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First published online 19 February 2008
doi: 10.1242/jcs.017681

Journal of Cell Science 121, 753-761 (2008)
Published by The Company of Biologists 2008
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Signal-dependent export of GABA transporter 1 from the ER-Golgi intermediate compartment is specified by a C-terminal motif

Hesso Farhan1, Veronika Reiterer1, Alexander Kriz2, Hans-Peter Hauri2, Margit Pavelka3, Harald H. Sitte1 and Michael Freissmuth1,*

1 Institute of Pharmacology, Center of Biomolecular Medicine and Pharmacology, Medical University of Vienna, Waehringer Str. 13a, 1090 Vienna, Austria
2 Department of Pharmacology and Neurobiology, Biozentrum, University of Basel, Klingelbergstrasse 70, CH-4056 Basel, Switzerland
3 Department of Cell Biology and Ultrastructure Research, Center for Anatomy and Cell Biology, Medical University of Vienna, Schwarzspanierstr. 17, A-1090 Vienna, Austria


Figure 1
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  		Fig. 1. Localization and transport of GAT1-SSS. (A) Schematic representation of GAT1 showing its 12 transmembrane segments and the C-terminus. The motif replaced by serines in the mutant GAT1-SSS is highlighted by the red box. (B) A HeLa cell expressing YFP-tagged GAT1-SSS. The image was captured 24 hours after transfection by confocal laser scanning microscopy (Leica SPE). (C) HeLa cells were co-transfected with plasmids encoding YFP-tagged GAT1-SSS and CFP-tagged reticulon 2 (CFP-RTN2). After 24 hours cells were fixed and imaged by confocal laser scanning microscopy (Leica SPE). Merged image is on the right. (D) HEK293 cells were transfected with plasmids encoding YFP-tagged GAT1-SSS (upper panels) or YFP-tagged GAT1-RL/AS (lower panels). Arrows indicate the bleached region. FRAP was recorded as described in the Materials and Methods. Fluorescence intensities were expressed as percentage of fluorescence measured prior to bleaching, and the data points were subjected to curvilinear regression using an equation for a monoexponential rise from a basal value to obtain the rate constant for fluorescence recovery and the mobile fraction. Data are means ± s.e.m. from four or five independent experiments. (E) YFP-GAT1-SSS, or GAT1-{Delta}37, and Sar1a-T39N were transiently co-expressed in HEK293 cells. On the next day, semi-intact cells were prepared and the in vitro budding assay was performed as described in the Materials and Methods. (F,G) Vesicle-budding assay performed in semi-intact cells transfected with Sar1a-T39N. Budding of endogenous ERGIC-53 (F) and CLIMP-63 (G) was determined as in panel E.

 

Figure 2
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  		Fig. 2. Ultrastructure of HEK293 cells expressing wild-type (WT) GAT1 (A) and GAT1-SSS (B). Cells grown on glass coverslips were fixed in 2.5% glutaraldehyde, pH 7.4, post-fixed in 1% veronal-acetate-buffered OsO4, dehydrated in a graded series of ethanol and embedded in Epon. The electron micrographs show parts of the paranuclear cytoplasm, in which cisternae of the ER, ER exit sites (arrows) and Golgi apparatus stacks (GA) are visible. Magnification was 33,000x (A,B).

 

Figure 3
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  		Fig. 3. Accumulation of GAT1-SSS in punctate structures requires the COPII-dependent ER-export machinery. (A) HEK293 cells were transfected with a plasmid encoding YFP-tagged GAT1-SSS. Cells were treated overnight with H89 (100 µM). Images were acquired 24 hours after transfection. (B,C) HEK293 cells were co-transfected with plasmids coding for CFP-tagged GAT1-SSS (1 µg) and Sar1-T39N (2 µg, B) or YFP-tagged Sec24D-VN (2 µg, C). Images were acquired 24 hours after transfection. (D) HEK293 cells were transfected with a plasmid coding for the double mutant YFP-tagged GAT1-RL/AS-SSS and images were captured 24 hours after transfection. (E) HEK293 cells were co-transfected with plasmids encoding SAR1A-T39N (2 µg) and either YFP-tagged wild-type GAT1 (wt; 1 µg) or YFP-tagged GAT1-SSS (sss; 1 µg). FRAP was recorded as outlined in the Materials and Methods. Data represent mean half-lives in seconds of fluorescence recovery from five independent experiments; error bars indicate s.e.m.

