FRINK Linked Data Fragments server

Matches in SemOpenAlex for { <https://semopenalex.org/work/W1976817149> ?p ?o ?g. }

• W1976817149 endingPage "44992" @default.
• W1976817149 startingPage "44984" @default.
• W1976817149 abstract "Differential targeting of neuronal proteins to axons and dendrites is essential for directional information flow within the brain, however, little is known about this protein-sorting process. Here, we investigate polarized targeting of lipid-anchored peripheral membrane proteins, postsynaptic density-95 (PSD-95) and growth-associated protein-43 (GAP-43). Whereas the N-terminal palmitoylated motif of PSD-95 is necessary but not sufficient for sorting to dendrites, the palmitoylation motif of GAP-43 is sufficient for axonal targeting and can redirect a PSD-95 chimera to axons. Systematic mutagenesis of the GAP-43 and PSD-95 palmitoylation motifs indicates that the spacing of the palmitoylated cysteines and the presence of nearby basic amino acids determine polarized targeting by these two motifs. Similarly, the axonal protein paralemmin contains a C-terminal palmitoylated domain, which resembles that of GAP-43 and also mediates axonal targeting. These axonally targeted palmitoylation motifs also mediate targeting to detergent-insoluble glycolipid-enriched complexes in heterologous cells, suggesting a possible role for specialized lipid domains in axonal sorting of peripheral membrane proteins. Differential targeting of neuronal proteins to axons and dendrites is essential for directional information flow within the brain, however, little is known about this protein-sorting process. Here, we investigate polarized targeting of lipid-anchored peripheral membrane proteins, postsynaptic density-95 (PSD-95) and growth-associated protein-43 (GAP-43). Whereas the N-terminal palmitoylated motif of PSD-95 is necessary but not sufficient for sorting to dendrites, the palmitoylation motif of GAP-43 is sufficient for axonal targeting and can redirect a PSD-95 chimera to axons. Systematic mutagenesis of the GAP-43 and PSD-95 palmitoylation motifs indicates that the spacing of the palmitoylated cysteines and the presence of nearby basic amino acids determine polarized targeting by these two motifs. Similarly, the axonal protein paralemmin contains a C-terminal palmitoylated domain, which resembles that of GAP-43 and also mediates axonal targeting. These axonally targeted palmitoylation motifs also mediate targeting to detergent-insoluble glycolipid-enriched complexes in heterologous cells, suggesting a possible role for specialized lipid domains in axonal sorting of peripheral membrane proteins. detergent-insoluble glycolipid-enriched complexes postsynaptic density growth-associated protein green fluorescent protein 150 mm NaCl, 20 mm Hepes, pH 7.4 microtubule-associated protein ratio of axonalversus dendritic expression analysis of variance 50 mm Tris-HCl, pH 7.4, 1 mm EDTA, 1 mm EGTA polyacrylamide gel electrophoresis synapse-associated protein paralemmin Proper neuronal function requires selective protein targeting to specialized cellular and plasma membrane domains including the nerve terminal, node of Ranvier, axon hillock, and postsynaptic density. An early step in this targeting decision tree involves a polarized sorting of proteins to either dendritic (postsynaptic) or axonal (presynaptic) domains. However, the mechanisms by which neurons target specific proteins to dendrites versus axons are poorly understood. Better characterized is protein sorting to apical versusbasolateral plasma membranes in polarized epithelial cells, which share certain features with axonal versus dendritic targeting in neurons (1Dotti C.G. Simons K. Cell. 1990; 62: 63-72Abstract Full Text PDF PubMed Scopus (358) Google Scholar, 2Jareb M. Banker G. Neuron. 1998; 20: 855-867Abstract Full Text Full Text PDF PubMed Scopus (154) Google Scholar). That is, short cytosolic C-terminal protein-sorting motifs are one route for both dendritic and basolateral targeting (2Jareb M. Banker G. Neuron. 1998; 20: 855-867Abstract Full Text Full Text PDF PubMed Scopus (154) Google Scholar), whereas specialized lipid rafts can mediate both axonal and apical sorting of certain transmembrane and glycosylphosphatidylinositol-anchored membrane proteins (3Ledesma M.D. Simons K. Dotti C.G. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 3966-3971Crossref PubMed Scopus (199) Google Scholar). The concept of specialized lipid rafts mediating polarized protein targeting emerged from observations that apical and basolateral cell membranes have different lipid compositions. Apical membranes are enriched in sphingolipids that aggregate with cholesterol to form packed raft-like domains within the fluid membrane bilayer. These rafts are insoluble in non-ionic detergents and, hence, are termed detergent-insoluble glycolipid-enriched complexes (DIGs).1 These complexes form in the trans-Golgi network and incorporate certain transmembrane, GPI-anchored, and dually acylated proteins, which are then targeted to the apical plasma membrane (4Simons K. Ikonen E. Nature. 1997; 387: 569-572Crossref PubMed Scopus (8157) Google Scholar, 5Melkonian K.A. Ostermeyer A.G. Chen J.Z. Roth M.G. Brown D.A. J. Biol. Chem. 1999; 274: 3910-3917Abstract Full Text Full Text PDF PubMed Scopus (555) Google Scholar). The inhibition of DIG formation by sphingolipid or cholesterol depletion disrupts this apical/axonal sorting pathway (3Ledesma M.D. Simons K. Dotti C.G. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 3966-3971Crossref PubMed Scopus (199) Google Scholar, 6Keller P. Simons K. J. Cell Biol. 1998; 140: 1357-1367Crossref PubMed Scopus (472) Google Scholar, 7Ledesma M.D. Brügger B. Bünning C. Wieland F.T. Dotti C.G. EMBO J. 1999; 18: 1761-1771Crossref PubMed Scopus (116) Google Scholar). However, the polarized targeting of cytosolic proteins via DIGs has not been explored. Postsynaptic density-95 (PSD-95) is a peripheral membrane protein that localizes exclusively to the PSD in hippocampal neurons and is believed to mediate the targeting and assembly of other synaptic proteins, including neurotransmitter receptors and signaling enzymes (8Hsueh Y.P. Sheng M. Prog. Brain Res. 1998; 116: 123-131Crossref PubMed Google Scholar, 9Kornau H.