Concentrative Export from the Endoplasmic Reticulum of the γ-Aminobutyric Acid Transporter 1 Requires Binding to SEC24D

Abstract

Re-uptake of γ-aminobutyric acid (GABA) into presynaptic specializations is mediated by the GABA transporter 1 (GAT1), a member of the SLC6 gene family. Here, we show that a motif in the COOH terminus of GAT1 (566RL567), which is conserved in SLC6 family members, is a binding site for the COPII coat component Sec24D. We also identified residues in Sec24D (733DD734) that are required to support the interaction with GAT1 and two additional family members, i.e. the transporters for serotonin and dopamine. We used three strategies to prevent recruitment of Sec24D to GAT1: knock-down of Sec24D by RNA interference, overexpression of Sec24D-VN (replacement of 733DD734 by 733VN734), and mutation of 566RL567 to 566AS567 (GAT1-RL/AS). In each instance, endoplasmic reticulum (ER) export of GAT1 was impaired: in the absence of Sec24D or upon coexpression of dominant negative Sec24D-VN, GAT1 failed to undergo concentrative ER export; GAT1-RL/AS also accumulated in the ER and exerted a dominant negative effect on cell surface targeting of wild type GAT1. Our observations show that concentrative ER-export is contingent on a direct interaction of GAT1 with Sec24D; this also provides a mechanistic explanation for the finding that oligomeric assembly of transporters is required for their ER export: transporter oligomerization supports efficient recruitment of COPII components. Re-uptake of γ-aminobutyric acid (GABA) into presynaptic specializations is mediated by the GABA transporter 1 (GAT1), a member of the SLC6 gene family. Here, we show that a motif in the COOH terminus of GAT1 (566RL567), which is conserved in SLC6 family members, is a binding site for the COPII coat component Sec24D. We also identified residues in Sec24D (733DD734) that are required to support the interaction with GAT1 and two additional family members, i.e. the transporters for serotonin and dopamine. We used three strategies to prevent recruitment of Sec24D to GAT1: knock-down of Sec24D by RNA interference, overexpression of Sec24D-VN (replacement of 733DD734 by 733VN734), and mutation of 566RL567 to 566AS567 (GAT1-RL/AS). In each instance, endoplasmic reticulum (ER) export of GAT1 was impaired: in the absence of Sec24D or upon coexpression of dominant negative Sec24D-VN, GAT1 failed to undergo concentrative ER export; GAT1-RL/AS also accumulated in the ER and exerted a dominant negative effect on cell surface targeting of wild type GAT1. Our observations show that concentrative ER-export is contingent on a direct interaction of GAT1 with Sec24D; this also provides a mechanistic explanation for the finding that oligomeric assembly of transporters is required for their ER export: transporter oligomerization supports efficient recruitment of COPII components. γ-Aminobutyric acid (GABA) 3The abbreviations used are: GABA, γ-aminobutyric acid; COPII, coat protein II; DAT, dopamine transporter; GAT1, GABA transporter 1; SERT, serotonin transporter; SLC6, solute carrier 6 family; VSVG, vesicular stomatitis virus G; GMP-P(NH)P, guanyl-5′-yl imidodiphosphate; FRET, fluorescence resonance energy transfer; FRAP, fluorescence recovery after photobleaching; ER, endoplasmic reticulum; YFP, yellow fluorescent protein; GFP, green fluorescent protein; siRNA, small interfering RNA; shRNA, short hairpin RNA; CFP, cyan fluorescent protein; GST, glutathione S-transferase. is the major inhibitory neurotransmitter in the mammalian brain. Its action is terminated by re-uptake into synaptic terminals. This is achieved by the GABA transporter 1 (GAT1), the predominant isoform in the central nervous system. GAT1 belongs to the Na+/Cl--dependent SLC6 gene family, which also includes transporters for serotonin (SERT), dopamine (DAT), and norepinephrine transporter. The turnover number of these transporters is low (i.e. in the range of 10 s-1). Accordingly, the number of active transporters at the cell surface is critical for the duration of synaptic transmission. This is