 

Figure 4
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  		Fig. 4. Colocalization of GAT1-SSS with subcellular markers. HeLa cells were transfected with plasmids encoding YFP-tagged GAT1-SSS. After 24 hours, cells were fixed and immunostained against the ERES marker Sec31 (A), the cis-Golgi marker GM130 (B), the late endosome/lysosome marker LAMP1 (C) and a marker for the intermediate compartment, ERGIC53 (D). (A,B,D) Boxed areas are shown as close-ups on the right. (A) Arrowheads highlight GAT1-SSS punctae that are closely associated with ERES. (C) The numbered images represent magnifications of region 2 (R.2), which were acquired in multiple optical slices. The numbers indicate how many µm the optical slices are from 0. To the right of these is a close-up of region 1 (R.1). Images were acquired using a confocal microscope (Leica SPE).

 

Figure 5
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  		Fig. 5. GAT1-SSS localizes to the ERGIC. (A) HEK293 cells co-expressing CFP-GAT1-SSS and GFP-ERGIC53 were incubated at 15°C for 2 hours. Cells were fixed and images were subsequently captured by confocal microscopy (Zeiss LSM 510). Overlays (right) were generated using the Zeiss LSM image browser. (B) LdlF cells were co-transfected with plasmids encoding CFP-tagged GAT1-SSS and GFP-tagged ERGIC53. Cells were maintained at 34°C for 20 hours. Thereafter, the temperature was shifted to 40°C for 5 hours; subsequently, the images were captured with a CCD camera, digitized and overlaid (right) using MetaMorph software. (C) HeLa cells were transfected with a plasmid encoding YFP-tagged GAT1-SSS. After 24 hours, cells were treated with BFA (5 µg/ml) for 10 minutes at 37°C. Afterwards, cells were fixed and GM130 was detected by immunofluorescence. Images were acquired using a confocal microscope (Leica SPE). The boxed area in the overlaid image is shown close-up on the right. (D) HeLa cells were co-transfected with plasmids encoding YFP-tagged GAT1-SSS and CFP-tagged RAB1. After 24 hours, cells were fixed. Images were acquired using a confocal microscope (Leica SP5). Raw images are displayed in grey to enhance visibility of the peripheral structures. Arrowheads highlight peripheral punctae showing colocalization. The lower panels show magnified views of the boxed region. (E) LdlF cells were co-transfected with plasmids encoding YFP-tagged wild-type GAT1 and myc-tagged ERGIC53. Cells were cultured at 34°C for 20 hours. Thereafter, the temperature was shifted to 40°C for 5 hours. Subsequently, cells were fixed and processed for immunofluorescence against the myc-epitope. (F) Cytosol from HEK293 cells (100 µg) was incubated with a fusion protein comprising GST and the GAT1 C-terminus (10 µg), either in its wild-type version (GST-wt) or that of mutated GAT1-SSS (GST-SSS). The GST-pull-down was performed as indicated in the Materials and Methods. The upper panel shows immunostaining for β-COP. In the lower panel, GST fusion proteins were visualized by staining with Ponceau red. The upper band represents the fusion protein of GST and C-terminus, whereas the lower band reflects proteolytic degradation and corresponds to the size of GST.

 

Figure 6
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  		Fig. 6. Transient colocalization of YFP-tagged VSVG-ts045 with CFP-tagged GAT1-SSS during its trafficking through the anterograde secretory pathway. HEK293 cells were co-transfected with plasmids encoding CFP-tagged GAT1-SSS and YFP-tagged VSVG-ts045. Cells were incubated for 20 hours at 40°C. Images were acquired using a confocal microscope before (A) and at 10 (B) and 30 (C) minutes after shifting to the permissive temperature. The two images on the right show close-ups of those in B. (C) Insets show immunoblots of CFP-tagged GAT1-SSS (left-hand panel) and YFP-tagged VSVG-ts045 (middle panel): cell extracts were prepared at 30 minutes after shifting to the permissive temperature and incubated overnight in the absence (–) or presence (+) of endoglycosidase H. Immunoreactive material was visualized by an antiserum directed against GFP.

 

Figure 7
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  		Fig. 7. Attaching a dilysine motif to GAT1-SSS supports its retrieval to the ER. (A) HEK293 cells were transfected with plasmids encoding CFP-tagged GAT1-SSS-KK. Images were acquired 24 hours later with a confocal microscope. (B,C) HEK293 cells expressing CFP-tagged GAT1-SSS-KK treated with 10 µg/ml BFA for 2 hours (B) and at 30 minutes after washout of BFA (C). (D,E) HEK293 cells co-expressing GAT1-SSS-KK and YFP-Golgi treated with 10 µg/ml BFA for 2 hours (D) and at 30 minutes after washout (E).

 

Figure 8
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  		Fig. 8. Localization of GAT1-SSS in hippocampal neurons. Hippocampal neurons were isolated from foetal rat brains (E18) and transfected after 7 days in culture using Lipofectamine 2000 with a plasmid encoding YFP-tagged GAT1-SSS. The transporter was visualized by confocal fluorescence microscopy 48 hours after transfection.

 

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© The Company of Biologists Ltd 2008