-C. Seeburg P.H. Kennedy M.B. Curr. Opin. Neurobiol. 1997; 7: 368-373Crossref PubMed Scopus (313) Google Scholar, 10Craven S.E. Bredt D.S. Cell. 1998; 93: 495-498Abstract Full Text Full Text PDF PubMed Scopus (429) Google Scholar, 11Garner C.C. Nash J. Huganir R.L. Trends Cell Biol. 2000; 10: 274-280Abstract Full Text Full Text PDF PubMed Scopus (481) Google Scholar). The N terminus of PSD-95 is posttranslationally modified with palmitate (12Topinka J.R. Bredt D.S. Neuron. 1998; 20: 125-134Abstract Full Text Full Text PDF PubMed Scopus (246) Google Scholar), a 16-carbon-saturated fatty acid linked via thioester bonds to specific cysteine residues (13Dunphy J.T. Linder M.E. Biochim. Biophys. Acta. 1998; 1436: 245-261Crossref PubMed Scopus (317) Google Scholar, 14Milligan G. Parenti M. Magee A.I. Trends Biochem. Sci. 1995; 20: 181-187Abstract Full Text PDF PubMed Scopus (285) Google Scholar, 15Mumby S.M. Curr. Opin. Cell Biol. 1997; 9: 148-154Crossref PubMed Scopus (240) Google Scholar). Dual palmitoylation of PSD-95 is necessary for appropriate postsynaptic localization (16Craven S.E. Husseini A.E. Bredt D.S. Neuron. 1999; 22: 497-509Abstract Full Text Full Text PDF PubMed Scopus (288) Google Scholar, 17El-Husseini A.E. Craven S.E. Chetkovich D.M. Firestein B.L. Schnell E. Aoki C. Bredt D.S. J. Cell Biol. 2000; 148: 159-172Crossref PubMed Scopus (242) Google Scholar). However, not all dually acylated proteins are found at postsynaptic membranes; GAP-43 is a dually palmitoylated protein that occurs predominantly at axonal membranes (18Goslin K. Schreyer D.J. Skene J.H. Banker G. Nature. 1988; 336: 672-674Crossref PubMed Scopus (315) Google Scholar). Both PSD-95 and GAP-43 accumulate in the secretory pathway in a palmitoylation-dependent manner (17El-Husseini A.E. Craven S.E. Chetkovich D.M. Firestein B.L. Schnell E. Aoki C. Bredt D.S. J. Cell Biol. 2000; 148: 159-172Crossref PubMed Scopus (242) Google Scholar,19Liu Y. Fisher D.A. Storm D.R. J. Neurosci. 1994; 14: 5807-5817Crossref PubMed Google Scholar), but it is unclear how they sort to separate vesicles destined for dendritic versus axonal membranes. To elucidate mechanisms for axonal versus dendritic sorting of peripheral membrane proteins, we analyzed the polarized targeting of PSD-95 and GAP-43 in hippocampal neurons. We find that the palmitoylation motif of PSD-95 is necessary but not sufficient for dendritic targeting, whereas the palmitoylation motif of GAP-43 is sufficient for axonal targeting. Systematic mutagenesis of these two palmitoylation motifs reveals that axonal targeting by the GAP-43 motif requires two adjacent cysteines as well as nearby basic residues, features that are conserved in other palmitoylated axonal proteins. Palmitoylation motifs that mediate axonal targeting also localize to DIGs in heterologous cells, indicating that lipid rafts probably mediate axonal targeting of certain cytosolic proteins. GW1 PSD-95, PSD-95(C3,5S), and PSD-95(1–26) fused to GFP were described previously (16Craven S.E. Husseini A.E. Bredt D.S. Neuron. 1999; 22: 497-509Abstract Full Text Full Text PDF PubMed Scopus (288) Google Scholar, 17El-Husseini A.E. Craven S.E. Chetkovich D.M. Firestein B.L. Schnell E. Aoki C. Bredt D.S. J. Cell Biol. 2000; 148: 159-172Crossref PubMed Scopus (242) Google Scholar). The mutations of the palmitoylation motif of PSD-95, the addition of the GAP-43 N-terminal palmitoylation motif to PSD-95, and the mutations within the GAP-43 palmitoylation motifs of the 43-PSD-95 chimera were constructed with oligos encoding the appropriate wild-type or mutated motif and restriction sites that were annealed and subcloned into GW1 PSD-95 GFP at a HindIII site upstream of the starter methionine and a silent KpnI site at amino acid 13 of PSD-95. The addition of the C-terminal prenyl-palmitoylation motif of paralemmin was added to the extreme C terminus of PSD-95(C3,5S) GFP with primers encoding the appropriate wild-type or mutated motif and restrictions sites, which were used to amplify the C-terminal GFP. Dr. David Sretavan (University of California, San Francisco) kindly provided wild-type GAP-43. Paralemmin was obtained by reverse transcriptase-polymerase chain reaction from mouse brain RNA and subcloned into pEGFP (CLONTECH) at theBglII and HindIII sites. Neuronal cultures were prepared from the hippocampi of E18/E19 rats. Hippocampi were dissociated by enzyme digestion with papain followed by brief mechanical trituration. Cells were plated on poly-d-lysine (Sigma)-treated glass coverslips (12 mm in diameter) and maintained in neurobasal media (Life Technologies, Inc.) supplemented with B27, penicillin, streptomycin, and l-glutamine as described in Brewer et al. (39Brewer G.J. Torricelli J.R. Evege E.K. Price P.J. J. Neurosci. Res. 1993; 35: 567-576Crossref PubMed Scopus (1912) Google Scholar). Hippocampal cultures were transfected by lipid-mediated gene transfer just before plating as described previously (20Kaech S. Kim J.B. Cariola M. Ralston E. Mol. Brain Res. 1996; 35: 344-348Crossref PubMed Scopus (41) Google Scholar). 