evident from the gene dosage effect in mice engineered to be GAT1-deficient. The absence of one Gat1-allele homozygous suffices to produce phenotypic consequences: heterozygous mice Gat1+/- consume more ethanol, are more responsive to locomotor activation by ethanol, and are more prone to ethanol dependence than their wild type counterparts (1Cai Y.Q. Cai G.Q. Liu G.X. Cai Q. Shi J.H. Shi J. Ma S.K. Sun X. Sheng Z.J. Mei Z.T. Cui D. Guo L. Wang Z. Fei J. J. Neurosci. Res. 2006; 84: 255-267Crossref PubMed Scopus (39) Google Scholar). Gat1-/- mice, however, suffer from a complex motor disorder that includes abnormal gait, reduced locomotion, and tremor (2Chiu C.S. Brickley S. Jensen K. Southwell A. Mckinney S. Cull-Candy S. Mody I. Lester H.A. J. Neurosci. 2005; 25: 3234-3245Crossref PubMed Scopus (187) Google Scholar). It has been widely appreciated that the number of regulatory membrane proteins (that is receptor, transporters, and ion channels) on the surface is controlled by the rate of endocytosis, recycling, and proteosomal/lysosomal degradation (see e.g. Refs. 3Ramamoorthy S. Blakely R.D. Science. 1999; 285: 763-766Crossref PubMed Scopus (313) Google Scholar and 4Holton K.L. Loder M.K. Melikian H.E. Nat. Neurosci. 2005; 8: 881-888Crossref PubMed Scopus (114) Google Scholar). In contrast, the role of ER export has been explored to a lesser extent but there is evidence that the steadystate level of membrane proteins can also be determined by a rate-limiting step at the level of the ER (5Snyder P.M. Endocrinology. 2005; 146: 5079-5085Crossref PubMed Scopus (196) Google Scholar). Originally, it was assumed that folding and quality control was the major determinant for ER (6Hurtley S.M. Helenius A. Annu. Rev. Cell Biol. 1989; 5: 277-307Crossref PubMed Scopus (778) Google Scholar). However, specific ER export motifs were shown to control exit of several membrane proteins from the ER. Two classes of ER export motifs have been identified: diacidic ((D/E)-X-(D/E)) and di-hydrophobic motifs (7Bonifacino J.S. Glick B.S. Cell. 2004; 116: 153-166Abstract Full Text Full Text PDF PubMed Scopus (1298) Google Scholar). More recently, a dibasic export motif ((R/K)X(R/K)) has been recognized in the cytoplasmic tail of glycosyltransferases (8Giraudo C.G. Macconi H.J. Mol. Biol. Cell. 2003; 14: 3757-3766Crossref Google Scholar). ER export motifs mediate interaction with components of the COPII coat, which is composed of the small G-protein, Sar1, the Sec23/24 complex, and the Sec13/31 complex (9Barlowe C. Orci L. Yeung T. Hosobuchi M. Hamamoto S. Salama N. Rexach M.F. Ravazzola M. Amherdt M. Schekman R. Cell. 1994; 77: 895-907Abstract Full Text PDF PubMed Scopus (1048) Google Scholar). Sec24 is the subunit that functions as a cargo receptor (9Barlowe C. Orci L. Yeung T. Hosobuchi M. Hamamoto S. Salama N. Rexach M.F. Ravazzola M. Amherdt M. Schekman R. Cell. 1994; 77: 895-907Abstract Full Text PDF PubMed Scopus (1048) Google Scholar). Mammals have 4 different isoforms termed Sec24A-D (9Barlowe C. Orci L. Yeung T. Hosobuchi M. Hamamoto S. Salama N. Rexach M.F. Ravazzola M. Amherdt M. Schekman R. Cell. 1994; 77: 895-907Abstract Full Text PDF PubMed Scopus (1048) Google Scholar). The differences between these subunits in terms of cargo selectivity are still unknown. This interaction leads to concentration of cargo into ER exit sites. These can be identified by their morphology, i.e. ribosome-free regions of the rough ER that are decorated with clusters of vesicles and tubules. In addition, they stain for components of the COPII coat. Efficient export from the ER is contingent on cargo concentration in ER exit sites: the vesicular stomatitis virus glycoprotein (VSVG) uses a di-acidic motif to exit the ER (10Nishimura N. Balch W.E. Science. 1997; 277: 556-558Crossref PubMed Scopus (397) Google Scholar). Mutation of the motif does not lead to complete retention but results in a 10-fold reduction of the ER export rate (11Nishimura N. Bannykh S. Slabough S. Matteson J. Altschuler Y. Hahn K. Balch W.E. J. Biol. