Article |
Address correspondence to Steinunn Baekkeskov, Hormone Research Institute, University of California San Francisco, 513 Parnassus Avenue, Room HSW 1090, San Francisco, CA 94143-0534. Tel.: (415) 476-6267. Fax: (415) 502-1447. E-mail: s_baekkeskov{at}biochem.ucsf.edu
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Abstract |
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Key Words: palmitoylation; Golgi targeting; post-Golgi targeting; polarized sorting; neurons
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Introduction |
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In this study, we have explored the trafficking signals that target the smaller isoform of the -aminobutyric acid (GABA)*synthesizing enzyme glutamate decarboxylase 65 (GAD65) to presynaptic termini in axons. GABA synthesized by GAD65 is required for fine tuning of GABA-ergic neurotransmission in response to a variety of environmental stimuli (Kash et al., 1997, 1999; Hensch et al., 1998). GAD65 is synthesized as a hydrophilic cytosolic protein, which undergoes hydrophobic posttranslational modifications in the NH2-terminal domain, resulting in anchoring to intracellular membranes (Christgau et al., 1991), including the Golgi compartment (Solimena et al., 1993, 1994). aa 183 of GAD65 were shown to mediate Golgi targeting of a soluble protein, ß galactosidase, whereas an interchange of aa 127 of GAD65 with aa 129 of a second isoform of glutamate decarboxylase, GAD67, mediated targeting of the latter to Golgi membranes (Solimena et al., 1994). However, because GAD67 appears to harbor intrinsic membrane anchoring properties (Kanaani et al., 1999), it is unclear whether the aa 127 region of GAD65 is sufficient for targeting of soluble proteins to Golgi membranes. Palmitoylation of cysteines 30 and 45 and phosphorylation of three of the serines 3, 6, 10, and 13 are exclusive properties of the membrane-anchored form of GAD65 (Christgau et al., 1992; Namchuk et al., 1997). However, neither modification is required for Golgi targeting or membrane association (Shi et al., 1994; Solimena et al., 1994; Namchuk et al., 1997). Instead, the results of Shi et al. (1994), suggest a critical role of aa 2431, or a subset thereof, in membrane anchoring. These residues may comprise a signal sequence for attaching a putative membrane anchor. In addition to the Golgi compartment, GAD65 in pancreatic ß cells occurs on the membrane of small vesicles, which are similar in size to synaptic-like microvesicles (Christgau et al., 1992). Immunofluorescence analysis of primary neuronal cultures shows that GAD65 targets to presynaptic termini, where it presumably resides on synaptic vesicles (Kanaani et al., 1999). The localization of GAD65 on the membrane of synaptic vesicles may be critical for rapid accumulation and secretion of GABA in response to environmental signals.
In this paper, we show that two separate signals are required for targeting of cytosolic GAD65 to Golgi membranes and that Golgi membranes constitute an essential sorting station and reservoir for GAD65 in route to axons. Although palmitoylation of a third trafficking signal in GAD65 is not required for targeting to the Golgi compartment, it is critical for trafficking of GAD65 from Golgi membranes to presynaptic sites.
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Results |
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The NH2-terminal region of GAD65 has been shown to contain a membrane targeting signal upstream of the palmitoylated cysteines (Shi et al., 1994; Solimena et al., 1994). The GAD65(2431A) mutation disrupted membrane association and yielded a soluble nonpalmitoylated protein in COS-7 cells (Fig. 1). This protein displayed a diffuse distribution in soma, dendrites, and axons, with no punctate staining (Fig. 4; Fig. S2, available at http://www.jcb.org/cgi/content/full/jcb.200205053/DC1). Thus membrane anchoring is a likely requirement for trafficking of GAD65 to presynaptic sites.
The palmitoylated aa 160 region of GAD65 mediates presynaptic clustering of soluble proteins and their associated partners
To test whether the signals for presynaptic clustering reside in the palmitoylated NH2-terminal region of GAD65, we first generated a chimera of aa 172 coupled to GFP. This hybrid protein was palmitoylated (Fig. 5) and displayed presynaptic clustering and relative dendritic exclusion, similar to that of full-length GAD65GFP (Figs. 4 and 6; Fig. S3 i, available at http://www.jcb.org/cgi/content/full/jcb.200205053/DC1). A C30,45A mutant of this protein, 172GAD65(C30,45A)GFP, was not palmitoylated (Fig. 5). This protein displayed a diffuse expression pattern in axons and dendrites (Fig. 6). In contrast to the full-length palmitoylation mutant, none of the 172GAD65 (C30,45A)GFP protein was targeted to presynaptic clusters (Fig. 4 B; Fig. 6). Several conclusions can be made based on these results. First, the palmitoylated aa 172 region of GAD65 contains the necessary signals for presynaptic clustering of a soluble protein, GFP. Second, palmitoylation is essential for this process. Third, the signal(s) involved in the targeting of a small fraction of the full-length palmitoylation-deficient mutant to presynaptic clusters is missing in the short form of the palmitoylation-deficient mutant.