2 μg of DNA and 10 μl of 1,2-dioleoyl-sn-glycero-3-trimethylammonium-propane (Roche Molecular Biochemicals) were mixed in 25 μl of HBS and added to the cells (1 million cells/0.25 ml) with immediate and gentle mixing. Cells were incubated for 1 h at 37 °C and then plated at a density of 600/mm2 on glass coverslips (Fisher) in 24-well plates (Falcon). To visualize transfected cells, coverslips were removed from the wells and mounted live onto slides (Frost Plus slides, Fisher) with Fluoromount-G (Southern Biotechnology Associates, Inc.). Transfection efficiency was never >0.01%, and on average, 15–30 transfected cells were obtained for each independent transfection. Coverslips were removed from culture wells and fixed in 4 °C paraformaldehyde for 15–20 min. The cells were washed with Tris-buffered saline containing 0.1% Triton X-100 Tris-buffered saline and blocked in Triton X-100 Tris-buffered saline with 3% normal goat serum for 1 h at room temperature. Primary antibodies against MAP-2 (monoclonal) (Pharmingen) or Thy-1 (MRC OX-7, Serotec) to stain dendrites or axons, respectively, were added to blocking solution for 1 h at room temperature followed by donkey anti-mouse or donkey anti-rabbit antibodies conjugated to Cy3 or 7-amino-4-methylcoumarin-3-acetic acid fluorophores (diluted 1:200 in blocking solution) for 1 h. at room temperature. Coverslips were then mounted on slides (Frost Plus) with Fluoromount-G, and images were taken under fluorescence microscopy with a × 60 objective affixed to a Zeiss-inverted microscope. Quantification of polarized protein expression in dendrites versus axons was performed on 15–50 neurons from 2–3 independent transfections. Images of neurons were acquired with a charge-coupled device camera and quantitated using Metamorph imaging software (Universal Imaging). The exposure time of the camera was adjusted to limit photobleaching, so that the maximum pixel intensity was approximately one-half to three-fourths saturating for cells with low to moderate expression levels as determined by total pixel counts. Because high protein expression can saturate targeting mechanisms, cells expressing to pixel saturation were not included in the analysis. The degree of polarized expression was determined by calculating the average pixel intensity in the axon versus that in the dendrites. The axon was identified as the thin process extending farthest from the cell body and not immunoreactive for MAP-2. The average pixel intensity was calculated by drawing a line through three dendrites and three sections of the axon at defined distances from the cell body. These averages were then converted into a ratio of axonalversus dendritic expression (A/D ratio) and compared with that for diffusely expressed GFP. The A/D ratio represents the percentage of protein in the axon as compared with that in the dendrites, and because the volume of the axon is considerably smaller than that of the dendrites, the ratio is always <1. Data were analyzed by one-way ANOVA with Bonferroni corrections for multiple comparisons with Prism software (GraphPAD, San Diego, CA). COS-7 cells were grown in Dulbecco's modified Eagle's medium containing 10% fetal bovine serum, penicillin, and streptomycin. Cells were transfected using LipofectAMINE reagent according to the manufacturer protocol (Life Technologies, Inc.). For studies of palmitoylation, transfected COS-7 cells were labeled in media containing 1 mCi/ml [3H]palmitic acid (50 Ci/mmol) (DuPont NEN). Cells were washed with ice-cold phosphate-buffered saline and resuspended in 0.4 ml of lysis buffer containing TEE, 150 mm NaCl, and 0.2% SDS. After extracting for 20 min at 4 °C, Triton X-100 was added to 1% to neutralize the SDS, and insoluble material was removed by centrifugation at 10,000 × g for 10 min. For immunoprecipitation experiments, the samples were then incubated with GFP antibodies (1:150 dilution, CLONTECH) for 1 h at 4 °C. After the addition of 20 μl of protein A-Sepharose beads (Amersham Pharmacia Biotech), samples were incubated for 1 h at 4 °C. Immunoprecipitates were washed three times with buffer containing TEE, 150 mm NaCl, and 1% Triton X-100, boiled in SDS-PAGE sample buffer with 1 mmdithiothreitol for 2 min, and analyzed by SDS-PAGE. For fluorography, protein samples were separated by SDS-PAGE and stained with Coomassie Blue. Gels were treated with Amplify (Amersham Pharmacia Biotech) for 30 min, dried under vacuum, and exposed to Hyperfilm-MP (Amersham Pharmacia Biotech) at −80 °C for 3–5 days. Detergent-insoluble fractions were prepared as described previously with modifications (21Brown D.A. Rose J.K. Cell. 1992; 68: 533-544Abstract Full Text PDF PubMed Scopus (2618) Google Scholar). COS-7 cells were grown in Dulbecco's modified Eagle's medium containing 10% fetal bovine serum, penicillin, and streptomycin. Cells were transfected using LipofectAMINE reagent according to the manufacturer protocol (Life Technologies, Inc.). Cells (50 × 106) were washed with ice-cold phosphate-buffered saline and resuspended in 1 ml of lysis buffer containing 25 mm Tris-HCl, pH 7.6, 5 mm EDTA, 150 mm NaCl, 0.5% Triton X-100, 1 mm phenylmethylsulfonyl fluoride, 10 μg/ml each of aprotinin and leupeptin. After extracting for 10 min at 4 °C, the homogenate was adjusted to 40% sucrose and placed at the bottom of an ultracentrifuge tube. A linear sucrose gradient (5–30%) was overlaid on top, and samples were centrifuged for 18 h at 200,000 ×g in a SwTi 50 rotor (46,245 rpm) at 4 °C. Sequential fractions across the gradient were collected, separated by SDS-PAGE, and immunoblotted using antibodies to caveolin-1 (Transduction Laboratories, Lexington, KY) and GFP (1:150 dilution,CLONTECH). PSD-95 and SAP-97 are highly homologous membrane-associated guanylate kinase proteins, however, PSD-95 is restricted to postsynaptic sites in forebrain neurons, whereas SAP-97 occurs both pre- and postsynaptically (11Garner C.C. Nash J. Huganir R.L. Trends Cell Biol. 2000; 10: 274-280Abstract Full Text Full Text PDF PubMed Scopus (481) Google Scholar). To test whether this polarized sorting can be reproduced in cell transfections, we expressed GFP fusions of PSD-95 or SAP-97 in hippocampal neurons (Table 1). As shown in Fig.1, we find that PSD-95 is restricted to postsynaptic clusters in the dendrites, whereas SAP-97 occurs both in dendrites and axon. As PSD-95 is palmitoylated and SAP-97 is not, we asked whether differential lipid modifications of these proteins account for their differential sorting to axons versusdendrites. Indeed, we found that mutating the palmitoylated cysteines of PSD-95 to serine (PSD-95(C3,5S)) disrupts axon exclusion and causes the mutant to enter the