip- minimal essential medium (Life Technologies, Paisley, UK) 16 h itation analysis. Putative complex formation was studied in UGT- prior to imaging. Confocal microscopy images were acquired using deficient mutant MDCK-RCAr cells, which theoretically should the Zeiss LSM 510 META laser scanning confocal microscope (Zeiss, cause all NGT molecules to interact with recombinant UGTs Jena, Germany). ImageJ software was used to analyze and process exclusively. After overexpression of both UGT splice variants in images (http://rsb.info.nih.gov/ij/). Time-resolved fluorescence MDCK-RCAr cells a complex formation was observed (Fig. 1C), imaging was performed with an LSM 510 microscope (Zeiss) demonstrating optimal conditions of cell lysis and coimmunopre- equipped with a TCSPC FLIM module (PicoQuant, Berlin, Germany). cipitation. Further we demonstrated that NGT and UGT2 as well The dishes with cells expressing fusion protein(s) were mounted in as NGT and UGT1 formed complexes (Fig. 1A and B). These a dedicated observation stage chamber (PeCon GmbH, Erbach, in vitro results for the first time revealed an interaction occurring

Germany) and left for gas (5% CO2) and temperature (37 °C) equil- between two different NSTs. However, coimmunoprecipitation ibration. Fluorescence of eGFP was excited at 470 nm with a pulsed performed on multitransmembrane proteins in the presence of laser (40 MHz) and collected through water immersion 63 Â LCI detergents may result in non-specific, false positive results. 4084 D. Maszczak-Seneczko et al. / FEBS Letters 586 (2012) 4082–4087

Fig. 1. Coimmunoprecipitation analysis of complexes formed between UDP-N-acetylglucosamine transporter (NGT) and UDP-galactose transporter (UGT). Combinations of (A and B) NGT-HA and both splicing variants of UGT (UGT1-c-myc and UGT2-c-myc), (C) UGT1-HA and UGT2-FLAG, (D) NGT-HA and B4-FLAG (UDP-xylose/N- acetylglucosamine transporter) or NGT-HA and UGT2-FLAG were overexpressed in MDCK-RCAr mutant cells. Cell lysate proteins were subjected to coimmunoprecipitation using anti-HA antibody and Protein A-SepharoseÒ 4B. Complexes formed were visualized using Western blotting with anti-c-myc (A and B) or anti-FLAG (C and D) antibodies and chemiluminescence staining (top panels). Protein staining with colloidal (bottom panels) served as a control of lysis efficiency and protein loading for coimmunoprecipitation. Arrowheads indicate coimmunoprecipitated complexes.

Therefore we carried out the additional pull-down experiment as to quantify FRET efficiency is based on measurement and compar- negative control. As shown in Fig. 1D we did not observe any ison of donor fluorescence lifetime in the presence and absence of interaction between NGT and UDP-xylose/N-acetylglucosamine acceptor by using fluorescence lifetime imaging microscopy (FLIM) multitransmembrane transporter SLC35B4 (Fig. 1D). [29]. Shortening of the donor fluorescence lifetime indicates FRET, however, consideration of protein–protein interaction requires fur- 3.2. FLIM–FRET analysis confirms complex formation between NGT ther analysis to identify the source of lifetime reduction. One can and UGT assume that in detection volume either a fraction of donor coupled with acceptor or all labeled molecules are interacting. The advan- To investigate protein–protein interactions in cells a variety of tage of FRET measurement by FLIM is the possibility to resolve be- microscopic techniques has been developed. Our previous studies tween these two elements accounting for the overall efficiency of demonstrated that after overexpression in MDCK-RCAr cells energy transfer. Performing analysis of FLIM–FRET data using mul- UGT1 colocalized with Golgi marker and UGT2 with ER marker ti-exponential decay models reveals both fractional contribution [16]. However, when UGT1 and UGT2 were overexpressed in par- and fluorescence lifetimes of interacting and non-interacting spe- allel, UGT1 colocalized with both ER and Golgi markers. This sug- cies. Therefore, to confirm interaction between NGT and UGT gests that subcellular localization of UGTs may depend not only in vivo, we employed a combined FLIM–FRET approach. on the presence of respective glycosyltransferase [25], but also NSTs are exceptionally suitable FRET partners due to their rela- on the presence of the partner splice variant. However, such stud- tively small size and convenient topology (both N and C termini are ies do not provide direct proof of interaction between proteins due directed to the cytosolic side of the membrane and therefore can to the insufficient resolving power of the fluorescent microscope. be easily tagged). FRET is more efficient when the acceptor is the The most powerful means to demonstrate interaction of proteins protein of higher stoichiometry, which minimizes the percentage in living cells is based on Förster resonance energy transfer (FRET), of unpaired donor molecules [30]. Our numerous preliminary which provides direct information that appropriately labeled do- experiments demonstrated that UGT2 and UGT1 are overexpressed nor and acceptor molecules are positioned in the nanometer-range in higher amounts compared with NGT (data not shown). There- proximity [29]. The efficiency of energy transfer between an ex- fore, eGFP (a FRET donor) attached to NGT and mRFP (a FRET cited donor and a nearby acceptor may be used as a molecular ruler acceptor) attached to UGT splice variants were used. Respective to determine the distance spacing proteins. One possible method tagged NSTs were overexpressed in MDCK-RCAr cells and D. Maszczak-Seneczko et al. / FEBS Letters 586 (2012) 4082–4087 4085

