The role of N-glycosylation in the stability, trafficking and GABA-uptake of GABA-transporter Terminal N-glycans facilitate efficient GABA-uptake activity of the GABA transporter Guoqiang Cai1,2, Petrus S Salonikidis3, Jian Fei1, Wolfgang Schwarz3, Ralf Schulein4, ă Werner Reutter2 and Hua Fan2 Institute of Biochemistry and Cell Biology, SIBS, CAS, Shanghai, China ´ Institut fur Molekularbiologie und Biochemie, CBF, Charite Universitatsmedizin Berlin, Berlin-Dahlem, Germany ă ă Max-Planck Institut fur Biophysik, Frankfurt, Germany ă Forschungsinstitut fur Molekulare Pharmakologie, Berlin-Buch, Germany ă Keywords GABA transporter; N-glycosylation; N-glycan trimming; membrane trafcking; patchclamp Correspondence H Fan, Institut fur Molekularbiologie und ă Biochemie, Campus Bejamin Franklin, Charite Universitatsmedicin Berlin, ă Arnimallee 22, D-14195 Berlin-Dahlem, Germany Fax: +49 30 84451541 Tel: +49 30 84451544 E-mail: hua.fan@charite.de (Received 17 July 2004, revised 24 January 2005, accepted February 2005) doi:10.1111/j.1742-4658.2005.04595.x Neurotransmitter transporters play a major role in achieving low concentrations of their respective transmitter in the synaptic cleft The GABA transporter GAT1 belongs to the family of Na+- and Cl–-coupled transport proteins which possess 12 putative transmembrane domains and three N-glycosylation sites in the extracellular loop between transmembrane domain and To study the significance of N-glycosylation, green fluorescence protein (GFP)-tagged wild type GAT1 (NNN) and N-glycosylation defective mutants (DDQ, DGN, DDN and DDG) were expressed in CHO cells Compared with the wild type, all N-glycosylation mutants showed strongly reduced protein stability and trafficking to the plasma membrane, which however were not affected by 1-deoxymannojirimycin (dMM) This indicates that N-glycosylation, but not terminal trimming of the N-glycans is involved in the attainment of a correctly folded and stable conformation of GAT1 All N-glycosylation mutants were expressed on the plasma membrane, but they displayed markedly reduced GABA-uptake activity Also, inhibition of oligosaccharide processing by dMM led to reduction of this activity Further experiments showed that both N-glycosylation mutations and dMM reduced the Vmax value, while not increasing the Km value for GABA uptake Electrical measurements revealed that the reduced transport activity can be partially attributed to a reduced apparent affinity for extracellular Na+ and slowed kinetics of the transport cycle This indicates that N-glycans, in particular their terminal trimming, are important for the GABA-uptake activity of GAT1 They play a regulatory role in the GABA translocation by affecting the affinity and the reaction steps associated with the sodium ion binding The cellular membrane transporter for the inhibitory neurotransmitter c-aminobutyric acid (GABA) belongs to a family of secondary active systems that are driven by electrochemica1 gradients of Na+ and Cl– [1] The main physiological function of the transporter is believed to be the control of the concentration and dwell time of GABA in the synaptic cleft Because the transport of one molecule of GABA is coupled to the Abbreviations CHO, Chinese hamster ovary; dMM, 1-deoxymannojirimycin; ER, endoplasmic reticulum; FACS, fluorescence activated cell sorting; GABA, c-aminobutyric acid; GAT1, GABA transporter type I; GFP, green fluorescence protein FEBS Journal 272 (2005) 1625–1638 ª 2005 FEBS 1625 Role of N-glycosylation and N-glycan trimming of GAT1 cotransport of two Na+ ions and one Cl– ion [2–4], the translocation across the membrane is associated with a current that can be measured by voltage clamping In the absence of GABA the transport cycle is not completed, but transient charge movements can be detected that reflect partial reactions associated with extracellular Na+ binding and hence provide kinetic information about the transport cycle [5–7] Four subtypes of GABA transporters (GAT1–4) have been found so far [8,9] GABA transporter type (GAT1) is a single polypeptide of about 67 kDa with 12 putative transmembrane domains Both N- and C-termini are located in the cytoplasm The large extracellular loop between transmembrane domains and contains three conserved N-glycosylation sites (Asn176, Asn181 and Asn184) It has been demonstrated that all three N-glycosylation sites are used in vivo and that no additional sites are present [10] N-glycosylation is a major post-translational modification in eukaryotic cells Recent results suggest that this post-translational modification may influence many of the physicochemical and biological properties of the proteins, such as protein folding, stability, targeting, dynamics and ligand binding, as well as cell-matrix and cell–cell interactions [11–16] It has been suggested that N-glycosylation is involved in the regulation of the transport activity and surface expression of neurotransmitter transporters [10,17] Functional expression of the GABA transporter is abolished by tunicamycin, a potent inhibitor of N-glycosylation [18] Experiments with HeLa transfectants showed that removal of one or two glycosylation sites by site-directed mutagenesis had little effect on the expression of GABA-uptake activity However, removal of all three N-glycosylation sites resulted in a reduction of GABA-uptake activity [10] Although such experiments indicate that N-glycosylation mutations lead to a reduction of GABA-uptake activity, we not know how the N-linked oligosaccharide side chains influence the function of this transporter Liu et al demonstrated in Xenopus oocytes that mutations of two of the three N-glycosylation sites led to a reduction in turnover rates and complex changes in the interaction of external Na+ with the transport protein as measured by voltage clamping [7] However, the question remained as to whether the reduction in function of the mutants was due to a change in the biochemical properties of this transporter or to a reduction in the number of GABA transporters per cell In order to clarify the functional significance of N-glycosylation and N-linked oligosaccharides in GAT1, green fluorescence protein (GFP)1626 G Cai et al tagged wild type GAT1 (NNN) and four glycosylation mutants (DDQ, DGN, DDN, and DND) were stably expressed in CHO cells lacking endogenous GAT1 The influence of N-glycosylation mutations and inhibition of N-glycosylation processing on biochemical properties and function were investigated In this work, we demonstrated that defective N-glycosylation resulted in reduction of the stability and a decrease in the cell surface expression of this protein The GAT1 mutants containing two N-glycosylation mutations showed a delayed intracellular translocation, but they targeted to the plasma membrane and showed reduced GAT1-specific GABA-uptake activity If all three N-glycosylation sites were eliminated, a decreased percentage of DDQ mutants was found on the cell surface However, the GABA-uptake activity could hardly be detected in this mutant Inhibition of N-glycosylation processing by 1-deoxymannojirimycin (dMM) affected neither the cell surface expression nor stability of this protein, but it resulted in marked reduction of GABA-uptake activity This suggests that N-glycans, in particular terminal structures of N-glycans, are involved in the GABA-uptake process of GAT1 Finally, we found that deficiency of N-glycosylation did not affect the affinity of GAT1 for GABA The observed reduction of GAT1-specific GABAuptake due to deficiency of N-glycans was attributed to a reduced apparent affinity for extracellular Na+ ions, resulting in a reduction of the kinetics of the transport cycle Results Expression of GAT1/GFP fusion proteins in CHO cells cDNAs of GFP tagged wild type (NNN) and mutants DND, DDN, DGN and DDQ were transfected into CHO cells, which not express endogenous GAT1 and GFP Stable transfectants were selected by fluorescence activated cell sorting (FACS) Flow cytometry analysis showed the expression of NNN and the mutants on the surface of transfected CHO cells (Fig 1A) Fluorescence and immunofluorescence microscopy showed that both GFP-fluorescence and antiGAT1 antibodies can be used to detect the expression of the GAT1 ⁄ GFP fusion protein (NNN) on surface and interior of CHO cells (Fig 1B,C) The expression of NNN was determined by Western blotting with either anti-GAT1 pAb (Fig 2A) or anti-GFP mAb (Fig 2B) following immunoprecipitation with anti-GFP pAb This GAT1 ⁄ GFP fusion protein showed several bands in SDS ⁄ PAGE, two FEBS Journal 272 (2005) 1625–1638 ª 2005 FEBS G Cai et al Role of N-glycosylation and N-glycan trimming of GAT1 Fig Protein expression and N-glycosylation processing of GFPtagged GAT1 in CHO cells NNN stable transfected CHO cells were incubated with and without dMM (1 mM) for 72 h The solubilized protein of transfected cells (1 · 107) was subjected to immunoprecipitation with anti-GFP Igs Aliquots of each immunoprecipitate were treated either with Endo H or PNGase F The resulting mixture and the other aliquots of the immunoprecipitate were analyzed by SDS ⁄ PAGE (7.5%) and immunoblotting with anti-GAT1 pAb (A) or anti-GFP mAb (B, C) Fig Flow cytometry, fluorescence microscopy and immunofluorescence microscopy of GFP-tagged GAT1 in transfected CHO cells (A) Flow cytometry of GFP-tagged GAT1 wild type and mutants The polyclonal anti-GAT1 IgG was used for immunostaining Visualization was performed with R-phycoerythrin-conjugated goat anti-(rabbit IgG) Ig NNN, GFP-tagged wild type GAT1 DND, DDN, DGN and DDQ, GFP-tagged N-glycosylation mutants (B) Fluorescence microscopy of NNN The fluorescence of GFP in GFP ⁄ GAT-fusion protein (NNN) was detected (C) Immunofluorescence microscopy of NNN Anti-GAT1 polyclonal antibodies were used for immunostaining after cell fixation and permeabilization Visualization was performed with R-phycoerythrin-conjugated goat anti-(rabbit IgG) Ig FEBS Journal 272 (2005) 1625–1638 ª 2005 FEBS monomeric forms running as a main band of about 108 kDa and a small band of about 96 kDa The 108 kDa polypeptide was resistant to Endo H digestion, while the 96 kDa polypeptide was converted into a polypeptide of 90 kDa after digestion with Endo H (Fig 2B) Digestion of both monomeric forms with PNGase F resulted in a single 90 kDa N-glycan-free peptide (Fig 2C, lane 2) This indicates that the 108 kDa peptide contains mature N-glycans of the complex type, while the 96 kDa peptide contains only N-glycans of the mannosidic type The 210 kDa band may represent a dimeric form or a protein aggregate In addition, inhibition of N-glycosylation processing of NNN by 1-deoxymannojirimycin (dMM) leads to the reduction of NNN molecule mass to 96 kDa (Fig 2C, lanes and 6) After digestion with either PNGase F or Endo H, this 96 kDa polypeptide was converted to a 90 kDa N-glycan-free polypeptide (Fig 2C, lanes and 5), indicating that the 96 kDa polypeptide contains only N-glycans of oligomannosidic type 1627 Role of N-glycosylation and N-glycan trimming of GAT1 G Cai et al Expression of N-glycosylation mutants on the plasma membrane of CHO cells The expression of NNN and N-glycosylation mutants DGN, DDN, DND and DDQ on the plasma membrane of CHO cells was investigated As shown in Fig 3A, N-glycosylation mutants exhibited a reduced molecular mass in comparison to that of the wild type NNN Nevertheless, all N-glycosylation mutants, as well as the wild type NNN, were expressed on the plasma membrane Although all three N-glycosylation sites are absent in DDQ, this mutant was also detected on the plasma membrane of CHO cells, suggesting that N-glycosylation or N-linked oligosaccharides are important, but not essential for the translocation of GAT1 to the cell surface All intracellular proteins of wild type as well as mutants (with the exception of DDQ) gave two bands, while plasma membrane proteins gave only one large band (Fig 3A) On the basis of the Endo H digestion (Fig 2B,C), the large band is assigned to proteins with N-glycans of mature complex type and the small band to the proteins with N-glycans of mannose-rich type All bands of the mutants had reduced molecular mass, compared with that of wild type NNN The reduced molecular masses of mutants are compatible with the absence of N-glycans at the two eliminated N-glycosylation sites, suggesting that the mutants in the cell interior contain N-glycans of both mannosidic and complex types, while those in the plasma membrane contain only N-glycans of the mature complex type The