axon. Conversely, appending the palmitoylated N terminus of PSD-95 to SAP-97 yields a chimera that is excluded from the axon (Fig. 1).Table ISummary of the chimeric constructs used in this studyPSD-95, full-length PSD-95 fused to GFP;PSD-95(C3,5S), the palmitoylation-deficient mutant of full-length PSD-95 fused to GFP;PSD-95(CC), full-length PSD-95(D2L,ΔL4) fused to GFP; PSD-95(T8R), full-length PSD-95(T8R) fused to GFP; PSD-95(CC-T8R), full-length PSD-95(D2L) deletion of L4-T8R fused to GFP; 43-PSD-95, N-terminal 11 residues of GAP-43 fused to Δ1–12PSD-95-GFP;43-PSD-95(CXC), N-terminal 11 residues of GAP-43 fused to Δ1–12PSD-95-GFP with an insertion of a leucine between Cys-3 and -4; 43-PSD-95(R6I,R7I), N-terminal 11 residues of GAP-43 with R6,7I fused to Δ1–12PSD-95-GFP;43-PSD-95(CXC-R6I,R7I), N-terminal 11 residues of GAP-43 with R6,7I fused to Δ1–12PSD-95-GFP with an insertion of a leucine between Cys-3 and -4; Par, full-length paralemmin fused to GFP; GFP-par, C-terminal 14 amino acids of paralemmin fused to GFP; PSD-95(C3,5S-par), C-terminal 14 amino acids of paralemmin fused to PSD-95(C3,5S)-GFP;C3,5S-par-palmitoyl, C-terminal 14 amino acids of paralemmin (C334,3366S) fused to PSD-95(C3,5S)-GFP; C3,5S-par-B, C-terminal 14 amino acids of paralemmin (amino acids 330, 331, 333, 335L) fused to PSD-95(C3,5S). In schematic, the palmitoylation motif (P) is indicated by a striped box. Palmitoylated cysteines are in italics, and prenylated cysteines are in bold. Open table in a new tab Figure 1Membrane-associated guanylate kinase palmitoylation is required for axonal exclusion. PSD-95 GFP in cultured hippocampal neurons clusters at synapses in the dendrites (PSD-95, green) and is excluded from the axon (arrow). Hence, all neurites that contain PSD-95 GFP are immunoreactive for MAP-2 (MAP-2, red). By contrast, SAP-97 GFP occurs both in dendrites and in the axon. A palmitoylation-deficient PSD-95 construct (PSD-95(C3,5S)) occurs both in dendrites and axon, whereas a SAP-97 chimera containing the palmitoylated N terminus of PSD-95 (95-SAP-97) is excluded from the axon. Scale bar = 10 μm.View Large Image Figure ViewerDownload (PPT) PSD-95, full-length PSD-95 fused to GFP;PSD-95(C3,5S), the palmitoylation-deficient mutant of full-length PSD-95 fused to GFP;PSD-95(CC), full-length PSD-95(D2L,ΔL4) fused to GFP; PSD-95(T8R), full-length PSD-95(T8R) fused to GFP; PSD-95(CC-T8R), full-length PSD-95(D2L) deletion of L4-T8R fused to GFP; 43-PSD-95, N-terminal 11 residues of GAP-43 fused to Δ1–12PSD-95-GFP;43-PSD-95(CXC), N-terminal 11 residues of GAP-43 fused to Δ1–12PSD-95-GFP with an insertion of a leucine between Cys-3 and -4; 43-PSD-95(R6I,R7I), N-terminal 11 residues of GAP-43 with R6,7I fused to Δ1–12PSD-95-GFP;43-PSD-95(CXC-R6I,R7I), N-terminal 11 residues of GAP-43 with R6,7I fused to Δ1–12PSD-95-GFP with an insertion of a leucine between Cys-3 and -4; Par, full-length paralemmin fused to GFP; GFP-par, C-terminal 14 amino acids of paralemmin fused to GFP; PSD-95(C3,5S-par), C-terminal 14 amino acids of paralemmin fused to PSD-95(C3,5S)-GFP;C3,5S-par-palmitoyl, C-terminal 14 amino acids of paralemmin (C334,3366S) fused to PSD-95(C3,5S)-GFP; C3,5S-par-B, C-terminal 14 amino acids of paralemmin (amino acids 330, 331, 333, 335L) fused to PSD-95(C3,5S). In schematic, the palmitoylation motif (P) is indicated by a striped box. Palmitoylated cysteines are in italics, and prenylated cysteines are in bold. Axons were unambiguously identified both by their morphology and by the absence of MAP-2 immunoreactivity (Fig. 1). To quantitate the extent of axonal targeting, the average fluorescent pixel intensity of GFP fusions in the axon versus the dendrites were determined (A/D ratio) (see under “Experimental Procedures”) and compared with the A/D ratio of GFP alone. This analysis revealed that wild-type PSD-95 protein and 95-SAP-97 are excluded from the axon, whereas SAP-97 and PSD-95(C3,5S) are present in the axon to a similar extent as is GFP alone (Fig. 2 C). Whereas palmitoylation of PSD-95 is required for axon exclusion, not all palmitoylated neuronal proteins are restricted to dendrites. For example, dually palmitoylated GAP-43 predominantly localizes to axonal membranes (18Goslin K. Schreyer D.J. Skene J.H. Banker G. Nature. 1988; 336: 672-674Crossref PubMed Scopus (315) Google Scholar). Given these different localizations, we asked whether the palmitoylation motifs play opposing roles in protein trafficking. To accomplish this task, we first replaced the dually palmitoylated N-terminal 13 amino acids of PSD-95 with those of GAP-43. The GAP-43 palmitoylation motif on PSD-95 (43-PSD-95) maintains palmitoylation and partially maintains postsynaptic targeting (17El-Husseini A.E. Craven S.E. Chetkovich D.M. Firestein B.L. Schnell E. Aoki C. Bredt D.S. J. Cell Biol. 2000; 148: 159-172Crossref PubMed Scopus (242) Google Scholar). However, the 43-PSD-95 chimera also dramatically targets to the axon, a localization not observed with wild-type PSD-95 (Fig. 2). Given the relocation of PSD-95 by the GAP-43 palmitoylation motif, we next assessed differential targeting mediated by these motifs. When the PSD-95 palmitoylation motif alone is fused to GFP, the A/D ratio of PSD-95(1–26) is not statistically different from GFP (Fig. 2). In contrast, the GAP-43 palmitoylation motif, fused to GFP (GAP-43(1–14)) though not exclusively axonal, is enriched in the axon compared with GFP alone (Fig. 2). These data suggest that the palmitoylation motif of PSD-95 is necessary but not sufficient for axon exclusion, whereas the palmitoylation motif of GAP-43 is sufficient for axonal targeting. We considered the possibility that axon exclusion of PSD-95 may simply result from its clustering at postsynaptic sites. However, we find that the 43-PSD-95 chimera is both more postsynaptically clustered and more axonally localized than PSD-95(C3,5S), indicating that postsynaptic clustering and axonal exclusion are separable processes. To help to understand the constituents of the GAP-43 