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First published online 29 January 2008
doi: 10.1242/jcs.011916

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A palmitoylation cycle dynamically regulates partitioning of the GABA-synthesizing enzyme GAD65 between ER-Golgi and post-Golgi membranes

¹ Department of Medicine and Diabetes Center, University of California San Francisco, 513 Parnassus Avenue, HSW 1090, San Francisco, CA 94143-0534, USA
² Department of Microbiology and Immunology, University of California San Francisco, 513 Parnassus Avenue, HSW 1090, San Francisco, CA 94143-0534, USA
³ Cell Biology and Metabolism Branch, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, MD 21218, USA

View larger version (11K): [in this window] [in a new window]   Fig. 1. Schematic model of post-translational modification and subcellular targeting of GAD65. Newly synthesized hydrophilic and soluble GAD65 (F1) undergoes an irreversible hydrophobic modification in the cytosol (Step A). The resulting hydrophobic GAD65 (F2) reversibly associates with Golgi membranes. In Golgi membranes, the protein undergoes palmitoylation (Step B) catalyzed by a palmitoyl transferase (PAT). The palmitoylated protein (F3) is targeted out of the Golgi compartment and to a pathway to synaptic vesicles and presynaptic termini in neurons and synaptic-like microvesicles in pancreatic β cells.

View larger version (56K): [in this window] [in a new window]   Fig. 2. Palmitoylation-deficient GAD65-GFP accumulates in ER and Golgi membranes whereas wt GAD65-GFP is efficiently cleared from the ER. (A-D) Projected confocal images of rat hippocampal neurons (A,B) and COS-7 cells (C,D) coexpressing the ER marker, DsRed2-ER and either GAD65(C30,45A)-GFP (A,C) or wt GAD65-GFP (B,D). Rat hippocampal neurons were transfected at DIV7, fixed 43 hours following transfection, and immunostained for GFP before confocal analyses. COS-7 cells were fixed 16 hours post transfection and analyzed by confocal microscopy. Palmitoylation-deficient GAD65-GFP is detected in Golgi membranes and shows an extensive colocalization with the ER marker DsRed2-ER in soma and dendrites (arrows) of neurons (A, enlarged frame) and in the cytosolic fine mesh of ER membranes in COS-7 cells (C, enlarged frame). By contrast, wt GAD65-GFP is not detected in ER membranes in neurons (B) and COS-7 cells (D). Instead, wt GAD65 is localized predominantly to Golgi membranes and vesicular structures in the soma, dendrites (arrows) (B, enlarged frame) and axons (arrowhead) of neurons, and in the Golgi membranes and vesicular structures in the cytosol of COS-7 cells (D, enlarged frame). (E) Correlation coefficient for colocalization of wt and palmitoylation-deficient GAD65-GFP with the DsRed2-ER marker. Results are presented as mean ± s.e. for 10 cells for each protein. *P<0.001.

View larger version (36K): [in this window] [in a new window]   Fig. 3. Palmitoylation is required for targeting of GAD65-GFP to vesicles. (A-F) Projected confocal images of rat hippocampal neurons (A,B), MDCK cells (C,D) and COS-7 cells (E,F), transiently expressing either wt GAD65-GFP (A,C,E) or the palmitoylation-deficient mutant GAD65(C30,45A)-GFP (B,D,F). Rat hippocampal neurons were transfected at DIV6, fixed 72 hours following transfection and immunostained for GFP before confocal analyses. COS-7 and MDCK cells were fixed 18 hours post-transfection and analyzed by confocal microscopy. In addition to Golgi localization, GAD65-GFP-containing vesicles were observed in soma, dendrites (arrows) and axons (arrowheads) of rat hippocampal neurons and in the cytoplasm of fixed COS-7 and MDCK cells (see also supplementary material Movies 1 and 2). By contrast, palmitoylation-deficient GAD65-GFP was detected in Golgi membranes and in a diffuse pattern throughout the cell but not in vesicles in any of the three cell types (see also supplementary material Movie 3). (G) Quantification of wt GAD65-GFP and GAD65(C30,45A)-GFP positive vesicles in the cytosol of COS-7 cells. Results are presented as mean ± s.e. for five cells for each protein. *P<0.001.