relative levels of surface vs intracellular GAT1 and mutants in a steady expression state were quantified The distributions between cell surface and cell interior of the mutants DGN, DND and DDN were not significantly different from that of wild type NNN About 46 ± 4.7% is found on the cell surface However, the percentage of the cell surface expression in mutant DDQ which lacks all three N-glycosylation sites was only 30 ± 4.4% in the steady expressed state (Fig 3B) N-Glycosylation mutations result in reduction of GABA-uptake activity For quantitative measurement of the specific activity of GABA-uptake, an aliquot of the stable CHO transfectants was used for the GABA-uptake assay, and another aliquot was used to determine the amount of the membrane-expressed wild type or mutant proteins The GABA-uptake activities were normalized to the same amount of cell surface proteins of wild type and mutants Compared with that of the wild type, the GABA-uptake activities of the N-glycosylation 1628 Fig Determination of expression of GFP-tagged GAT1 mutants on the surface of transfected CHO cells and measurement of GABA-uptake by GFP-tagged GAT1 wild type and mutants in transfected CHO cells (A) Cell surface and intracellular expression of GFP-tagged GAT1 wild type (NNN) and mutants (DGN, DND, DDN, and DDQ) were analyzed by biotin labelling and Western blotting Anti-GAT1 serum or anti-GFP mAb MAB2510 were used for immunostaining I, intracellular expression; M, plasma membrane expression (B) The protein bands obtained in western blotting were analyzed by phosphoimager scanning Each value represents the mean ± SEM of three separate experiments The total protein of the cell surface and intracellular bands of each wild type or mutant were set at 100% (C) Measurement of GABA-uptake by GFPtagged GAT1 wild type and mutants in transfected CHO cells The measured GABA-uptake activity was normalized to the amount of GAT1 or mutant protein expressed on the plasma membrane The activity of GABA-uptake by NNN was set at 100% All other values were expressed relative to this value The values represent the mean ± SEM of four separate experiments mutants were reduced significantly Figure 3C shows that the GABA-uptake activities of mutants with double N-glycosylation mutations, DND, DGN and FEBS Journal 272 (2005) 1625–1638 ª 2005 FEBS G Cai et al Role of N-glycosylation and N-glycan trimming of GAT1 DDN, were reduced to 64% (±5.6%), 42% (±12.4%) and 32% (±8.2%) of that of NNN, respectively GAT1-mediated transport could hardly be detected in the mutant DDQ, although this mutant was expressed on the plasma membrane Mutant DDQ does not exhibit any N-glycosylation site Because all values were normalized to the transporter proteins in the plasma membrane, the reduced specific activities of the mutants are not due to a reduced number of GABA transporters per cell, but to a reduced transport rate This suggests that N-linked oligosaccharide side-chains are important for the GABA transport activity 1-Deoxymannojirimycin inhibits the GABA-uptake of GAT1 In order to gain further insight into the role of the terminal structures of the N-glycans of GAT1, N-glycosylation processing of NNN was inhibited by 1-deoxymannojirimycin (dMM) Inhibition by dMM leads to the formation of NNN molecules containing N-glycans of oligomannosidic type Figure 4A shows that after treatment with dMM (1 mm) for 72 h, the amount of plasma membrane NNN containing mannosidic N-glycans was in the same range as that of NNN containing mature complex N-glycans without treatment with dMM However, the activity of GABAuptake was reduced to 37% after treatment with dMM (Fig 4B) This indicates that the terminal trimming of N-oligosaccharides is not involved in the regulation of plasma membrane trafficking of GAT1, but in the regulation of GABA uptake As well as wild type, mutant DND, DGN and DDN exhibited only one small band on SDS ⁄ PAGE after treatment with dMM (data not shown), indicating that, like wild type, they contain only mannosidic N-glycans The level of cell surface expression was similar with and without dMM treatment for both wild type and mutants (Figs 4A and 5A) However, their GABA-uptake activity was reduced to half after treatment with dMM (1 mm) for 48 h (Fig 5B) Although mutant DND, DGN and DDN contain only one N-glycosylation site, deficiency of terminal trimming of their N-oligosaccharides strongly affected their GABAuptake activity These indicate that the terminal structure of the oligosaccharides facilitate efficient GABA-uptake activity of the GABA transporter Defective N-glycosylation results in reduction of the stability of GAT1 In order to study the influence of the N-glycosylation and N-linked oligosaccharides on protein stability, the FEBS Journal 272 (2005) 1625–1638 ª 2005 FEBS Fig Influence of dMM on plasma membrane trafficking and GABA-uptake of GFP-tagged GAT1 wild type NNN stable transfected CHO cells were incubated with and without dMM (1 mM) for 72 h (A) Aliquots of cells were used for membrane biotinylation After solubilization, 300 lg total proteins of cell lysates were precipitated with streptavidin beads The eluates were analyzed by Western blotting using anti-GFP mAb (B) Another aliquot of cells was used for measurement of GABA-uptake as described above The values represent the mean ± SEM of three separate experiments intracellular decay time of the GAT1, GAT1 treated with dMM, and the mutants was determined by pulsechase experiments (Fig 6) Figure 6B shows that wild type NNN was very stable with an exponential half-life of about 22 h, whereas the half-life of mutant DDN containing two N-glycosylation mutations was reduced to 12 h The half-life of DDQ containing all three N-glycosylation mutations was reduced even more, compared with that of DDN, showing a value of only 5.5 h In contrast, the stability of NNN containing only mannosidic N-glycans after treatment with dMM 1629 Role of N-glycosylation and N-glycan trimming of GAT1 G Cai et al Fig Analysis of the cell surface expression and GABA-uptake activity of GFP-tagged GAT1 wild type and mutants after treatment with dMM CHO stable transfectants were incubated with and without dMM (1 mM) for 48 h (A) Cell surface expression by FACS analysis Anti-GAT IgG was used