palmitoylation motif necessary for axonal targeting, we compared the sequences of the PSD-95 and GAP-43 palmitoylation motifs. The palmitoylated cysteines of the GAP-43 motif are adjacent, whereas those in PSD-95 are separated by a leucine. Strikingly, a PSD-95 mutant containing adjacent cysteines (PSD-95(CC)) is not exclusively dendritic; the protein also localizes to the axon though not to the degree of 43-PSD-95 (Fig.3). On the other hand, the addition of a single amino acid between the contiguous cysteines of the GAP-43 palmitoylation motif (43-PSD-95(CXC)) does not affect axonal targeting of 43-PSD-95, and the A/D ratio is unchanged (Fig. 3). These mutated constructs are all efficiently palmitoylated (Fig. 5), so changes in protein targeting are not attributed to alterations in protein palmitoylation. These results suggest that the spacing of the cysteines is important but is not the only feature of these motifs that determines targeting.Figure 5Palmitoylation of PSD-95 GFP fusion constructs. COS cells were transiently transfected with various PSD-95 constructs described in Figs. Figure 1, Figure 2, Figure 3, Figure 4. Cells were lysed in radioimmune precipitation buffer, and the solubilized material was immunoprecipitated with an antibody to GFP. Immunoprecipitates were loaded onto duplicate gels that were analyzed for [3H]palmitate by fluorography (upper gel) or were immunoblotted for GFP (lower gel).View Large Image Figure ViewerDownload (PPT) Additionally, the GAP-43 motif contains two basic amino acids that is one residue away from the palmitoylated cysteines, whereas the PSD-95 motif does not have basic residues near the cysteines. Remarkably, adding a single basic amino acid to PSD-95 two amino acids away from the cysteines (PSD-95(T8R)) redistributes the mutant to the axon with almost half the efficiency of 43-PSD-95 (Fig.3). Conversely, mutating the basic amino acids of the GAP-43 palmitoylation motif to isoleucines (43-PSD-95(R6I,R7I)) does not alter protein palmitoylation but reduces axonal targeting of 43-PSD-95 by approximately half (Fig. 3). These results suggest that the basic amino acids are critically involved but do not entirely explain the targeting differences between these motifs. Finally, we combined alternations in spacing between the cysteines and mutations in the basic amino acids. A PSD-95 palmitoylation motif with juxtaposed cysteines and the T8R mutation (PSD-95(CC-T8R) targets PSD-95 to the axon similar to 43-PSD-95 (Fig. 3). In addition, a GAP-43 palmitoylation motif both with cysteines separated and with mutations in the basics (43-PSD-95(CXC-R6I,R7I)) no longer targets 43-PSD-95 to the axon, but rather the protein localizes solely to the postsynaptic membrane similar to wild-type PSD-95 (Fig. 3). These results suggest that these two features account entirely for differential targeting by these domains. We next asked whether the adjacent cysteine/basic amino acid motif might play a general role in the axonal sorting of palmitoylated proteins. Paralemmin is a neuronal prenyl-palmitoyl-anchored protein that is found at axonal membranes (22Kutzleb C. Sanders G. Yamamoto R. Wang X. Lichte B. Petrasch-Parwez E. Kilimann M.W. J. Cell Biol. 1998; 143: 795-813Crossref PubMed Scopus (62) Google Scholar). The C-terminal palmitoylation motif of paralemmin contains adjacent prenylated/palmitoylated cysteines and nearby basic amino acids and thereby resembles the lipidated domain of GAP-43 (Fig.4). GFP-tagged paralemmin is significantly targeted to the axon, similar to GAP-43. Furthermore, the isolated prenyl-palmitoylation motif of paralemmin fused to GFP (GFP-par) is sufficient for axonal targeting (Fig. 4), and the addition of this motif to the C terminus of palmitoylation-deficient PSD-95 targets the chimera (PSD-95(C3,5S-par)) to the axon (Fig. 4). We next determined whether axonal targeting by the paralemmin motif requires similar features as the GAP-43 motif. Adjusting the spacing of the cysteines interferes with prenylation and subsequent palmitoylation (data not shown), so we were unable to assess the importance of cysteine residue spacing in protein trafficking. However, mutating the palmitoylated cysteines to serines (PSD-95(C3,5S-par-palmitoyl)) retains prenylation but blocks palmitoylation (Fig.5) and disrupts axon targeting (Fig. 4). In addition, mutations in the basic amino acids maintain lipidation but reduce axonal targeting, consistent with a requirement for nearby basic amino acids in axonal targeting (Fig. 4). Dually acylated proteins can be incorporated into DIGs, and these complexes have been implicated in targeting to axonal membranes (4Simons K. Ikonen E. Nature. 1997; 387: 569-572Crossref PubMed Scopus (8157) Google Scholar, 7Ledesma M.D. Brügger B. Bünning C. Wieland F.T. Dotti C.G. EMBO J. 1999; 18: 1761-1771Crossref PubMed Scopus (116) Google Scholar, 23Winckler B. Mellman I. Neuron. 1999; 23: 637-640Abstract Full Text Full Text PDF PubMed Scopus (116) Google Scholar). We, therefore, asked whether the palmitoylation motifs of GAP-43 and PSD-95 might differentially associate with DIGs. Resident proteins of DIGs float in sucrose gradients and are found in light membrane fractions together with α-caveolin (24Rothberg K.G. Heuser J.E. Donzell W.C. Ying Y.S. Glenney J.R. Anderson R.G. Cell. 1992; 68: 673-682Abstract Full Text PDF PubMed Scopus (1873) Google Scholar). As previously published, only a small amount of PSD-95 associates with DIGs, and this is independent of palmitoylation (Fig. 6) (25Perez A.S. Bredt D.S. Neurosci. Lett. 1998; 258: 121-123Crossref PubMed Scopus (50) Google Scholar, 26Wu C. Butz S. Ying Y. Anderson R.G. J. Biol. Chem. 1997; 272: 3554-3559Abstract Full Text Full Text PDF PubMed Scopus (239) Google Scholar). In contrast, the palmitoylation motif of GAP-43 efficiently targets a GFP reporter to DIGs (Fig. 6) (40Arni S. Keilbaugh S.A. Ostermeyer A.G. Brown D.A. J. Biol. Chem. 1998; 273: 28478-28485Abstract Full Text Full Text PDF PubMed Scopus (128) Google Scholar) as does the axonally targeted prenyl-palmitoylation motif of paralemmin (Fig. 6). Furthermore, when the palmitoylation motif of PSD-95 is replaced with that of GAP-43 or paralemmin, these chimeras also associate with DIGs, whereas the 43-PSD-95(R6I,R7I) shows an intermediate degree of DIG partitioning (Fig. 6, and data not shown). Thus, there is a strong correlation between the ability of palmitoylation motifs to target proteins to DIGs and to axonal membranes. This analysis of polarized sorting of peripheral membrane proteins demonstrates that palmitoylation motifs can mediate either dendritic/postsynaptic or axonal targeting. Previous work shows that dual palmitoylation is necessary for targeting PSD-95 to postsynaptic membranes (16Craven S.E. Husseini A.E. Bredt D.S. Neuron. 