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar). We and that ER export of neurotransmitter transporters is contingent on their oligomeric assembly M. J. Biol. 277: Full Text Full Text PDF PubMed Scopus Google A. K. C. A. J. Biol. 2003; Full Text Full Text PDF PubMed Scopus Google M.K. D. Blakely R.D. J. Neurosci. 2003; PubMed Google T. S. A. J. Biol. 2003; Full Text Full Text PDF PubMed Scopus Google M. J. Biol. 2004; Full Text Full Text PDF PubMed Scopus Google Scholar). We also a composed of 10 in the GAT1 which mediated interaction with Sec24D M. J. Biol. 2004; Full Text Full Text PDF PubMed Scopus Google Scholar). We that the to a di-hydrophobic ER export (7Bonifacino J.S. Glick B.S. Cell. 2004; 116: 153-166Abstract Full Text Full Text PDF PubMed Scopus (1298) Google Scholar). However, in the we identified 566RL567 as the residues that mediate binding of GAT1 to This interaction of GAT1 with Sec24D was for cargo concentration and determined the level of the transporter at the cell We also show that a which is to with a dominant negative effect on the surface of the wild type transporter. These observations are with the that ER export oligomeric assembly of transporters M. J. Biol. 277: Full Text Full Text PDF PubMed Scopus Google for a mechanistic explanation by to efficient recruitment of the COPII and be for membrane proteins as A. S. M. S. M. J. Biol. 2004; Full Text Full Text PDF PubMed Scopus Google Scholar). and was a from R. The was a from Balch of Cell The In and were by the to a to M. J. Biol. 277: Full Text Full Text PDF PubMed Scopus Google this is in oligomerization to the by A. K. C. A. J. Biol. 2003; Full Text Full Text PDF PubMed Scopus Google Scholar). was also the was from was a from of of was from was was to Sec24D number was from Cell and for were in with and were on were with the in the of were was with and the were with the The was on the was after The was also of was determined as in M. J. Biol. 2004; Full Text Full Text PDF PubMed Scopus Google Scholar). In were into after after the were in the of for was as the of cell determined in the of 10 RNA and was the to the RNA was used for the with the with to the were at with a with a The fluorescence used on this were from were with a with a fluorescence resonance energy were with of GAT1 and with Sec24D as in the resonance energy was by the extent to which fluorescence by after of recovery after the were into M. J. Biol. 2004; Full Text Full Text PDF PubMed Scopus Google Scholar). recovery after was also on a was with a were with regions of were for and a was the of was with at at were in with of The fluorescence were and the these were and to to the the to a to the fluorescence after and to the that is at of and to the different of the GAT1 COOH terminus as as the COOH terminus of were into the This was used to of were to of at was by of for were by at for The cell was in 1 was to for on of and and a the was and on for a The was by at for 1 and the was a The was with with by 1 were with glutathione was by of concentration and in a were at were with Sec24D or were and by in of the was by for was with on to of was and were for were by and with were by of and for at of the was a In in were with the in the were by a in and these are to as The was to and J. Cell Biol. 2004; PubMed Scopus Google with the the of of of of of of of and 10 of were at with for were at for in of to cell and and at for The the vesicular the ER, were by on a and a The membrane was with in for 1 at in was for 1 the membrane was with in with for 1 at were by from the of by was to be the were with the that of was used of and the was in the of 4 1 The in these was from control and from by in of The was by at of the Sec24 in the COOH of we that mutation of the motif to to retention of the transporter M. J. Biol. 2004; Full Text Full Text PDF PubMed Scopus Google Scholar). we that the However, the results of that still with Sec24D In for a conserved to the we the 566RL567 motif that was conserved of the Na+/Cl--dependent neurotransmitter transporter family Mutation of 566RL567 to and the interaction with Sec24D the transporter accumulated