Table 1 In vivo FLIM–FRET analysis of interactions between UDP-N-acetylglucosamine transporter (NGT) and UDP-galactose transporter (UGT).

2 FRET combinations n sa (ns) ss (ns) fs sl (ns) v eGFP-NGT alone 14 2.68 ± 0.02 – 1.00 2.68 ± 0.02 1.00 ± 0.01 eGFP-NGT + mRFP-UGT2 13 2.45 ± 0.04 1.06 ± 0.11 0.18 ± 0.03 2.73 ± 0.1 1.00 ± 0.03 eGFP-NGT + mRFP-UGT1 15 2.53 ± 0.03 1.02 ± 0.18 0.1 ± 0.01 2.69 ± 0.02 1.00 ± 0.05 eGFP-NGT + mRFP-NGT 19 2.53 ± 0.05 0.98 ± 0.12 0.11 ± 0.02 2.73 ± 0.04 0.99 ± 0.03

Abbreviations used: n, number of cells examined; sa, average fluorescence lifetime ± standard deviation (SD); ss, fluorescence lifetime of the interacting donor fraction ± SD; s,

fluorescence lifetime of the non-interacting donor fraction ± SD; fs, the interacting donor fraction ± SD; eGFP, enhanced green fluorescent protein; mRFP, monomeric red fluorescent protein; NGT, UDP-N-acetylglucosamine transporter; UGT, UDP-galactose transporter.

fluorescence intensity was examined, followed by the eGFP life- NGT directs UGT2 to the Golgi apparatus despite the ER retention time measurements in the absence and presence of the acceptor motif present within the C terminus of the latter. As expected, (Table 1 and Fig. 2). First we examined subcellular localization of within other combinations tested (eGFP-NGT + mRFP-UGT1; fluorescently labeled NSTs in living cells. eGFP-NGT (Fig. 2A) and Fig. 2I and J, and eGFP-NGT + mRFP-NGT; data not shown) all NSTs mRFP-UGT1 (data not shown) localized exclusively to the Golgi retained their initial Golgi localization. apparatus when expressed alone, while mRFP-UGT2 resided in Subsequent FLIM measurements revealed that in the absence of the ER only (Fig. 2B). These results are consistent with data ob- an acceptor fluorophore (referred to as control), lifetime of eGFP- tained using immunofluorescence analysis [16]. Interestingly, tagged NGT was 2.68 ns (Fig. 2C and D and Table 1). This lifetime simultaneous overexpression of eGFP-NGT and mRFP-UGT2 re- was significantly shortened upon coexpression with mRFP-UGT2 sulted in partial relocalization of both recombinant proteins and (Fig. 2G and H and Table 1), mRFP-UGT1 (Fig. 2K and L and Table a remarkable amount of mRFP-UGT2 could be detected in the Golgi 1), and mRFP-NGT (Table 1). A more thorough analysis of FLIM data apparatus (Fig. 2F), where the majority of eGFP-NGT resided acquired for the fluorescently labeled NST combinations (NGT– (Fig. 2E). Differences in UGT isoforms are confined to the dilysine NGT, NGT–UGT1, NGT–UGT2) allowed distinguishing two distinct motif present at the C terminus of UGT2, responsible for its ER lifetime components: a longer one (2.7 ns), corresponding to that localization [31]. It seems, however, that complex formation with of control and an additional shorter one (1 ns) (Table 1). We