1999; 22: 497-509Abstract Full Text Full Text PDF PubMed Scopus (288) Google Scholar, 17El-Husseini A.E. Craven S.E. Chetkovich D.M. Firestein B.L. Schnell E. Aoki C. Bredt D.S. J. Cell Biol. 2000; 148: 159-172Crossref PubMed Scopus (242) Google Scholar). We now find that palmitoylation is also necessary to exclude PSD-95 from axons, although the isolated palmitoylation motif is not sufficient for axonal exclusion. In contrast, the dually palmitoylated motifs of the axonal proteins GAP-43 and paralemmin are sufficient to mediate protein targeting to axonal membranes and, therefore, are the first identified axonal targeting motifs for peripheral membrane proteins. Differential sorting of the PSD-95 and GAP-43 palmitoylation motifs depend on two features, the spacing of the cysteine residues and the presence of nearby basic amino acids. The PSD-95 and GAP-43 palmitoylation motifs also differ in their capacity to associate with DIGs; PSD-95 is only faintly incorporated into DIGs, whereas the isolated palmitoylation motif of GAP-43 is sufficient for association with these complexes. These data suggest that the incorporation of peripheral membrane proteins into lipid rafts may mediate axonal trafficking. Whether dendritic sorting of PSD-95 results from active dendritic targeting or axonal exclusion is unclear. Interestingly, when axonally targeted palmitoylation motifs from GAP-43 and paralemmin are added to PSD-95, the resulting chimeras are not as polarized as their isolated palmitoylation motifs, nor are they as well associated with DIGs. These results suggest that additional dendritic targeting or axonal exclusion signals reside within PSD-95. Consistent with this finding, the palmitoylation motif of PSD-95 is insufficient for dendritic targeting. Yet because unpalmitoylated PSD-95(C3,5S) is distributed similar to GFP, the expression of the dendritic targeting signal within the body of PSD-95 probably requires an association with membranes via palmitoylation. The identity of the additional region(s) of PSD-95 involved in dendritic targeting/axonal exclusion remains to be uncovered. In contrast to the absolute polarization of dendritically targeted PSD-95, axonally targeted constructs, such as GAP-43 and paralemmin, are also expressed in dendrites. These proteins are considered axonal because of their enhanced density in the axon compared with diffusely expressed GFP. Previous studies have also found that exogenous expression of axonal proteins often yields some protein in dendrites (30Stowell J.N. Craig A.M. Neuron. 1999; 22: 525-536Abstract Full Text Full Text PDF PubMed Scopus (133) Google Scholar). This dendritic expression may occur for a number of reasons, including inadequate axonal retention or missorting due to saturation of targeting mechanisms. Alternatively, dendritic localization may be explained by the presence of dendritic targeting signals and/or the absence of dendritic exclusion signals, as axonal proteins can also occur in dendritic/postsynaptic localizations (22Kutzleb C. Sanders G. Yamamoto R. Wang X. Lichte B. Petrasch-Parwez E. Kilimann M.W. J. Cell Biol. 1998; 143: 795-813Crossref PubMed Scopus (62) Google Scholar). Indeed, we found that PSD-95 constructs containing GAP-43 or paralemmin palmitoylation motifs concentrate in the axon but also occur at the PSD. PSD-95 and related membrane-associated guanylate kinases display complex expression patterns that depend on the specific protein isoform and neuronal cell type. In hippocampal neurons, PSD-95 and PSD-93 are excluded from axons, whereas SAP-97 and SAP-102 occur both in axons and dendrites (17El-Husseini A.E. Craven S.E. Chetkovich D.M. Firestein B.L. Schnell E. Aoki C. Bredt D.S. J. Cell Biol. 2000; 148: 159-172Crossref PubMed Scopus (242) Google Scholar). As PSD-95 and PSD-93 are palmitoylated and SAP-97, and SAP-102 are not (16Craven S.E. Husseini A.E. Bredt D.S. Neuron. 1999; 22: 497-509Abstract Full Text Full Text PDF PubMed Scopus (288) Google Scholar), this differential targeting in vivomay reflect the lipid-dependent mechanisms as described here. Unlike its dendritic localization in hippocampal and other forebrain neurons, PSD-95 occurs prominently in axons of cerebellar basket cells (27Kistner U. Wenzel B.M. Veh R.W. Cases-Langhoff C. Garner A.M. Appeltauer U. Voss B. Gundelfinger E.D. Garner C.C. J. Biol. Chem. 1993; 268: 4580-4583Abstract Full Text PDF PubMed Google Scholar). Interestingly, basket cells are unusual in that their axons are devoid of microtubules (28Matus A.I. Ng M. Jones D.H. J. Neurocytol. 1979; 8: 513-525Crossref PubMed Scopus (39) Google Scholar). Axons of hippocampal and most other neurons contain plus end distal microtubules, and dendrites contain both plus and minus end-directed microtubules (29Baas P.W. Neuron. 1999; 22: 23-31Abstract Full Text Full Text PDF PubMed Scopus (165) Google Scholar). Therefore, dendritic localization of PSD-95 may reflect a selective association with minus end-directed microtubule motors that cooperate with palmitoylation to target PSD-95 into dendrites and to exclude PSD-95 from axons. Thus, the presence of PSD-95 in basket cell axons is potentially explained by the loss of microtubule-dependent axon exclusion. A recent study (30Stowell J.N. Craig A.M. Neuron. 