Polarized Targeting of Peripheral Membrane Proteins in Neurons

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 were in μl of and added to the with and Cells were for 1 at and then plated at a density of on glass coverslips in To transfected cells, coverslips were from the and with was and on transfected were obtained for were from and in for The were with and in with for 1 at or to dendrites or were added to for 1 at followed by or to or acid in for 1 at were then on with and were with a to a of polarized protein expression in dendrites versus axons was on neurons from of neurons were with a and The of the was to that the was to for with to expression as by protein expression can targeting to were not in the The of polarized expression was by the in the axon versus that in the The axon was as the from the cell and not for The was by a dendrites and of the axon at from the cell These were then into a ratio of axonalversus dendritic expression and with that for GFP. The ratio the of protein in the axon as with that in the dendrites, and the of the axon is that of the dendrites, the ratio is were analyzed by with for with San were in modified penicillin, and Cells were transfected to the (Life Technologies, of transfected were in media 1 acid Cells were with and in of 150 mm NaCl, and for 20 at was added to to the and insoluble was by at for 10 the were then with GFP for 1 at the addition of 20 μl of protein were for 1 at were with 150 mm NaCl, and in with 1 for 2 and analyzed by protein were by and with were with for and to at for were prepared as described previously with D.A. Cell. Full Text PDF PubMed Scopus Google Scholar). were in modified penicillin, and Cells were transfected to the (Life Technologies, Cells were with and in 1 of mm Tris-HCl, pH mm EDTA, 150 mm NaCl, 1 mm 10 of and for 10 at the was to and at the of was on and were for at in a 50 at the were by and to and GFP PSD-95 and are proteins, however, PSD-95 is to postsynaptic sites in whereas occurs both and 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 this polarized sorting can in cell we GFP of PSD-95 or in hippocampal neurons in we find that PSD-95 is to postsynaptic in the dendrites, whereas occurs both in dendrites and PSD-95 is palmitoylated and is we lipid of these proteins for sorting to axons we found that the palmitoylated cysteines of PSD-95 to disrupts axon and the to the the palmitoylated N terminus of PSD-95 to a chimera that is from the axon of the used in this PSD-95 fused to the of PSD-95 fused to fused to fused to of fused to N-terminal residues of GAP-43 fused to N-terminal residues of GAP-43 fused to with of a and N-terminal residues of GAP-43 with fused to N-terminal residues of GAP-43 with fused to with of a and paralemmin fused to C-terminal amino acids of paralemmin fused to C-terminal amino acids of paralemmin fused to C-terminal amino acids of paralemmin fused to C-terminal amino acids of paralemmin acids fused to the palmitoylation motif is by a cysteines are in and cysteines are in in a palmitoylation is for axonal PSD-95 GFP in hippocampal neurons at in the dendrites and is from the axon all that PSD-95 GFP are for GFP occurs both in dendrites and in the PSD-95 occurs both in dendrites and whereas a chimera the palmitoylated N terminus of PSD-95 is from the 10 PSD-95, PSD-95 fused to the of PSD-95 fused to fused to fused to of fused to N-terminal residues of GAP-43 fused to N-terminal residues of GAP-43 fused to with of a and N-terminal residues of GAP-43 with fused to N-terminal residues of GAP-43 with fused to with of a and paralemmin fused to C-terminal amino acids of paralemmin fused to C-terminal amino acids of paralemmin fused to C-terminal amino acids of paralemmin fused to C-terminal amino acids of paralemmin acids fused to the palmitoylation motif is by a cysteines are in and cysteines are in were both by and by the of To the of axonal targeting, the fluorescent of GFP in the axon versus the dendrites were and with the ratio of GFP analysis that wild-type PSD-95 protein and are from the whereas and PSD-95(C3,5S) are in the axon to a as is GFP 2 Whereas palmitoylation of PSD-95 is for axon not all palmitoylated neuronal proteins are to 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). these different we the palmitoylation motifs in protein To this we the dually palmitoylated N-terminal 13 amino acids of PSD-95 with of GAP-43. The GAP-43 palmitoylation motif on PSD-95 palmitoylation and 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 to the a localization not with wild-type PSD-95 the of PSD-95 by the GAP-43 palmitoylation we targeting by these motifs. the PSD-95 palmitoylation motif is fused to the ratio of PSD-95(1–26) is not different from GFP the GAP-43 palmitoylation fused to GFP not exclusively is enriched in the