Fig. 2. In vivo FLIM–FRET analysis of interactions between UDP-N-acetylglucosamine transporter (NGT) and UDP-galactose transporter (UGT). Confocal intensity-resolved and time-resolved imaging of NGT-eGFP interaction with UGT2-mRFP (middle panel) and UGT1-mRFP (bottom panel) in MDCK-RCAr cells in comparison with cells expressing NGT-eGFP only (top panel, except B). Image taken from cells expressing mRFP-UGT2 only (B) is presented to emphasize partial relocalization of the transporter upon eGFP- NGT coexpression. Bar = 10 lm. Arrowheads indicate areas of NGT (E) and UGT2 (F) colocalization within the Golgi apparatus. (C), (G), (K) show typical pseudocolored FLIM data representing average donor lifetimes across images. Red-to-blue color changes reflect shortening of the fluorescence lifetime. Average fluorescence lifetime values obtained from corresponding FLIM measurements were plotted as frequency distribution histograms (D), (H), (L). The rainbow scale bar placed above each histogram represents fluorescence lifetime range between 2.4 ns (blue) and 3.1 ns (red). 4086 D. Maszczak-Seneczko et al. / FEBS Letters 586 (2012) 4082–4087 assumed that the longer lifetime component corresponds to a non- References interacting donor fraction, while the shorter one results from the energy transfer between the donor and acceptor. The lifetime of [1] Gerardy-Schahn, R., Oelmann, S. and Bakker, H. (2001) Nucleotide sugar transporters: biological and functional aspects. Biochimie 83, 75–82. the shorter component suggests comparably tight association be- [2] Caffaro, C.E. and Hirschberg, C.B. (2006) Nucleotide sugar transporters of the tween NGT and both UGT splice variants as well as between two Golgi apparatus: from basic science to diseases. Acc. Chem. Res. 39, 805– NGT monomers. However, its fractional contribution was relatively 812. low (0.1–0.18), which may be explained by the reasons mentioned [3] Brandli, A.W., Hansson, G.C., Rodriguez-Boulan, E. and Simons, K. (1988) A polarized epithelial cell mutant deficient in translocation of UDP-galactose below. One may assume that NSTs have a tendency to dimerize in into the Golgi complex. J. Biol. Chem. 263, 16283–16290. an as yet undetermined orientation. We successfully demonstrated [4] Toma, L., Pinhal, M.A., Dietrich, C.P., Nader, H.B. and Hirschberg, C.B. (1996) that similar to UGT1 [20], also NGT oligomerizes in vivo (Table 1). Transport of UDP-galactose into the Golgi lumen regulates the biosynthesis of proteoglycans. J. Biol. Chem. 271, 3897–3901. Therefore, additional interactions are highly possible to occur, [5] Oelmann, S., Stanley, P. and Gerardy-Schahn, R. (2001) Point mutations resulting in the formation of one or more of the following identified in Lec8 Chinese hamster ovary glycosylation mutants that inactivate homo- and heterologous complexes: (1) eGFP-NGT/eGFP-NGT; both the UDP-galactose and CMP-sialic acid transporters. J. Biol. Chem. 276, 26291–26300. (2) mRFP-NGT/mRFP-NGT; (3) eGFP-NGT/endogenous NGT; (4) [6] Stanley, P. (1983) Lectin-resistant CHO cells: selection of new mutant mRFP-NGT/endogenous NGT; (5) mRFP-UGT/endogenous NGT; phenotypes. Somatic Cell Genet. 9, 593–608. (6) mRFP-UGT/mRFP-UGT. Obviously, none of these interactions [7] Ishida, N., Yoshioka, S., Iida, M., Sudo, K., Miura, N., Aoki, K. and Kawakita, M. (1999) Indispensability of transmembrane domains of Golgi UDP-galactose would support FRET, instead reducing amounts of donor and transporter as revealed by analysis of genetic defects in UDP-galactose acceptor molecules available for interactions resulting in energy transporter-deficient murine Had-1 mutant cell lines and construction of transfer. Another possible explanation is based on the assumption deletion mutants. J. Biochem. (Tokyo) 126, 1107–1117. [8] Briles, E.B., Li, E. and Kornfeld, S. (1977) Isolation of wheat germ agglutinin- that NGT and UGT are not the sole constituents of discovered resistant clones of Chinese hamster ovary cells deficient in membrane sialic complexes. In such a scenario, the extent of interactions between acid and galactose. J. Biol. Chem. 252, 1107–1116. overexpressed NSTs would largely depend on the availability of [9] Ishida, N., Miura, N., Yoshioka, S. and Kawakita, M. (1996) Molecular cloning and endogenous complementary proteins. Lastly, a high contribution characterization of a novel isoform of the human UDP-galactose transporter, and of related complementary DNAs belonging to the nucleotide-sugar transporter of the non-interacting donor fraction might result in some cases gene family. J. Biochem. (Tokyo) 120, 1074–1078. from the incomplete folding of the acceptor fluorophore as well [10] Miura, N., Ishida, N., Hoshino, M., Yamauchi, M., Hara, T., Ayusawa, D. and as from the suboptimal donor orientation towards the acceptor. Kawakita, M. (1996) Human UDP-galactose translocator: molecular cloning of a complementary DNA that complements the genetic defect of a mutant Surprisingly, the size of the interacting donor fraction was cell line deficient in UDP-galactose translocator. J. Biochem. (Tokyo) 