1999; 22: 525-536Abstract Full Text Full Text PDF PubMed Scopus (133) Google Scholar) has determined the axonal exclusion signal for a metabotropic glutamate receptor. This work shows that dendritic/axonal targeting of these transmembrane proteins relies on signals within their cytoplasmic C termini. By contrast, our analysis of palmitoylation-dependent sorting of peripheral membrane proteins implicates a role for DIGs. These complexes are rich in glycosphingolipids and cholesterol, and their formation in the secretory pathway is thought to serve as a sorting platform to direct proteins to the apical membrane of epithelial cells (4Simons K. Ikonen E. Nature. 1997; 387: 569-572Crossref PubMed Scopus (8157) Google Scholar). Polarized sorting of proteins to axonal membranes has been compared with this apical sorting (23Winckler B. Mellman I. Neuron. 1999; 23: 637-640Abstract Full Text Full Text PDF PubMed Scopus (116) Google Scholar). Indeed, an incorporation into sphingolipid-cholesterol rafts appears to mediate axonal targeting of the GPI-anchored protein, Thy-1 (3Ledesma M.D. Simons K. Dotti C.G. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 3966-3971Crossref PubMed Scopus (199) Google Scholar, 7Ledesma M.D. Brügger B. Bünning C. Wieland F.T. Dotti C.G. EMBO J. 1999; 18: 1761-1771Crossref PubMed Scopus (116) Google Scholar). We find that dual palmitoylation motifs that associate with these rafts can also target proteins to axonal membranes. In contrast, the dual palmitoylation motif of PSD-95 is not incorporated into DIGs, and rather than mediating axonal targeting, it plays a role in dendritic targeting/axonal exclusion. To uncover a more direct relationship between DIG association and axonal targeting, we attempted to disrupt DIG formation using lovastatin and methyl-β-cyclodextrin to deplete cholesterol. However, this treatment did not affect axonal targeting of endogenous Thy-1, 2A. E. El-Husseini, S. E. Craven, S. C. Brock, and D. S. Bredt, unpublished results. suggesting that cholesterol depletion was not successful in our cultures. Both GAP-43 and PSD-95 are found in the secretory pathway (17El-Husseini A.E. Craven S.E. Chetkovich D.M. Firestein B.L. Schnell E. Aoki C. Bredt D.S. J. Cell Biol. 2000; 148: 159-172Crossref PubMed Scopus (242) Google Scholar, 19Liu Y. Fisher D.A. Storm D.R. J. Neurosci. 1994; 14: 5807-5817Crossref PubMed Google Scholar) where lipid rafts are first formed. And it is here that these two proteins may be segregated to separate secretory vesicles for transport to dendritic versus axonal membranes. The palmitoylation motif of GAP-43 can mediate lipid raft association for protein trafficking to the axonal membrane. Indeed, previous studies have found that GAP-43 is enriched in DIGs from brain homogenates (31Maekawa S. Kumanogoh H. Funatsu N. Takei N. Inoue K. Endo Y. Hamada K. Sokawa Y. Biochim. Biophys. Acta. 1997; 1323: 1-5Crossref PubMed Scopus (54) Google Scholar) and is transported to axons on vesicles derived from the secretory pathway (32Ferreira A. Niclas J. Vale R.D. Banker G. Kosik K.S. J. Cell Biol. 1992; 117: 595-606Crossref PubMed Scopus (167) Google Scholar). On the other hand, signals within the body of PSD-95 working together with palmitoylation may mediate its association with dendritic targeting vesicles and its exclusion from rafts. Near its C terminus, PSD-95 possesses a tyrosine-based protein-trafficking motif that is sufficient to mediate protein endocytosis via clathrin-coated vesicles and that is required for postsynaptic targeting (33Craven S.E. Bredt D.S. J. Biol. Chem. 2000; 275: 20045-20051Abstract Full Text Full Text PDF PubMed Scopus (28) Google Scholar). As an exogenous axonally targeted lipid raft protein, influenza virus hemagglutinin is efficiently excluded from clathrin-coated endocytotic vesicles (34Bretscher M.S. Thomson J.N. Pearse B.M. Proc. Natl. Acad. Sci. U. S. A. 1980; 77: 4156-4159Crossref PubMed Scopus (121) Google Scholar,35Roth M.G. Doyle C. Sambrook J. Gething M.J. J. Cell Biol. 1986; 102: 1271-1283Crossref PubMed Scopus (70) Google Scholar). Adding an internalization motif to hemagglutinin induces endocytosis (35Roth M.G. Doyle C. Sambrook J. Gething M.J. J. Cell Biol. 1986; 102: 1271-1283Crossref PubMed Scopus (70) Google Scholar, 36Lazarovits J. Roth M. Cell. 1988; 53: 743-752Abstract Full Text PDF PubMed Scopus (141) Google Scholar) and potentially blocks association with DIGs (37Lazarovits J. Naim H.Y. Rodriguez A.C. Wang R.H. Fire E. Bird C. Henis Y.I. Roth M.G. J. Cell Biol. 1996; 134: 339-348Crossref PubMed Scopus (22) Google Scholar,38Scheiffele P. Roth M.G. Simons K. EMBP J. 1997; 16: 5501-5508Crossref PubMed Scopus (571) Google Scholar). Thus, the inclusion of PSD-95 in clathrin-coated pits may aid in its exclusion from lipid rafts. Alternatively, PSD-95 and GAP-43 may differentially associate with DIGs because different palmitoyl-transferase enzymes recognize their distinct palmitoylation motifs. Although such transferase enzymes have not yet been isolated, the sequence specificity for palmitoylation identified here suggests that this is indeed an enzymatic process. For instance, palmitoylation motifs similar to GAP-43 and paralemmin that contain adjacent cysteines with nearby basic amino acids may be recognized by a palmitoyl-transferase enzyme in thetrans-Golgi network followed by the incorporation into lipid rafts for axonal trafficking. In contrast, the palmitoylation motif of PSD-95 may be recognized by a separate enzyme that is cytosolic or associated with membranes other than the trans-Golgi network, and thus PSD-95 is not incorporated into DIGs. Identification of the putative palmitoyl-transferase enzyme(s) that mediate palmitoylation of neuronal proteins will help to clarify these issues. Furthermore, elucidating how protein palmitoylation contributes to polarized trafficking may provide fundamental insights for understanding many different sorting decisions in the cell." @default.