axon with GFP These that the palmitoylation motif of PSD-95 is necessary but not sufficient for axon whereas the palmitoylation motif of GAP-43 is sufficient for axonal targeting. We the that axon of PSD-95 from at postsynaptic sites. However, we find that the 43-PSD-95 chimera is both and axonally PSD-95(C3,5S), indicating that postsynaptic and axonal are To to the of the GAP-43 palmitoylation motif necessary for axonal targeting, we the of the PSD-95 and GAP-43 palmitoylation motifs. The palmitoylated cysteines of the GAP-43 motif are whereas in PSD-95 are by a a PSD-95 adjacent cysteines is not exclusively the protein also localizes to the axon not to the of 43-PSD-95 the other the addition of a amino acid the cysteines of the GAP-43 palmitoylation motif not axonal targeting of and the ratio is These mutated are all palmitoylated in protein targeting are not to in protein These that the spacing of the cysteines is but is not the of these motifs that of PSD-95 GFP were transfected with PSD-95 described in Cells were in and the was with to GFP. were that were analyzed for by or were for GFP the GAP-43 motif contains two basic amino acids that is one from the palmitoylated whereas the PSD-95 motif not have basic residues the a basic amino acid to PSD-95 two amino acids from the cysteines the to the axon with the of 43-PSD-95 the basic amino acids of the GAP-43 palmitoylation motif to not protein palmitoylation but axonal targeting of 43-PSD-95 by These that the basic amino acids are but not the targeting these motifs. we in spacing the cysteines and mutations in the basic amino PSD-95 palmitoylation motif with cysteines and the PSD-95 to the axon to 43-PSD-95 a GAP-43 palmitoylation motif both with cysteines and with mutations in the 43-PSD-95 to the but the protein localizes to the postsynaptic membrane to wild-type PSD-95 These that these two features for targeting by these domains. We the adjacent amino acid motif a role in the axonal sorting of palmitoylated proteins. Paralemmin is a neuronal protein that is found at axonal membranes C. G. B. E. J. Cell Biol. 1998; PubMed Scopus Google Scholar). The C-terminal palmitoylation motif of paralemmin contains adjacent cysteines and nearby basic amino acids and resembles the of GAP-43 paralemmin is targeted to the to GAP-43. the prenyl-palmitoylation motif of paralemmin fused to GFP is sufficient for axonal targeting and the addition of this motif to the C terminus of PSD-95 the chimera to the axon We axonal targeting by the paralemmin motif requires features as the GAP-43 the spacing of the cysteines with and palmitoylation not we were to the of cysteine spacing in protein However, the palmitoylated cysteines to but palmitoylation and disrupts axon targeting mutations in the basic amino acids but axonal targeting, with a for nearby basic amino acids in axonal targeting acylated proteins can into and these complexes have been 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, B. Neuron. 1999; Full Text Full Text PDF PubMed Scopus (116) Google Scholar). the palmitoylation motifs of GAP-43 and PSD-95 with proteins of DIGs in and are found in membrane with J.R. Cell. Full Text PDF PubMed Scopus Google Scholar). previously a of PSD-95 with and this is of palmitoylation Bredt D.S. Neurosci. 1998; PubMed Scopus Google Scholar, C. S. Y. J. Biol. Chem. 1997; Full Text Full Text PDF PubMed Scopus Google Scholar). the palmitoylation motif of GAP-43 a GFP to DIGs S. Ostermeyer A.G. Brown D.A. J. Biol. Chem. 1998; Full Text Full Text PDF PubMed Scopus Google as the axonally targeted prenyl-palmitoylation motif of paralemmin the palmitoylation motif of PSD-95 is with that of GAP-43 or these also with whereas the of DIG and not is a the of palmitoylation motifs to target proteins to DIGs and to axonal membranes. analysis of polarized sorting of peripheral membrane proteins that palmitoylation motifs can mediate either or axonal targeting. that 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 find that palmitoylation is also necessary to PSD-95 from the palmitoylation motif is not sufficient for axonal the dually palmitoylated motifs of the axonal proteins GAP-43 and paralemmin are sufficient to mediate protein targeting to axonal membranes and, are the axonal targeting motifs for peripheral membrane proteins. Differential sorting of the PSD-95 and GAP-43 palmitoylation motifs on two the spacing of the cysteine residues and the presence of nearby basic amino The PSD-95 and GAP-43 palmitoylation motifs also in to with PSD-95 is into whereas the palmitoylation motif of GAP-43 is sufficient for with these These that the of peripheral membrane proteins into lipid rafts mediate axonal dendritic sorting of PSD-95 from dendritic targeting or axonal is axonally targeted palmitoylation motifs from GAP-43 and paralemmin are added to PSD-95, the are not as polarized as palmitoylation are they as well with These that dendritic targeting or axonal within PSD-95. with this the palmitoylation motif of PSD-95 is for dendritic targeting. PSD-95(C3,5S) is to the expression of the dendritic targeting within the of PSD-95 probably requires with membranes via The of the of PSD-95 in dendritic to to the of targeted PSD-95, axonally targeted as GAP-43 and are also in These proteins are axonal of density in the axon with GFP. have also found that expression of axonal proteins protein in dendrites Neuron. 