120, significantly higher for UGT2-bound acceptor compared with 236–241. UGT1 (Table 1). This could be explained by differences in UGTs [11] Maszczak-Seneczko, D., Olczak, T., Wunderlich, L. and Olczak, M. (2011) Comparative analysis of involvement of UGT1 and UGT2 splice variants of overexpression levels [11]. However, one cannot exclude the UDP-galactose transporter in glycosylation of macromolecules in MDCK and possibility of differences resulting from dissimilarities between CHO cell lines. Glycoconj. J. 28, 481–492. C-terminal sequences of UGTs, accounting for slightly altered [12] Guillen, E., Abeijon, C. and Hirschberg, C.B. (1998) Mammalian Golgi apparatus UDP-N-acetylglucosamine transporter: molecular cloning by phenotypic affinities towards NGT. correction of a yeast mutant. Proc. Natl. Acad. Sci. USA 95, 7888–7892. [13] Ishida, N., Yoshioka, S., Chiba, Y., Takeuchi, M. and Kawakita, M. (1999) Molecular cloning and functional expression of the human Golgi UDP-N- 4. Conclusions acetylglucosamine transporter. J. Biochem. (Tokyo) 126, 68–77. [14] Suda, T., Kamiyama, S., Suzuki, M., Kikuchi, N., Nakayama, K., Narimatsu, H., To date, several NSTs have been characterized; however, their Jigami, Y., Aoki, T. and Nishihara, S. (2004) Molecular cloning and characterization of a human multisubstrate specific nucleotide-sugar function is still not clear. Since NGT and UGT are evolutionarily re- transporter homologous to Drosophila fringe connection. J. Biol. Chem. 279, lated, and overexpression of NGT in UGT-defective cells may re- 26469–26474. store galactosylation, we hypothesize that NGT specificity is [15] Ashikov, A., Routier, F., Fuhlrott, J., Helmus, Y., Wild, M., Gerardy-Schahn, R. and Bakker, H. (2005) The human solute carrier gene SLC35B4 encodes a broader than previously believed. We postulate that heterologous bifunctional nucleotide sugar transporter with specificity for UDP-xylose and NGT/UGT complexes might mediate transport of both UDP-Gal UDP-N-acetylglucosamine. J. Biol. Chem. 280, 27230–27235. and UDP-GlcNAc. Alternatively, NGT/UGT heterodimers could sim- [16] Maszczak-Seneczko, D., Olczak, T. and Olczak, M. (2011) Subcellular localization of UDP-GlcNAc, UDP-Gal and SLC35B4 transporters. Acta ply bring NGT and UGT homodimers together (providing that high- Biochim. Pol. 58, 413–419. er order oligomers are likely to occur as well). In either case, [17] Maszczak-Seneczko, D., Olczak, T., Jakimowicz, P. and Olczak, M. (2011) however, the ability of NGT and UGT to interact with each other Overexpression of UDP-GlcNAc transporter partially corrects galactosylation defect caused by UDP-Gal transporter mutation. FEBS Lett. 585, 3090–3094. would comprise one of the mechanisms involved in precise spatio- [18] Martinez-Duncker, I., Mollicone, R., Codogno, P. and Oriol, R. (2003) The temporal regulation of N-glycan biosynthesis in the Golgi appara- nucleotide-sugar transporter family: a phylogenetic approach. Biochimie 85, tus by ensuring sufficient amounts of UDP-GlcNAc and UDP-Gal 245–260. [19] Liu, L., Xu, Y.X. and Hirschberg, C.B. (2010) The role of nucleotide sugar to respective glycosyltransferases. Taking all together, results transporters in development of eukaryotes. Semin. Cell Dev. Biol. 21, 600– gained in this study add to understanding of glycosylation process, 608. one of the basic posttranslational modifications, which affects the [20] Olczak, M. and Guillen, E. (2006) Characterization of a mutation and an r majority of macromolecules. alternative splicing of UDP-galactose transporter in MDCK-RCA mutant cell line. Biochim. Biophys. Acta 1763, 82–92. [21] Eckhardt, M., Gotza, B. and Gerardy-Schahn, R. (1999) Membrane topology of Acknowledgment the mammalian CMP-sialic acid transporter. J. Biol. Chem. 274, 8779–8787. [22] Puglielli, L., Mandon, E., Rancour, D.M., Menon, A.K. and Hirschberg, C.B. (1999) Identification and purification of the rat liver Golgi membrane UDP-N- This work was supported by Grant No. 2011/03/B/NZ1/02084 acetylgalactosamine transporter. J. Biol. Chem. 274, 4474–4479. from the National Science Center (NCN), Krakow, Poland. [23] Gao, X.D. and Dean, N. (2000) Distinct protein domains of the yeast Golgi GDP- mannose transporter mediate oligomer assembly and export from the endoplasmic reticulum. J. Biol. Chem. 275, 17718–17727. [24] Hong, K., Ma, D., Beverley, S.M. and Turco, S.J. (2000) The Leishmania GDP- Appendix A. Supplementary data mannose transporter is an autonomous, multi-specific, hexameric complex of LPG2 subunits. Biochemistry 39, 2013–2022. Supplementary data associated with this article can be found, in [25] Sprong, H., Degroote, S., Nilsson, T., Kawakita, M., Ishida, N., van der Sluijs, P. and van Meer, G. (2003) Association of the Golgi UDP-galactose transporter the online version, at http://dx.doi.org/10.1016/j.febslet.2012. with UDP-galactose:ceramide galactosyltransferase allows UDP-galactose 10.016. import in the endoplasmic reticulum. Mol. Biol. Cell 14, 3482–3493. D. Maszczak-Seneczko et al. / FEBS Letters 586 (2012) 4082–4087 4087