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aa 123 in GAD65 contain a Golgi targeting signal and are required for presynaptic clustering
Neither palmitoylation nor the first 23 aa of GAD65 are required for membrane anchoring (Christgau et al., 1992; Shi et al., 1994), whereas aa 2431, or a subset of this region, may harbor the putative membrane anchor (Shi et al., 1994). We tested whether the first 23 aa of the aa 160 region are required for targeting to presynaptic clusters by generating a chimera of aa 2460 of GAD65 and GFP (2460GAD65GFP). The 2460GAD65GFP protein was palmitoylated (Fig. 5) and membrane anchored in COS-7 cells (Fig. 1). In primary hippocampal neurons, the 2460GAD65GFP protein was detected in a punctate pattern in the soma and along both dendrites and axons, but completely failed to reach presynaptic clusters (Fig. 7; Fig. S3 iii). Thus, the first 23 aa are not required for membrane anchoring, but are indispensable for the targeting of GAD65 to presynaptic clusters.
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The 172GAD65GFP, 172GAD65(C30,45A)GFP, and 160GAD65/113PSD-95GFP proteins were also highly concentrated in a perinuclear region where they colocalized with Golgi matrix protein 130 (GM130) (Fig. 8; Fig. S5). The immunolabeled 2460GAD65GFP protein, however, was detected in a punctate pattern throughout the cytosol and did not concentrate in a perinuclear region defined by GM130 (Fig. 8). Thus, the 2460GAD65GFP protein mainly targets to membranes distinct from the Golgi compartment. These results suggest that membrane association and Golgi targeting are mediated by separate signals and that the intact aa 123 region is critical for the latter. Furthermore, the failure of the 2460GAD65GFP protein to target to presynaptic termini suggests that the Golgi compartment is an obligatory sorting station in route to axons.
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Discussion |
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Golgi targeting is mediated by two trafficking signals distinct from the palmitoylation motif and is required, but not sufficient, for presynaptic clustering
A Golgi targeting signal resides in aa 123, and a lack of this signal results in a protein, 2460GAD65GFP, that anchors to intracellular membranes distinct from the Golgi compartment and fails to target to axon termini. Loss of the membrane anchoring signal yields a protein, GAD65(2431A)GFP, that is soluble and distributed equally throughout the neuron. Finally, loss of palmitoylation of cysteines 30 and 45 results in a protein that is membrane anchored and targets to the Golgi compartment but fails to enter a post-Golgi trafficking pathway for efficient and selective targeting to presynaptic termini. The targeting of the palmitoylation-deficient mutant to Golgi membranes separates Golgi localization from axonal transport in that only the latter demonstrably requires palmitoylation. Although palmitoylation can function as an essential part of a Golgi localization signal in some proteins (Lutjens et al., 2000), a lack of such a role is not without precedent; palmitoylation of lymphoma proprotein convertase is not essential for its TGN localization (van de Loo et al., 2000), and dual palmitoylation of H-ras is not required for its Golgi targeting but rather for exit from the Golgi compartment and trafficking to the plasma membrane (Apolloni et al., 2000). These results demonstrate that the aa context of a palmitoylated trafficking signal, and/or the combination with other trafficking signals, is a critical parameter for determining membrane destination.
Anchoring to the cytosolic leaflet of Golgi membranes appears to provide a mechanism by which a soluble protein like GAD65 can gain access to a major sorting compartment for polarized proteins (Keller et al., 2001) and subsequently enter a post-Golgi trafficking pathway to axon termini, which requires membrane association. The 2460GAD65GFP protein does not localize to the Golgi compartment and yet is palmitoylated in COS-7 cells. It is therefore possible that newly synthesized GAD65 first anchors to the cytoplasmic leaflet of pre-Golgi membranes, possibly the ER or the ERGolgi intermendiate compartment, and that palmitoylation precedes trafficking to and accumulation in the Golgi compartment. The lack of a Golgi targeting signal in the 2460GAD65GFP protein would result in a palmitoylated protein that diverges from wt GAD65 in pre-Golgi membranes and bypasses the Golgi compartment. There is evidence to suggest the presence of a palmitoyl transferase in the ER. Thus, newly synthesized cytosolic H-ras undergoes farnesylation and carboxymethylation to become anchored to the cytoplasmic leaflet of the ER (Choy et al., 1999; Apolloni et al., 2000), where it appears to undergo palmitoylation before its trafficking to the Golgi compartment (Apolloni et al., 2000). An alternative possibility is that the palmitoyl transferase responsible for palmitoylation of cysteines 30 and 45 of GAD65 is in more than one location and that newly synthesized 2460GAD65GFP and wt GAD65GFP undergo palmitoylation in two distinct membrane compartments.