• W1976817149 created "2016-06-24" @default.
• W1976817149 creator A5010435284 @default.
• W1976817149 creator A5028788852 @default.
• W1976817149 creator A5065657841 @default.
• W1976817149 creator A5076894976 @default.
• W1976817149 date "2001-11-01" @default.
• W1976817149 modified "2023-09-27" @default.
• W1976817149 title "Polarized Targeting of Peripheral Membrane Proteins in Neurons" @default.
• W1976817149 cites W1489215358 @default.
• W1976817149 cites W1591314372 @default.
• W1976817149 cites W1608213943 @default.
• W1976817149 cites W1983199685 @default.
• W1976817149 cites W1983558778 @default.
• W1976817149 cites W1996988785 @default.
• W1976817149 cites W1997200059 @default.
• W1976817149 cites W1998865212 @default.
• W1976817149 cites W1999175477 @default.
• W1976817149 cites W2006376420 @default.
• W1976817149 cites W2014800457 @default.
• W1976817149 cites W2018581402 @default.
• W1976817149 cites W2031775944 @default.
• W1976817149 cites W2032582749 @default.
• W1976817149 cites W2034310728 @default.
• W1976817149 cites W2039132353 @default.
• W1976817149 cites W2040694980 @default.
• W1976817149 cites W2041611018 @default.
• W1976817149 cites W2043706080 @default.
• W1976817149 cites W2044343226 @default.
• W1976817149 cites W2045765706 @default.
• W1976817149 cites W2053426757 @default.
• W1976817149 cites W2053854358 @default.
• W1976817149 cites W2060705097 @default.
• W1976817149 cites W2062474690 @default.
• W1976817149 cites W2069444054 @default.
• W1976817149 cites W2072240979 @default.
• W1976817149 cites W2078837666 @default.
• W1976817149 cites W2080494149 @default.
• W1976817149 cites W2084886317 @default.
• W1976817149 cites W2086651259 @default.
• W1976817149 cites W2089643815 @default.
• W1976817149 cites W2091754306 @default.
• W1976817149 cites W2110045854 @default.
• W1976817149 cites W2114871465 @default.
• W1976817149 cites W2121936687 @default.
• W1976817149 cites W2133388060 @default.
• W1976817149 cites W2138059603 @default.
• W1976817149 cites W2148376621 @default.
• W1976817149 cites W73391000 @default.
• W1976817149 doi "https://doi.org/10.1074/jbc.m103049200" @default.
• W1976817149 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/11546762" @default.
• W1976817149 hasPublicationYear "2001" @default.
• W1976817149 type Work @default.
• W1976817149 sameAs 1976817149 @default.
• W1976817149 citedByCount "73" @default.
• W1976817149 countsByYear W19768171492012 @default.
• W1976817149 countsByYear W19768171492013 @default.
• W1976817149 countsByYear W19768171492015 @default.
• W1976817149 countsByYear W19768171492016 @default.
• W1976817149 countsByYear W19768171492017 @default.
• W1976817149 countsByYear W19768171492018 @default.
• W1976817149 countsByYear W19768171492019 @default.
• W1976817149 countsByYear W19768171492021 @default.
• W1976817149 countsByYear W19768171492022 @default.
• W1976817149 countsByYear W19768171492023 @default.
• W1976817149 crossrefType "journal-article" @default.
• W1976817149 hasAuthorship W1976817149A5010435284 @default.
• W1976817149 hasAuthorship W1976817149A5028788852 @default.
• W1976817149 hasAuthorship W1976817149A5065657841 @default.
• W1976817149 hasAuthorship W1976817149A5076894976 @default.
• W1976817149 hasBestOaLocation W19768171491 @default.
• W1976817149 hasConcept C12554922 @default.
• W1976817149 hasConcept C126322002 @default.
• W1976817149 hasConcept C144647389 @default.
• W1976817149 hasConcept C161200384 @default.
• W1976817149 hasConcept C185592680 @default.
• W1976817149 hasConcept C41625074 @default.
• W1976817149 hasConcept C46762472 @default.
• W1976817149 hasConcept C55493867 @default.
• W1976817149 hasConcept C560191 @default.
• W1976817149 hasConcept C71924100 @default.
• W1976817149 hasConcept C86803240 @default.
• W1976817149 hasConcept C95444343 @default.
• W1976817149 hasConceptScore W1976817149C12554922 @default.
• W1976817149 hasConceptScore W1976817149C126322002 @default.
• W1976817149 hasConceptScore W1976817149C144647389 @default.
• W1976817149 hasConceptScore W1976817149C161200384 @default.
• W1976817149 hasConceptScore W1976817149C185592680 @default.
• W1976817149 hasConceptScore W1976817149C41625074 @default.
• W1976817149 hasConceptScore W1976817149C46762472 @default.
• W1976817149 hasConceptScore W1976817149C55493867 @default.
• W1976817149 hasConceptScore W1976817149C560191 @default.
• W1976817149 hasConceptScore W1976817149C71924100 @default.
• W1976817149 hasConceptScore W1976817149C86803240 @default.
• W1976817149 hasConceptScore W1976817149C95444343 @default.
• W1976817149 hasIssue "48" @default.
• W1976817149 hasLocation W19768171491 @default.
• W1976817149 hasOpenAccess W1976817149 @default.

• next