for the immunostaining Visualization was performed with R-phycoerythrin-conjugated goat anti-(rabbit IgG) Ig (B) GABA-uptake activity The GABA-uptake activities were normalized to the amount of GAT1 or mutant protein expressed on the plasma membrane The activity of GABA-uptake by NNN was set at 100% The values represent the mean ± SEM of five separate experiments is similar to that of NNN containing N-glycans of the mature complex type The results suggest that N-glycosylation is important for the stability of this protein, but the terminal structure of the N-glycans is not Defective N-glycosylation reduces the trafficking of GAT1 to the plasma membrane In order to study the influence of N-glycosylation on plasma membrane trafficking of GAT1, the distribution of wild type and mutants on the cell surface and in the cell interior was kinetically analyzed by pulsechase experiments Figure shows that after a 40 chase, 34% of total wild type (NNN) proteins, whereas only 18 and 12% of total mutant DDN and DDQ proteins, respectively, were expressed on the plasma membrane After a 120-min chase, the membrane expression of the NNN was increased to 50%, whereas that of mutant DDN and DDQ was increased only to 40% and 15%, respectively This result suggests that 1630 deficiency of N-glycosylation impairs the plasma membrane trafficking of GAT1 Defective N-glycosylation or dMM treatment did not increase the Km GABA values of GAT1 The above results show that both N-glycosylation mutations and terminal structures of N-linked oligosaccharide side chains have a measurable effect on the GABA-uptake activity of GAT1 To determine whether the N-linked oligosaccharide side chains of GAT1 influence the affinity of GAT1 for GABA, concentration dependencies were analyzed on the basis of the MichaelisMenten equation V ẳ Vmax GABA ẵGABA Km GABA ỵ ẵGABA and the parameters for NNN with and without treatment with dMM, and for N-glycosylation mutant DDN were determined As shown in Fig 8, the Vmax FEBS Journal 272 (2005) 1625–1638 ª 2005 FEBS G Cai et al Fig Biological stability of GFP-tagged GAT1 wild type and mutants in transfected CHO cells (A) CHO stable transfectants (2 · 106 cells per dish) were preincubated with and without dMM (1 mM) for 72 h, then pulse-labelled with 3.7 · 106 Bq per dish [35S]methionine for h and immediately chased for the stated times Immunoprecipitates of cell lysates obtained at the indicated chase-times were analyzed by SDS ⁄ PAGE (B) The results of the pulse-chase experiments were analyzed by phosphoimager scanning The radioactivities obtained by immunoprecipitation of the pulse-labelled cells without chase were set at 100% All other values were expressed relative to this value Each time point represents the mean ± SEM of three separate experiments Solid lines represent the exponential fit with half-lives of 22 and 23 h for wild type GAT1 in the absence and presence of dMM, respectively, and of 12 and 5.5 h for the DDN and DDQ mutants, respectively GABA values of NNN treated with dMM, and of mutant DDN were reduced significantly The Vmax GABA value of NNN without dMM is 1.21 pmolỈlgỈprotein)1Ỉmin)1, whereas the value for mutant DDN was only 0.29 pmolỈlgỈprotein)1Ỉmin)1 After treatment of NNN with dMM, the Vmax GABA value of NNN containing mannose-rich N-glycans was strongly reduced to 0.55 pmolỈlgỈprotein)1Ỉmin)1 Although mutations at N-glycosylation sites, as well as N-glycans of the oligomannosidic type reduced the Vmax value of rate of GABA uptake markedly, the Km FEBS Journal 272 (2005) 1625–1638 ª 2005 FEBS Role of N-glycosylation and N-glycan trimming of GAT1 Fig Plasma membrane trafficking of GFP-tagged GAT1 wild type and mutants in transfected CHO cells (A) CHO stable transfectants were pulse-labelled with 3.7 · 106 Bq per dish [35S]methionine for h and chased for min, 40 min, 80 min, 120 and 180 Membrane biotinylation was performed after chase After cell solubilization, total GFP-tagged GAT1 wild type and mutant proteins were immunoprecipitated with anti-GFP pAb and eluted with 100 lL sample buffer containing 0.5% SDS The eluates were diluted with NaCl ⁄ Pi buffer to 400 lL The biotin-labelled membrane proteins were isolated from the diluted eluates with streptavidin beads After removal of all membrane proteins, the intracellular proteins were immunoprecipitated with anti-GFP antibodies Both M (membrane) and I (intracellular) precipitates were eluted and analyzed by SDS ⁄ PAGE (B) The results of the pulse-chase experiments were analyzed by phosphoimager scanning The total radioactivity of membrane and intracellular fractions obtained by immunoprecipitation at each chase time were set at 100% Each value represents the mean ± SEM of membrane fractions derived from three separate experiments GABA values were not affected The data in Fig were fitted with a common Km GABA value of 4.1 lm These results suggest that the defect of N-linked oligosaccharides did not reduce the binding affinity of GAT1 to GABA As treatment with dMM did not affect GAT1 protein translocation to the plasma membrane, the decreased GABA-uptake activity of NNN 1631 Role of N-glycosylation and N-glycan trimming of GAT1 G Cai et al Fig Kinetic analysis of GABA-uptake by GFP-tagged GAT1 wild type (NNN) with and without dMM and N-glycosylation mutant (DDN) Kinetic analysis of GABA-uptake by GFP-tagged GAT1 wild type (NNN) with and without dMM and N-glycosylation mutant (DDN) GABA-uptake assays of wild type NNN pre-incubated with and without dMM (1 mM) and mutant DDN were performed with different GABA concentrations All values presented were calculated after subtraction of the mock values The data were fitted by a Michealis–Menten equation with a common Km value of 4.1 lM and Vmax values of 1.21, 0.55 and 0.29 pmolỈlg protein)1Ỉmin)1 for wild type without and with dMM and for DDN, respectively The values represent the mean ± SEM of three separate experiments after treatment with dMM may be caused by the reduction in substrate translocation by GAT1 (turnover rate) Defective N-glycosylation results in reduced GAT1-mediated currents and reduced rate of external Na+ interaction To obtain additional information on the mechanism of the reduced rate of GABA-uptake due to the mutations, we performed electrical