How does palmitoylation control trafficking of GAD65?
The results of cholesterol depletion experiments suggest that trafficking of palmitoylated GAD65 to presynaptic clusters is sensitive to cellular cholesterol levels. GAD65 is soluble in mild nonionic detergents and does not share the detergent insolubility of many proteins that stably associate with lipid rafts (Brown and Rose, 1992; Pralle et al., 2000). However, it was recently shown that palmitoylated H-ras has a reversible interaction with lipid rafts, which is not associated with detergent insolubility (Prior et al., 2001) and may represent a low-affinity mechanism afforded to other palmitoylated proteins. We propose that palmitoylation of GAD65 mediates its attachment to specialized membrane microdomains in the TGN, resulting in lateral segregation from the nonpalmitoylated protein before the formation of transport vesicles. In a separate study, we have shown that palmitoylation of GAD65 regulates its entry into a post-Golgi trafficking pathway to axon termini, which involves early endosomes and Rab5a and is shared with several synaptic vesicle proteins (unpublished data). Thus, the trafficking of GAD65 appears to involve sorting steps in both the Golgi compartment and in early endosomes.
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Materials and methods |
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DNA construction
All constructs used in this study, except GAD65(S3,6,10,13A), are shown in Fig. 1. Chimeras containing EGFP at the COOH-terminal end of either wt human GAD65, GAD65(S3,6,10,13A), or GAD65(2431A) were generated by PCR and subcloned into the KpnI-BamHI sites in a pEGFP-N3 vector (CLONTECH Laboratories, Inc.). wt rat GAD65 and a palmitoylation-deficient mutant, GAD65(C30,45A), were generated by PCR and subcloned into the HindIII-KpnI sites in frame with and in front of EGFP in pEGFP-N3. The plasmids encoding wt and mutant GAD65 that served as templates in PCR reactions were described previously (Shi et al., 1994; Namchuk et al., 1997). wt PSD-95GFP and PSD-95(C3,5S)GFP in pGW1 were previously described (Craven et al., 1999). Fusions of aa 2460 of wt GAD65, aa 172 of wt GAD65, the palmitoylation-deficient 172GAD65(C30,45A), or the soluble 172GAD65(2431A) with the NH2 terminus of EGFP were performed by PCR using primers encoding the 5' and 3' ends of aa 2460 of wt GAD65 or aa 172 of wt GAD65, or the mutant forms, and the restriction sites HindIII-KpnI. Amplified fragments were digested and subcloned into the HindIII-KpnI sites of pEGFP-N3. To generate a chimera of the palmitoylated NH2-terminal domain of GAD65 and a palmitoylation-deficient PSD-95, a construct was generated by PCR using primers encoding the 5' and 3' ends of aa 160 of GAD65 and HindIII-KpnI restriction sites. The amplified fragments were digested and subcloned into GW1 PSD-95GFP at a HindIII site upstream of the starter methionine and a silent KpnI site at aa 13 of PSD-95.
COS-7 cell experiments
Culture, transfection, and labeling with [3H]palmitic acid of COS-7 cells were performed as described previously (Christgau et al., 1992; Shi et al., 1994; Namchuk et al., 1997). Cells were extracted in hypotonic Hepes buffer (10 mM Hepes/NaOH, pH 7.4, 1 mM MgCl2, 1 mM 2-aminoethylisothiouronium bromide, 0.2 mM pyridoxal 5'-phosphate, 1 mM phenylmethylsulfonylfluoride, 1% Triton X-114). The 100,000 g supernatant was immunoprecipitated with guinea pig anti-GFP antibodies and immunocomplexes were isolated and processed for SDS-PAGE followed by fluorography as previously described (Namchuk et al., 1997). For Western blotting, resolved proteins were electroblotted onto Protran BA nitrocellulose transfer membranes (Applied Scientific), probed with a primary antibody against GFP (monoclonal mouse; BABCO) and HRP-conjugated secondary antibodies (Amersham Biosciences), and visualized by ECL reagent (Amersham Biosciences).