View larger version (51K): [in this window] [in a new window]   Fig. 4. Palmitoylation results in spatially distinct localization of GAD65-GFP in Golgi membranes of rat hippocampal neurons and increased colocalization with the trans-Golgi marker TGN38. High-resolution projected confocal images of rat hippocampal neurons at DIV10 transiently expressing either palmitoylation-deficient (A,B) or wild-type (C,D) GAD65-GFP. Neurons were double immunolabeled for GFP and either the trans-Golgi network protein TGN38 (A,C) or cis-Golgi matrix protein GM130 (B,D). Palmitoylation-deficient GAD65 segregates from both TGN38 (A) and GM130 (B) and appears to be oriented more proximal to the cytosol than the two markers. Wild-type GAD65-GFP, however, colocalizes with TGN38 (C) but segregates from GM130 (D) albeit to a distinct face of GM130 than palmitoylation-deficient GAD65. GAD65-GFP-containing vesicles are observed for wt GAD65 (arrowheads) but not palmitoylation-deficient GAD65. (E) Correlation coefficient (r) for colocalization of palmitoylation-deficient and wt GAD65-GFP with TGN38. Only wt GAD65-GFP colocalizes significantly with TGN38 (r=0.80±0.02). Results are presented as mean ± s.d. for 10 cells for each protein. *P<0.001.

View larger version (57K): [in this window] [in a new window]   Fig. 5. Dispersion of the Golgi pool of palmitoylation-deficient GAD65-GFP but not wt GAD65-GFP following treatment with BFA. Selected single-layer confocal images obtained from a live-cell imaging time series recorded immediately after the addition of BFA (5 µg/ml) to COS-7 cells coexpressing GalT-mRFP1 and either the palmitoylation-deficient GAD65(C30,45A)-GFP (A) or wt GAD65-GFP (B). (A) BFA treatment has resulted in the complete dissociation of the Golgi pools of both GAD65(C30,45A)-GFP and GalT-mRFP1 at the 15 minute time-point following treatment with BFA (see also supplementary material Movie 4). Note that the cell in the lower right corner has protein aggregates because of overexpression, precluding analyses of the effect of BFA. (B) The Golgi pool of GalT-mRFP1 has redistributed to the ER at the 15 minute time-point following exposure to BFA (5 µg/ml). By contrast, only a fraction of the Golgi pool of wild-type GAD65-GFP has dispersed at 15 and 30 minutes, and a prominent fraction of the protein is still detected in punctate structures in the perinuclear region at those timepoints (see also supplementary material Movie 5). The effect of BFA is global and the cells shown in A and B are representative of cells transfected with palmitoylation-deficient GAD65-GFP and wt GAD65-GFP, respectively. Note that GAD65-containing cytosolic vesicles are not visible in the single confocal layer selected for imaging of the Golgi compartment.

View larger version (40K): [in this window] [in a new window]   Fig. 6. FRAP analyses reveal two pools of wt GAD65-GFP replenishing the Golgi in primary rat hippocampal neurons. The Golgi-associated pool of palmitoylation-deficient (A) and wt GAD65-GFP (B) in living rat hippocampal neurons at DIV10 were selectively photobleached (t=0) and fluorescence recovery was monitored over time at 37°C. (C) Kinetics of fluorescence recovery after bleaching of the Golgi-associated pool of wt GAD65-GFP (red circles, n=10 neurons) and GAD65(C30,45A)-GFP (blue circles, n=10 neurons). Recovery of GFP fluorescence into the photobleached Golgi compartment was recorded at low laser power and normalized taking into consideration the loss of GFP fluorescence in the cell during the recording. Data are mean ± s.d. for a representative experiment. (D) Half-times of Golgi fluorescence recovery following photobleaching for wild-type and palmitoylation-deficient GAD65-GFP. (E) Percentage recovery of Golgi fluorescence following photobleaching. Data for D and E are the mean ± s.e. from five independent experiments (5-10 cells per experiment).

View larger version (52K): [in this window] [in a new window]   Fig. 7. FRAP analyses reveal two pools of wt GAD65-GFP replenishing the Golgi in COS-7 cells. The Golgi-associated pool of palmitoylation-deficient (A) and wt GAD65-GFP (B) in living COS-7 cells were selectively photobleached (circle, t=0) and fluorescence recovery was monitored over time at 37°C. Note the presence of wt GAD65-GFP in cytosolic punctate structures, whereas the mutant protein is localized in the ER network of membranes. (C) Kinetics of fluorescence recovery after bleaching of the entire Golgi-associated pool of wild-type GAD65-GFP (red circles, n=10 cells) and GAD65(C30,45A)-GFP (blue circles, n=10 cells). Recovery of GFP fluorescence into the photobleached Golgi compartment was recorded at low laser power and normalized taking into consideration the loss of GFP fluorescence in the whole cell during the recording. Data are presented as mean ± s.d. from a representative experiment. (D) Half-times of Golgi fluorescence recovery following photobleaching for wild-type and palmitoylation-deficient GAD65-GFP. (E) Percentage recovery of Golgi fluorescence following photobleaching. Data for D and E are presented as mean ± s.e. from four independent experiments (5-10 cells per experiment).