measurements under voltage clamp for wild type and mutant DDN and DGN The number of transporters was calculated from the transient charge movement in the absence of extracellular Na+ [5–7] Figure 9A shows the dependence of the GAT1-mediated current, expressed as charges translocated per functional transporter on the cell surface per second, on the extracellular Na+ concentration The results were similar to those for the GABA uptake, in that the current produced by a single transporter was reduced by the mutation to 46 and 57% for DGN and DDN, respectively, which is close to the reduced GABA uptake seen in the flux measurements A signal from DDQ could hardly be detected Though treatment with dMM makes the CHO cells very unstable for the patch-clamp method, we were, nevertheless, able to obtain evidence for a reduced GAT1-mediated current (data not shown) The 1632 Fig Electrophysiological characterization of GAT1-mediated steady-state and transient currents CHO transient transfectants were subjected to whole-cell patch clamp (A) Steady-state, GABAinduced currents were determined during voltage pulses from a holding potential of )30 mV to )100 mV at different extracellular Na+ concentrations The solid lines represent fits of the Hill equation with Hill coefficients of n ¼ 1.3 for the wild type GAT1 and n ¼ for the mutants (as used previously by Liu et al 1998) and Km Na values of 40 and about 130 mM for wild type and the mutants, respectively (B) The rate constants of the GAT1-mediated current decline in response to a voltage jump from +100 mV to )30 mV were determined at different extracellular Na+ concentrations All data are averages of to determinations ± SEM dependence on Na+ concentration reveals that mutation of the two N-glycosylation sites reduced the apparent affinity from 24 m)1 to about m)1 The transient currents in the absence of GABA were analyzed for jumps in potential to the holding potential of )30 mV The kinetics of the reaction step associated with the extracellular Na+ binding was slowed down by both mutations (Fig 9B) All the rate constants slightly increased with increasing Na+ concentration, FEBS Journal 272 (2005) 1625–1638 ª 2005 FEBS G Cai et al the value for the wild type NNN being about twice that for the mutants Discussion There is increasing evidence that cotranslational N-glycosylation crucially influences the three-dimensional structure, the biological half-life and intracellular trafficking of proteins It is also essential for many recognition processes [13,14,16] Previous studies showed that the mutation of N-glycosylation sites resulted in a reduction of GABA-uptake activity by GAT1 [7,10] However, the possibility that this reduction in function results from a decrease in the number of GABA transporters per cell was not excluded In order to clarify whether N-glycans are directly involved in the GABAuptake process and whether the modulation of N-glycosylation influences the biochemical properties of this protein, quantitative and kinetic analysis of GABA transport expression and activity was performed using stable CHO transfectants Both our own and commercially available anti-GAT1 antibodies were unsuitable for the quantitative analysis of GAT1, as they bind very weakly to this protein Therefore, wild type GAT1 and N-glycosylation defective mutants were tagged with GFP, which has been reported not to influence the intracellular distribution of GAT1; moreover, the tag does not modulate the relevant functions of GAT1 [20] For the quantitative analysis of GABA transport activity, the cell surface expression of GAT1 wild type and N-glycosylation mutants was determined by cell surface biotinylation and the resulting values were used for normalization This is a well established method for the quantitative analysis of cell surface proteins, in which the biotinylation reagent does not react with intracellular proteins [21] We found that all N-glycosylation mutants were expressed on the cell surface, even when all three N-glycosylation sites have been removed, e.g in mutant DDQ (Fig 3A), indicating that N-glycosylation is not essential for the plasma membrane trafficking of GAT1 However, the mutant DDQ was expressed at lower levels on the cell surface, indicating that deficiency of N-glycosylation impairs plasma membrane translocation of GAT1 It was reported that deficiency of N-glycosylation influenced the intracellular trafficking of some glycoproteins [22–25] In order to examine whether the N-glycosylation of GAT1 has influence on its plasma membrane trafficking, a kinetic analysis by pulse-chase experiments was performed These experiments revealed that the plasma membrane trafficking of N-glycosylation mutant proteins was reduced (Fig 7), although the distributions between FEBS Journal 272 (2005) 1625–1638 ª 2005 FEBS Role of N-glycosylation and N-glycan trimming of GAT1 cell surface and cell interior of the mutants DGN, DND and DDN in the steady expression state were not significantly different from that of wild type NNN (Fig 3B) It has been reported that, in the endoplasmic reticulum (ER) and in the early secretory pathway, the N-glycans play a pivotal role in protein folding, oligomerization, quality control, sorting, and transport Thus defective N-glycosylation may lead to a misfolding of this protein, resulting in its partial retention in the ER and its rapid digestion thereafter [16,19] Accordingly, our results indicate that the intracellular trafficking of N-glycosylation mutants was delayed, and ⁄ or partly inhibited due to retention of some mutant protein in the ER, followed by digestion The transfectants of GAT1 wild type (NNN) and mutant DGN, DND and DDN exhibited in SDS ⁄ PAGE (Fig 3A) two intracellular bands, whereas only one large band was found in the plasma membrane fraction However, treatment of the transfectants with dMM, which inhibits N-glycosylation processing, resulted in only small band in the SDS ⁄ PAGE for both the wild type (NNN) (Figs 2C and 4A) and mutant DNG, DND and DDN (data not shown) The mutant DDQ, which does not possess any N-glycosylation site, expressed only one N-glycan-free band of 90 kDa in both the intracellular and the plasma membrane compartments (Fig 3A) The 108 kDa large band of NNN was Endo H resistant, whereas