1999; 22: Full Text Full Text PDF PubMed Scopus Google Scholar). dendritic expression for a of including axonal or to of targeting dendritic localization by the presence of dendritic targeting the of dendritic as axonal proteins can also in C. G. B. E. J. Cell Biol. 1998; PubMed Scopus Google Scholar). we found that PSD-95 GAP-43 or paralemmin palmitoylation motifs in the axon but also at the PSD-95 and expression that on the specific protein and neuronal cell hippocampal PSD-95 and are from whereas and 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). PSD-95 and are palmitoylated and and 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 targeting in the mechanisms as described dendritic localization in hippocampal and other PSD-95 occurs in axons of U. C. U. B. C.C. J. Biol. Chem. 1993; Full Text PDF PubMed Google Scholar). are in that axons are of A.I. M. J. PubMed Scopus Google Scholar). of hippocampal and other neurons and dendrites both and Neuron. 1999; 22: Full Text Full Text PDF PubMed Scopus Google Scholar). dendritic localization of PSD-95 a selective with that with palmitoylation to target PSD-95 into dendrites and to PSD-95 from axons. the presence of PSD-95 in cell axons is by the of axon Neuron. 1999; 22: Full Text Full Text PDF PubMed Scopus Google has the axonal for a that targeting of these transmembrane proteins on within C analysis of palmitoylation-dependent sorting of peripheral membrane proteins a role for These complexes are in and and formation in the secretory pathway is to as a sorting to proteins to the apical membrane of epithelial (4Simons K. Ikonen E. Nature. 1997; 387: 569-572Crossref PubMed Scopus (8157) Google Scholar). sorting of proteins to axonal membranes has been with this apical sorting B. Neuron. 1999; Full Text Full Text PDF PubMed Scopus (116) Google Scholar). into rafts to mediate axonal targeting of the (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 palmitoylation motifs that with these rafts can also target proteins to axonal membranes. the palmitoylation motif of PSD-95 is not into and mediating axonal targeting, it a role in dendritic To a DIG and axonal targeting, we to DIG formation and to However, this not axonal targeting of E. S. E. S. C. and S. suggesting that cholesterol depletion was not in 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, Y. Fisher D.A. Storm D.R. J. Neurosci. 1994; 14: 5807-5817Crossref PubMed Google lipid rafts are it is that these two proteins to separate secretory vesicles for to dendritic versus axonal membranes. The palmitoylation motif of GAP-43 can mediate lipid for protein to the axonal have found that GAP-43 is enriched in DIGs from brain S. K. Y. K. Y. Biochim. Biophys. Acta. 1997; PubMed Scopus Google and is to axons on vesicles from the secretory pathway A. J. Banker G. J. Cell Biol. PubMed Scopus Google Scholar). the other within the of PSD-95 with palmitoylation mediate with dendritic targeting vesicles and from C PSD-95 a motif that is sufficient to mediate protein via vesicles and that is for postsynaptic targeting S.E. Bredt D.S. J. Biol. Chem. 2000; Full Text Full Text PDF PubMed Scopus Google Scholar). axonally targeted lipid is from vesicles Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google M.G. C. J. J. Cell Biol. PubMed Scopus Google Scholar). motif to M.G. C. J. J. Cell Biol. PubMed Scopus Google Scholar, J. Roth M. Cell. 1988; Full Text PDF PubMed Scopus Google and with DIGs J. E. C. Roth M.G. J. Cell Biol. 1996; PubMed Scopus Google P. Roth M.G. Simons K. J. 1997; PubMed Scopus Google Scholar). the of PSD-95 in in from lipid PSD-95 and GAP-43 with DIGs different enzymes palmitoylation motifs. enzymes have not been the for palmitoylation that this is process. palmitoylation motifs to GAP-43 and paralemmin that adjacent cysteines with nearby basic amino acids by a enzyme in network followed by the into lipid rafts for axonal the palmitoylation motif of PSD-95 by a separate enzyme that is cytosolic or with membranes other the trans-Golgi and PSD-95 is not into of the that mediate palmitoylation of neuronal proteins to these how protein palmitoylation to polarized for different sorting in the

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