Hippocampal neurons
Primary hippocampal neurons were prepared from E18/E19 rat brains as described by Craven et al. (1999). Neurons were transfected at day in vitro (DIV) 67 by lipid-mediated gene transfer using Effectene transfection reagent according to the manufacturer's protocol (QIAGEN). After 35 h of incubation at 37°C, the transfection solution was replaced with a 50:50 solution of fresh/conditioned medium (replacement medium). Neurons were fixed 96 h after transfection. For experiments with 2-bromopalmitate, neurons were washed twice in replacement medium and then cultured in the same medium containing 10 µM of either palmitate or 2-bromopalmitate. Cells were fixed 48 h after transfection. Cholesterol depletion experiments and staining of neurons with filipin were performed essentially as described by Simons et al. (1998). Transfected neurons were cultured in replacement medium containing 4 µM lovastatin (A.G. Scientific, Inc.) and 0.25 mM mevalonate (Sigma-Aldrich) for 4 d. Cells were incubated for 20 min with 5 mM methyl-ß-cyclodextrin (Sigma-Aldrich) in fresh neurobasal medium, washed twice with the same medium, and fixed. For analyses of cholesterol levels, fixed neurons were incubated with filipin III (Cayman Chemical) at 125 µg/ml in PBS.
Immunofluorescence analyses
For indirect immunofluorescence, neuronal cultures were fixed in either 2% paraformaldehyde in PBS, pH 7.4, or methanol (-20°C) (staining for synaptophysin and GKAP). COS-7 cells were fixed 1824 h after transfection with 2% paraformaldehyde. A guinea pig anti-GFP antibody (CLONTECH Laboratories, Inc.) was used as a primary antibody to enhance the signal of GFP chimeras in neuronal transfections. After incubation with primary and secondary antibodies, fluorescent images were obtained using either a ZEISS inverted microscope or a Leica TCS NT laser scanning confocal microscope with a kryptonargon laser.
Quantitative measurement of polarized protein expression
Quantification of polarized protein sorting was performed on 520 neurons from two to three independent transfections. Images of neurons were acquired with a CCD digital camera and quantitated using Metamorph imaging software (Universal Imaging Corp.) (El-Husseini et al., 2001). The degree of presynaptic clustering or polarized expression of GAD65 was determined by calculating the average pixel intensity in the axon, axon puncta, and dendrites. The average pixel intensity was obtained by tracing five representative sections of both dendrites and axons, beginning at least 40 microns from the cell body. For constructs present in presynaptic clusters, lines were drawn through the axons including puncta. These measurements were averaged and used to calculate a ratio of axonal intensity (with or without puncta) versus dendritic intensity or axonal puncta intensity versus axonal background to compare distribution of GAD65GFP and other constructs. These ratios are representative of the relative amounts of protein present in axons versus dendrites and in presynaptic clusters versus diffuse in axons, respectively. Results were analyzed by a t test using a two-tailed distribution and two-sample equal variance.
Quantitative measurement of cholesterol levels
Fluorescence images of neurons incubated in either normal conditions or in cholesterol-depleting conditions and then fixed and incubated with the cholesterol-binding fluorescent antibiotic filipin III were captured on an Olympus IX-70 fluorescence microscope. Filipin IIIstained cholesterol was excited with light at 365395 nm. Fluorescence emissions at 425465 nm were collected using a CCD digital camera. The amounts of background-subtracted fluorescence were determined for the perinuclear area in the soma and for areas in the neurites in each neuron and quantitated using Metamorph imaging software. Data collection parameters were maintained constant for all images and amounts of fluorescence from 50 neurons for each condition were averaged. Statistically significant differences were determined using a nonpaired, two-tailed t test.
Online supplemental material
The supplemental figures (Figs. S1S5) for this article are available at http://www.jcb.org/cgi/content/full/jcb.200205053/DC1. These figures show confocal analysis of primary hippocampal neurons transfected with (a) wt GAD65GFP and cultured in the presence of either 10 mM 2-bromopalmitate or 10 mM palmitate (Fig. S1); (b) GAD65(2431A)GFP (Fig. S2); (c) 172GAD65GFP, 160GAD65/D113PSD-95GFP, or 2460GAD65GFP and immunolabeled for GFP and endogenous synaptophysin (Fig. S3); (d) the 160GAD65/D113PSD-95GFP chimera and double immunolabeled for GFP and GKAP (Fig. S4); and (e) wt or mutant GAD65GFP and double immunolabeled for GFP and the Golgi marker protein GM130 (Fig. S5).
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Footnotes |
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* Abbreviations used in this paper: GABA,
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Acknowledgments |
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Support by grants from the Nora Eccles Treadwell Foundation (S. Baekkeskov) and the National Institutes of Health (S. Baekkeskov and D.S. Bredt), the American Heart Association (D.S. Bredt), and the Medical Research Council of Canada (A.E. El-Husseini) is gratefully acknowledged.
Submitted: 13 May 2002
Revised: 31 July 2002
Accepted: 7 August 2002
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