digestion with PNGase F converted it to a 90 kDa N-glycan free polypeptide (Fig 2C), indicating a mature N-glycan of complex type However, the 96 kDa small band of NNN was converted to a 90 kDa polypeptide after either Endo H- or PNGase F-digestion (Fig 2B,C), indicating an N-glycan of the mannosidic type The N-glycosylation mutants DGN, DDN and DND exhibited a reduced molecular mass in accordance with the absence of N-glycans at the two eliminated N-gylcosylation sites in those proteins This suggests that the mutants DGN, DND and DDN, as well as wild type NNN, were N-glycosylated in CHO cells and their N-glycans were processed before they arrived at the cell surface To determine the influence of N-glycosylation on the quality control of GAT1, the half-life of GAT1 wild type and mutants was also investigated by kinetic analysis of pulse-chase experiments We found that the half-life of mutants containing either double (DDN) or triple (DDQ) N-glycosylation mutations was remarkably reduced (Fig 6) Keynan et al reported that the functional expression of the GABA transporter in HeLa cells was abolished by tunicamycin, a potent inhibitor of N-glycosylation [18] We found that inhibition of N-glycosylation processing by dMM did not 1633 Role of N-glycosylation and N-glycan trimming of GAT1 affect either the protein stability (Fig 6) or intracellular trafficking (Figs 4A and 5A) This suggests that cotranslational N-glycosylation, but not the terminal trimming of N-glycans is involved in the regulation of the stability and trafficking of GAT1 It has been reported that a variety of molecular chaperones and folding enzymes assist the folding of newly synthesized proteins in the ER If N-glycosylation is inhibited, some glycoproteins fail to fold or assemble efficiently, resulting in a prolonged retention in the ER and an increased proteolytic breakdown [26–29] Our results indicate that the impaired plasma membrane trafficking and reduced stability of the N-glycosylation mutants of GAT1 must be a result of misfolding of these proteins The deficiency of N-glycosylation results in a markedly reduced GABA-uptake activity (Fig 3C) as well as a GAT1-mediated current in CHO cells (Fig 9A) This is in accordance with our previous work using the expression system of the Xenopus oocyte [7] In order to exclude the possibility that the reduction in function in the mutants could be due to a reduction in the number of GABA transporters per cell, values for the transport activity were normalized for the surface protein of these mutants Double N-glycosylation mutants showed a marked reduction of GABA-uptake activity of 60–40% of that of the wild type GAT-mediated GABA transport activity could hardly be detected in the mutant lacking all three N-glycosylation sites (DDQ), despite the fact that this protein was expressed on the surface of CHO cells (Fig 3A) The N-glycosylation processing inhibitor 1-deoxymannojirimycin (dMM) also strongly inhibited GABA-uptake (Figs 4B and 5B), although the amount of cell surface expression and the intracellular trafficking of GAT1 were not affected by dMM (Figs 4A and 5A) This indicates that the observed reduction of GABA-uptake activity is a result of a deficiency of N-glycans The possibility that the reduced GAT1 activity could be due to a general effect of the inhibitor on other glycoproteins required for GAT1 activity is very unlikely It has been demonstrated that GAT transport function can be reconstructed in liposomes and that no other proteins are needed for GABA-uptake activity [30] Our results suggest that N-glycans, in particular their terminal structure, are involved in the GABA-uptake process of GAT1 However, the GABA-uptake tolerates the modification of neuraminic acid to N-propanoyl neuraminic acid, as incubation with N-propanoylmannosamine (P-NAP), a synthetic precursor of N-propanoyl neuraminic acid [31–33], did not significantly change the GABA-uptake activity of GAT1 (data not shown) 1634 G Cai et al How the oligosaccharides of GAT1 influence GABA-uptake? In order to clarify the functional mechanism of oligosaccharide side chains in GABA-uptake, a kinetic analysis was performed Deficient N-glycosylation decreased the Vmax values of GABA-uptake by GAT1, while the Km GABA values were not affected Similar results were also obtained after treatment with dMM (Fig 8) Our results indicate that the turnover rate of the transporter is affected, but not the substrate binding process This provides strong evidence that N-glycans, in particular their terminal structures, are involved in regulating the GABA translocation of GAT1, but not in binding of GAT1 to GABA Transport of GABA by GAT1 across the cell membrane is driven by an electrochemical gradient of Na+ and Cl– [1,34] with a stoichiometry that results in an electrogenic substrate transport Voltage-clamp experiments suggest that deficient N-glycosylation reduces the affinity of GAT1 for Na+ [7] The present work revealed that the reduced transport activity can at least partially be attributed to a reduced apparent affinity of GAT1 for extracellular Na+ and slowed kinetics of the transport cycle (Fig 9) This was observed in both wild type and mutants after inhibition with dMM As the GABA transport process is driven by the gradient of Na+, it is reasonable to deduce that the affinity of GAT1 for Na+ determines the turnover rate of GABA transport As the data presented in Fig are for a single, functional transporter expressed on the cell surface, the reduced GABA-uptake cannot be due to reduced cell surface expression of transporters In this event the oligosaccharides of GAT1 play a role in the regulation of GABA-uptake by affecting the affinity for sodium ions In conclusion, cotranslational N-glycosylation is important for the correct folding of GAT1 to a functional conformation Defective N-glycosylation leads to decreased protein stability and disturbed intracellular trafficking N-Linked oligosaccharides, in particular their terminal structures, are involved in the regulation of GABA-transport of GAT1 by influence on its affinity for sodium ions Experimental procedures Construction of N-glycosylation mutants of GAT1 and of GAT1/GFP fusion proteins The mutants were based on the neuronal wild type GABA transporter