in the ER, i.e. the site of Sec24D recruitment (see 566RL567 is conserved SLC6 family members, family to Sec24D. The interaction between cargo and Sec24D in the ER. Accordingly, the transporter of be in the the in of GAT1 is by with the is in oligomerization and in the ER M. J. Biol. 277: Full Text Full Text PDF PubMed Scopus Google Scholar). This still with Sec24D M. J. Biol. 2004; Full Text Full Text PDF PubMed Scopus Google Scholar). We mutation in the of the of the of also leads to retention of the in the ER A. K. C. A. J. Biol. 2003; Full Text Full Text PDF PubMed Scopus Google and We as ER of to that binding of Sec24D was not to GAT1 but a more We a between Sec24D and was in to that with We the interaction by GAT1 COOH type and were with from In with the wild type and the COOH terminus Sec24D from In contrast, the COOH terminus the mutation not Sec24D. The COOH terminus of the with that of GAT1 with the of a motif that is of the motif in GAT1 The COOH terminus of also with Sec24D We that the of Sec24D binding by was to a of the COOH terminus by with a protein that with the three of GAT1 S. S. Mol. Cell. Neurosci. 2004; PubMed Scopus Google Scholar). as as with of the on Sec24D for the with the GAT1 COOH cargo binding have been in from Hamamoto S. Orci L. Schekman R. Cell. 2003; Full Text Full Text PDF PubMed Scopus Google J. Cell. 2003; Full Text Full Text PDF PubMed Scopus Google Scholar). the interaction motif on we the of and Sec24D and used the for We that a interaction motif to on the surface of the of the and as it has to with in the The motif was in which to 733DD734 in Sec24D and these residues to and in a reduction in the to with wild type Sec24D with This is to a effect of the as it the as wild type Sec24D (i.e. still ER exit In to Sec24D-VN wild type with the of Sec24D for ER of GAT1 in of cargo with Sec24 is to export from the ER. ER export of GAT1 was on Sec24D we in In mammalian these are it is to of cargo in the ER. The is this is efficient in the ER at the This is not for GAT1. We to a that on of This of COPII in a dominant negative were with wild type GAT1 and which retention of the transporter in the ER. The were to the and This was by from (i.e. wild type and to a on or at results in the of small which are not by Accordingly, for i.e. GAT1, is in the the was on low of GAT1 were in the at GAT1 was into the a we the protein with in the of was also at the was with from that Sec24D-VN, of GAT1 into the was reduced of the was on or at We that the action of Sec24D-VN was to a action by effect on of ER export of is controlled by a di-acidic which the interaction with the complex (11Nishimura N. Bannykh S. Slabough S. Matteson J. Altschuler Y. Hahn K. Balch W.E. J. Biol. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar). it is that a di-acidic motif to the 733DD734 motif in Sec24D. Accordingly, was from the ER at at a level with the with control that the di-acidic motif of Sec24D (733DD734) is in mammalian Sec24 This that binding to the COOH terminus of GAT1 is not to Sec24D. and isoforms can for we in of Sec24D. Sec24D was that not of and We that the in Sec24D into a of protein to Sec24D are not Accordingly, we and the by The reduced of the the control not the was with a of of GAT1 was reduced with from with a control of into the of these of and this the We that the effect in the was to by a Sec24D as The can be into by the on the for the siRNA, we that the Sec24D was from in which Sec24D was by shRNA, of GAT1 from the ER to a extent than from control Sec24D for ER of GAT1 in is that there is a major rate-limiting step in the of the ER In this the in vesicular which we in be of to the of GAT1 at the We the in Sec24D of GAT1 in wild type GAT1 the Accordingly, it accumulated at the membrane the of Sec24D were by of the a shRNA, GAT1 was the cell This was also upon of the dominant negative of Sec24D which the Sec24D binding was the cell and it with a for the endoplasmic reticulum In the DAT, mutation of is in the to in also leads to retention in the ER, but after the transporter to at the membrane