View larger version (40K): [in this window] [in a new window]   Fig. 8. The slowly recovering pool of GAD65-GFP in Golgi membranes is dependent on palmitoylation in both neurons and COS-7 cells. FRAP analysis of the Golgi-associated pool of GAD65(C30,45A)-GFP and wild-type GAD65-GFP in hippocampal neurons (A-D) and COS-7 cells (E-H). Transfected cells were treated for 5 hours at 37°C with either 25 µM 2-BP or DMSO followed by selective photobleaching of the Golgi-associated pools. (A,B,E,F) Kinetics of recovery of GFP fluorescence for palmitoylation-deficient GAD65(C30,45A)-GFP (A,E) and wt GAD65-GFP (B,F). Data for neurons are presented as mean ± s.d. (n=10 cells for each condition). Data for COS-7 cells are presented as mean ± s.d. (n=5 cells for each condition). (C,G) Half-times of recovery of Golgi fluorescence following photobleaching of wild-type and palmitoylation-deficient GAD65-GFP in the presence and absence of 2-BP. The slow phase of GAD65-GFP recovery in Golgi membranes is abolished in the presence of 2-BP. (D,H) Percentage recovery of Golgi fluorescence following photobleaching. Data for C and D are presented as mean ± s.e. (n=10 neurons for each condition). Data for G and H are presented as mean ± s.e. (n=5 COS-7 cells for each condition).

View larger version (57K): [in this window] [in a new window]   Fig. 9. GAD65-GFP accumulates in the ER and Golgi and is absent from cytosolic vesicles in the presence of 2-BP. (A-C) High-resolution projected confocal images of COS-7 cells expressing the ER-marker DsRed2-ER together with either wild-type (A,B) or palmitoylation-deficient (C) GAD65-GFP. Transfected COS-7 cells were allowed to express the protein for 16 hours and then treated with 25 µM 2-BP (B) or DMSO (A,C) for 5 hours at 37°C before fixation and immunofluorescence analysis. Wild-type GAD65-GFP is present in cytosolic vesicles and Golgi in the absence of 2-BP and does not colocalize with DsRed2-ER (A, enlarged frames a and b). However, inhibition of palmitoylation results in the disappearance of wt GAD65-GFP from cytosolic vesicles and accumulation in ER membranes (B, enlarged frames c and d), a localization similar to that shown for the palmitoylation-deficient GAD65-GFP (C, enlarged frames e and f). (D) Correlation coefficient for colocalization of wt GAD65-GFP with the DsRed2-ER marker following incubation with a solvent (DMSO) and 2-BP, respectively. Results are presented as mean ± s.e. (n=10 cells per condition). *P<0.001. (E) Quantification of wt GAD65-GFP positive vesicles in the cytosol of COS-7 cells treated with solvent (DMSO) and 2-BP. Results are presented as mean ± s.e. (n=5 cells per condition). *P<0.001.

View larger version (22K): [in this window] [in a new window]   Fig. 10. Schematic model of GAD65 cycling between the cytosol, the ER-Golgi and a vesicular pathway. Newly synthesized hydrophilic and soluble GAD65 (F1) undergoes an irreversible hydrophobic modification in the cytosol (Step A). The resulting hydrophobic GAD65 (F2) reversibly associates with ER and Golgi membranes, establishing an equilibrium between membrane and cytosolic pools. In Golgi membranes, the protein undergoes palmitoylation (Step B) catalyzed by a palmitoyl transferase (PAT). The palmitoylated protein (F3) is targeted to the TGN, shifting the equilibrium from the ER towards the Golgi. From the TGN, the protein is targeted to cytosolic vesicles and a pathway to synaptic vesicles and presynaptic termini in neurons and synaptic-like microvesicles in pancreatic β cells. A depalmitoylation along this pathway (Step C) results in a reversal to F2, trafficking back to ER-Golgi membranes by a microtubule-independent pathway, and a new cycle of palmitoylation and targeting to the vesicular pathway.

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