type of mouse (mGAT1) The cDNAs of N-glycosylation mutants DGN (N176D, N181G), DDN (N176D, N181D) and DND (N176D, N184D) were constructed earlier [7] In each mutant, two of three FEBS Journal 272 (2005) 1625–1638 ª 2005 FEBS G Cai et al N-glycosylation sites Asn (N) were mutated to Asp (D) or Gly (G) The mutant DDQ (N176D, N181D and N184Q) was constructed by site-directed mutagenesis in DDN with synthetic oligonucleotides bearing the designated mutation: Forward, 5¢-ACCACCCAAATGACCAGC-3¢; reverse, 5¢-GCTGGTCATTTGGGTGGT-3¢ The reaction was performed using the QuikChangeTM site-directed mutagenesis kit from Stratagene (Heidelberg, Germany) All mutants were cloned into the expression vector pCDNA3 (Invitrogen, Karlsruhe, Germany) and confirmed by sequence analysis To construct the GAT1 ⁄ GFP fusion proteins, HindIII— StuI fragments containing the cDNAs of GAT1 wild type or mutants were cut from the pAlter-1 vector, then cloned into HindIII and SmaI sites of pEGFP-N1 vector (Clontech, Heidelberg, Germany) containing the cDNA encoding the red-shifted GFP-variant In this construct, the cDNAs of GAT1 wild type or mutants were ligated with cDNA of GFP with an identical reading frame, which was confirmed by sequence analysis Preparation of polyclonal anti-GAT and anti-GFP sera Four different oligopeptides: LPWKQCDNPWNTDR (159–172), MHQMTDGLDKPGQIRC(197–211), DEYPRLLRNRRELFC(409–423) and SEDIVRPENGPEQPQAC (584–599), corresponding to sequences in the extracellular and intracellular loops of GAT1, were synthesized and used for immunization of rabbits Specificity of the antiserum was verified by immunoblotting with single or mixed peptides A polyclonal anti-GFP serum was prepared as described previously [35] Transfection of CHO cells and selection of stable transfectants In order to produce stable transfectans, each plasmid DNA (2–4 lg) was transfected into · 105 CHO cells using the Eppendorf Multiporator and an appropriate Eppendorf protocol (Wesseling-Berzdorf, Germany) Transfectants were cultured in six-well plates in alpha-modified Eagle’s medium (MEM alpha) containing 440 mgỈL)1 l-glutamine and 10% (v ⁄ v) fetal bovine serum for days, then selected with 400 mgỈL)1 geneticin G418 for 1–3 weeks The expression of GAT1 ⁄ GFP fusion proteins was detected by flow cytometry, fluorescence microscopy and immunofluorescence microscopy The stable transfectants expressing GAT1 ⁄ GFP fusion proteins were selected by flow cytometry with the FACS Vantage cell-sorter (Becton Dickinson, Erembodegem, Belgium) Higher transfection efficiencies were necessary for the electrophysiological investigations These were achieved with transiently transfected CHO cells The transient transfection was performed using Superfect (Qiagen, Hilden, FEBS Journal 272 (2005) 1625–1638 ª 2005 FEBS Role of N-glycosylation and N-glycan trimming of GAT1 Germany) or FuGENE6 (Roche, Mannheim, Germany) as reagents according to the protocols of Qiagen or Roche, respectively The cells were used for electrophysiological experiments 1–2 days after transfection Flow cytometry and immunofluorescence microscopy For analysis of surface expression of GFP tagged GAT1 and mutants, the anti-GAT1 peptide-specific polyclonal IgG was used for immunostaining transfectants at room temperature for 30 After washing with NaCl ⁄ Pi cells were incubated with R-phycoerythrin-conjugated goat anti(rabbit IgG) Ig at room temperature for 20 and then analyzed by flow cytometry For immunofluorescence microscopy cells were fixed with 3% (v ⁄ v) formaldehyde in NaCl ⁄ Pi at room temperature for 10 After permeabilization with 0.1% (v ⁄ v) Triton X-100 in NaCl ⁄ Pi at room temperature for min, cells were then extensively washed with NaCl ⁄ Pi and blocked with 5% bovine serum albumin and 0.1 m glycine in NaCl ⁄ Pi for 30 and washed with NaCl ⁄ Pi again Polyclonal antibodies against GAT1 were used for immunostaining at room temperature for h After further washing with NaCl ⁄ Pi, the cells were incubated with R-phycoerythrin conjugated goat anti-(rabbit IgG) Ig (diluted : 200) at room temperature for h The cells were extensively washed again with NaCl ⁄ Pi and then mounted with glycerol ⁄ NaCl ⁄ Pi (10 : 1, by volume) for fluorescence microscopy Immunoprecipitation and western blotting analysis Harvested cells were solubilized; followed by centrifugation at 40 000 g for 30 Aliquots of the supernatant were subjected to Western blotting as described previously [36] For immunoprecipitation experiments, the supernatant was incubated with protein A-Sepharose-bound anti-GFP polyclonal antibodies for 12 h at °C After intensive washing, immunoprecipitates were eluted by boiling for in SDS sample buffer SDS ⁄ PAGE was performed according to Laemmli [37] After electrophoresis, the separated proteins were transferred onto a nitrocellulose membrane The anti-GAT1 polyclonal antiserum or anti-GFP mAb MAB2510 (Chemicon, Temecula, CA, USA) was used for immunostaining Visualization was performed with peroxidase-conjugated goat anti-(rabbit IgG) or rabbit anti(mouse IgG) Ig (Sigma, St Louis, MD, USA) and the chemiluminescent reagent luminol Pulse-chase experiments Pulse-chase experiments were performed as described earlier [26] Cells (2 · 106 cells per dish) were incubated at 37 °C 1635 Role of N-glycosylation and N-glycan trimming of GAT1 for h in Dulbecco’s modified essential medium lacking cysteine and methionine The cells were pulse-labelled with [35S]methionine (ICN, Irvine, CA, USA) for the indicated times, using 3.7 · 106 Bq per dish After different chase times, cells were solubilized The expressed GAT1 ⁄ GFP fusion proteins were immunoprecipitated with polyclonal anti-GFP antiserum and analyzed by SDS ⁄ PAGE (7.5%) Quantification of radio-labeled protein was carried out on a PhosphorImagerTM (Molecular Dynamics, Sunnyvale, CA, USA) using iplabgel software The total protein of the cell surface and intracellular bands of each wild type or mutant were set at 100% G Cai et al tants using streptavidin beads (see above) The intracellular GAT1 ⁄ GFP fusion proteins in remaining fractions were isolated by immunoprecipitation with anti-GFP Igs Both the plasma membrane and intracellular proteins were eluted by boiling for in SDS sample buffer, and then analyzed by SDS ⁄ PAGE and western