M. T. A. J. Biol. 2004; Full Text Full Text PDF PubMed Scopus Google Scholar). These observations be for by a of concentrative ER which interaction with this were and also to GAT1, surface of GAT1-RL/AS to with We the number of in which GAT1-RL/AS was at the cell and that this number after We that the in on of the of a cell was that transporter was at the cell this not into the level of transporter at the cell GAT1 surface we of GABA does not the membrane and can be by transporters at the cell GABA a of the level of GAT1 surface in the of after of that determined after by a of in GAT1-RL/AS of In contrast, in wild type GAT1 of in the to by GAT1-RL/AS was than that of wild type this is to a which can be the retention of the transporter is into In contrast, the of GAT1-RL/AS is with the wild type transporter This that the mutation does not and surface of GAT1-RL/AS and the wild type transporter. were with a wild type GAT1 or The of in which fluorescence was at the cell was determined and after to of in two to three were with a wild type GAT1 or GAT1-RL/AS of GABA was determined and after for differences in by that the wild type transporter was used as a and This to or are for GABA in of Sec24D for the ER of SLC6 motif of GAT1 is conserved SLC6 family and Sec24D with and (see Sec24D-VN is to a dominant negative effect and the of these two and were at the membrane not M. M. J. Biol. 2006; Full Text Full Text PDF PubMed Scopus Google Scholar). Sec24D-VN was of and were the cell and In contrast, the was at the membrane than in which failed to the Sec24D-VN and the transporter by in In of wild type Sec24D ER retention of of of GAT1 from of than from of the in the are two that can be to of GAT1-RL/AS to the cell surface is S. J. J. Nat. Cell Biol. PubMed Scopus Google the of the transporter to with Sec24D does not it to into ER exit sites. The mutation a folding that a of GAT1-RL/AS with ER the GAT1-RL/AS is to ER retention of binding to Sec24D. this we the of GAT1-RL/AS by rate The of proteins on they are in the membrane or the ER. we wild type transporter GAT1 in the ER by coexpression of additional we GAT1-RL/AS with this not the of GAT1-RL/AS but controlled for membrane effect from overexpression of can be in there was in of the wild type and GAT1-RL/AS in the ER and were with that in GAT1-RL/AS and and We to that GAT1-RL/AS is a cargo that to undergo the concentration in the this was achieved by a cargo concentration the is to the it on the of the cargo with dominant negative This leads to retention of the proteins and of the cargo of in the after of the cargo concentration into ER exit is by of wild type can be in in the of wild type GAT1 was the cell of after of to GAT1 was into In contrast, we not upon of the with wild type This is with the that GAT1-RL/AS is SLC6 family M. J. 2003; PubMed Scopus Google Scholar). The that ER export motifs have to be in oligomeric M. 2006; Scopus Google Scholar). Accordingly, GAT1-RL/AS surface of the wild type transporter We this by with wild type GAT1 or with We cell surface by of can be mediated by transporters at the of GAT1-RL/AS reduced for the of wild type GAT1 at the cell surface The upon coexpression of wild type GAT1 and GAT1-RL/AS in were not than the of mediated by each transporter by the in but also than mediated by the wild type GAT1 of in We have that the COOH terminus of GAT1 of the transporter it two a that ER export and a at the which the interaction with the and at the membrane M. J. Biol. 2004; Full Text Full Text PDF PubMed Scopus Google Scholar). In the we identified the ER export which is conserved of the SLC6 transporter family (see in and a mechanistic are three observations that support the that 566RL567 is the ER export a mutation that this motif in ER retention and concentrative ER The motif was required to Sec24D in and in mutation in Sec24D exerted a dominant negative effect on ER export of GAT1. results show that of GAT1 by the COPII leads to cargo concentration that the transporters are in the and M. J. Biol. 277: Full Text Full Text PDF PubMed Scopus Google Sec24D but they