[26] Stanley, P., Sudo, T. and Carver, J.P. (1980) Differential involvement of cell aminyltransferase and alpha1,3-fucosyltransferase. J. Biol. Chem. 280, surface sialic acid residues in wheat germ agglutinin binding to parental and 12810–12819. wheat germ agglutinin-resistant Chinese hamster ovary cells. J. Cell. Biol. 85, [29] Padilla-Parra, S. and Tramier, M. (2012) FRET microscopy in the living cell: 60–69. different approaches, strengths and weaknesses. Bioassays 34, 369–376. [27] Hara, T., Endo, T., Furukawa, K., Kawakita, M. and Kobata, A. (1989) Elucidation [30] Snapp, E.L. and Hedge, R.S. (2006) Rational design and evaluation of FRET of the phenotypic change on the surface of Had-1 cell, a mutant cell line of experiments to measure protein proximities in cells. Curr. Protoc. Cell Biol.. mouse FM3A carcinoma cells selected by resistance to Newcastle Disease Unit-17.9. virus infection. J. Biochem. 106, 236–247. [31] Kabuss, R., Ashikov, A., Oelmann, S., Gerardy-Schahn, R. and Bakker, H. (2005) [28] Kawar, Z.S., Haslam, S.M., Morris, H.R., Dell, A. and Cummings, R.D. (2005) Endoplasmic reticulum retention of the large splice variant of UDP-galactose Novel poly-GalNAcbeta1-4GlcNAc (LacdiNAc) and fucosylated poly-LacdiNAc transporter is caused by a dilysine motif. Glycobiology 15, 905–911. N-glycans from mammalian cells expressing beta1,4-N-acetylgalactos-