blotting Either antiGFP mAb MAB2510 or anti-GAT1 pAb was used for the immunostaining The protein bands obtained in western blotting were analyzed by phosphoimager scanning The total protein of the cell surface and intracellular bands of each wild type or mutant were set at 100% Measurement of [3H]GABA uptake Endoglycosidase H treatment Immunoprecipitates were eluted by boiling for in buffer containing 0.4% SDS, 1% 2-mercaptoethanol and 40 mm EDTA Endoglycosidase H (Endo H, Boehringer Mannheim) treatment was performed with Endo H (0.02 U ⁄ 80 lL) at 37 °C for 16 h in 50 mm sodium acetate containing 0.5 lL protease inhibitor cocktail (Sigma) at pH 5.5 PNGase F treatment Immunoprecipitates were eluted by boiling for in buffer containing 0.5% (v ⁄ v) SDS, 50 mm 2-mercaptoethanol PNGase F (Roche) treatment was performed with PNGase F (15 40 lL)1) at 37 °C 16 h in 500 mm NaCl ⁄ Pi containing 0.5% (w ⁄ v) Mega 10 and 0.5 lL protease inhibitor cocktail (Sigma) at pH 7.5 Labelling of cell surface proteins with sulfo-NHSbiotin and isolation of membrane proteins Cell surface proteins were labelled with NHS-LC-biotin according to the protocol of Chen et al [38] Cells were washed three times with ice-cold NaCl ⁄ Pi (pH 8.0) and incubated with a freshly prepared solution of NHS-LC-biotin for 20 at °C [1.5 mgỈmL)1 EZ-LinkTM sulfo-NHSLC-biotin (Pierce, Rockford, IL, USA) in 10 mm Hepes buffer pH 9.0, mm CaCl2, 150 mm NaCl] Cells were washed with 100 mm glycine then incubated in 100 mm glycine for 20 The supernatant of the solubilized cells was incubated with 50 lL streptavidin beads (Pierce) at °C for 10 h After extensive washing, all the membrane proteins attached to beads were eluted by boiling for in SDS sample buffer, then subjected to western blotting analysis To determine the transport activity, uptake of [3H]GABA (Amersham-Pharmacia Biotech, Freiburg, Germany) was measured in the presence of 128 mm external Na+ and 10 lm total GABA Cells incubated in 96-well tissue culture plates were washed three times with wash buffer (128 mm NaCl, 5.2 mm KCl, 2.1 mm CaCl2, 2.9 mm MgSO4, mm dextrose and 10 mm Hepes) and then incubated with 200 lL wash buffer containing 3.7 · 104 Bq [3H]GABA, 10 lm cold GABA, 3.7 · 104 Bq [14C]sucrose (AmershamPharmacia Biotech) and 100 lm cold sucrose for 15 at room temperature The sucrose was used to detect leaky cell batches, which were then excluded from the analysis The uptake was stopped by washing cells three times with cold wash buffer, followed by solubilization of the cells with 100 lL of 0.5% (w ⁄ v) SDS solution for h at °C Aliquots were used for measurement of the remaining [3H]GABA and [14C]sucrose The protein concentration in the supernatant was determined using the bicinchoninic acid protein assay reagent (Pierce, USA) Unspecific uptake was determined in mock transfected cells The GABAuptake activity was measured as pmỈlg protein)1Ỉmin)1 For quantitative measurement of the activity of GABAuptake, an aliquot of the stable CHO transfectants was used for GABA-uptake assay, and another aliquot was used to determine the quantity of plasma membrane proteins of GAT1 and mutants (see above) The relative amounts of plasma membrane proteins of GAT1 or mutants were analyzed by imager scanning of western blots The GABA-uptake activity was normalized with the same amount of plasma membrane proteins of wild type and mutants The activity of GABA-uptake by NNN was set at 100% All other values were expressed relative to this value Patch-clamp experiments Analysis of plasma membrane and intracellular expressions of GAT1/GFP fusion proteins After biotinylation, cell solubilization and centrifugation, all the membrane proteins were isolated from the superna- 1636 Voltage-clamp experiments were performed on CHO transient transfectants in the whole-cell patch-clamp configuration Steady-state and transient currents were measured in response to rectangular voltage jumps from a holding FEBS Journal 272 (2005) 1625–1638 ª 2005 FEBS G Cai et al potential of )30 mV to potentials of )100 or +40 mV, using the EPC9 patch-clamp system and pulse software (HEKA, Lambrecht, Germany) From transient charge movements in the absence of GABA, the amount and voltage dependence of external Na+ interaction with the transporter can be determined [7] From the time course of the exponential current decline, the rate constant for a step associated with extracellular Na+ binding can be determined In addition, the total amount of charge Qmax moved by the transporters and the effective valency of the charge z* moved by a single transporter can be calculated Hence, the number of transporters N is given by N ¼ Qmax ⁄ z* and the turnover rate k of the charges transported across the membrane is given by k ¼ I ⁄ Qmax, where I is the steadystate current generated by the single functioning GAT1 molecule on the cell surface in the presence of GABA The external bath solution contained 150 mm NaCl, mm MgCl2, mm CaCl2 and mm Mops (pH ¼ 7.2) For solutions with reduced Na+, the NaCl was replaced by equimolar concentrations of TMACl The pipette solution in contact with the cell interior contained 150 mm K-d-gluconate, mm MgCl2, mm EGTA and mm Mops (pH ¼ 7.2) Acknowledgements This work was supported in part by the Special Funds for Major State Basic Research of China (Grant G1999053907), a grant from the Chinese Academy of Sciences, a grant from the Deutsche 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bacteriophage T4 Nature 227, 680–685 Chen JG, Liu-Chen S & Rudnick G (1997) External cysteine residues in the serotonin transporter Biochemistry 36, 1479–1486 FEBS Journal 272 (2005) 1625–1638 ª 2005 FEBS ... side-chains are important for the GABA transport activity 1- Deoxymannojirimycin inhibits the GABA- uptake of GAT1 In order to gain further insight into the role of the terminal structures of the N-glycans. .. N-glycans, in particular their terminal structures, are involved in regulating the GABA translocation of GAT1, but not in binding of GAT1 to GABA Transport of GABA by GAT1 across the cell membrane... trimming of their N-oligosaccharides strongly affected their GABAuptake activity These indicate that the terminal structure of the oligosaccharides facilitate efficient GABA- uptake activity of the