are not from the ER. This is with that oligomerization is required for ER export M. J. Biol. 277: Full Text Full Text PDF PubMed Scopus Google M. 2006; Scopus Google Scholar). We a which and the in the of into K. A. Mol. Biol. Cell. 2003; 14: PubMed Scopus Google K. A. J. Biol. 2004; Full Text Full Text PDF PubMed Scopus Google it that assembly of the COPII coat is by the of recruitment in the transporter transporters Sec24D the motif but to exit the ER the coat is not with COOH the M. J. Biol. 2004; Full Text Full Text PDF PubMed Scopus Google but to undergo concentrative they not Sec24D. This that that the motif a dominant negative effect on surface of the wild type This was for GAT1-RL/AS in the Our (i.e. that oligomerization recruitment of also be to proteins with e.g. the export of which is also contingent on in the ER A. S. M. S. M. J. Biol. 2004; Full Text Full Text PDF PubMed Scopus Google Scholar). In GAT1, the with the ER export motif in glycosyltransferases (8Giraudo C.G. Macconi H.J. Mol. Biol. Cell. 2003; 14: 3757-3766Crossref Google Scholar). However, it is that 566RL567 is to a dibasic motif for two the are conserved in SLC6 members, and (see In contrast, the by the motif is not this is by a a and a and in norepinephrine and The 566RL567 motif in the COOH terminus of GAT1 recruitment of the COPII component Sec24D. In contrast, the motif of with (8Giraudo C.G. Macconi H.J. Mol. Biol. Cell. 2003; 14: 3757-3766Crossref Google Scholar). with Sar1, for which cargo binding cargo binding to has been three binding for cargo have been on different of the which are to as and Hamamoto S. Orci L. Schekman R. Cell. 2003; Full Text Full Text PDF PubMed Scopus Google J. Cell. 2003; Full Text Full Text PDF PubMed Scopus Google Scholar). Our motif in Sec24D that this interaction This motif is conserved in mammalian Sec24 isoforms and in from S. However, the additional Sec24 which are in not for the level of the protein was reduced by RNA the of is the to show that a cargo for the membrane to a specific Sec24 isoform and between the we are at a to the of Sec24D for GAT1. explanation is that there are additional the different Sec24 that the of the these additional there is evidence to that the conserved ER export motif is also in the SLC6 family we that two additional members, i.e. and SERT, Sec24D. In addition, the motif has been in which on the of and in DAT, of to in by in a transporter that was in the ER and that was to the membrane M. T. A. J. Biol. 2004; Full Text Full Text PDF PubMed Scopus Google Scholar). this was to in the that in which to in GAT1, in retention 2006; PubMed Scopus Google Scholar). The of these however, is by the that mutation of does not the protein in the ER but also binding of that the protein is not It is also evident that the COOH terminus of is required to the several the COOH terminus binding C. K. J. Neurosci. 2004; PubMed Scopus Google Scholar). In contrast, of the COOH terminus retention of GAT1 in the endoplasmic it does not to or in vesicular M. J. Biol. 2004; Full Text Full Text PDF PubMed Scopus Google Scholar). GAT1-RL/AS the cell the transporter with with the wild the reduction in was for the reduction in cell surface we also of the which we to the ER export of this motif to does not the of the transporter to and to with but the in the the of observations for the that the cell surface level of a membrane protein is determined by to a COPII i.e. Sec24D. of GAT1 to with COPII not complete retention in the ER but in a in the ER export which into a reduction of surface is that ER export of a neurotransmitter transporter is to a mutation in of the norepinephrine transporter J. I. Blakely R.D. D. N. J. PubMed Scopus Google to retention of the protein in the ER M.K. D. Blakely R.D. J. Neurosci. 2003; PubMed Google Scholar). Mutation of one the dominant effect of the mutation is for by the that ER export observations also that the cell surface level of transporters can be by their ER export on these we that there also be in transporter also from in Sec24D This is to be of the of Sec24D is upon M. and C.