STOP-like Protein 21 Is a Novel Member of the STOP Family, Revealing a Golgi Localization of STOP Proteins

Abstract

Neuronal microtubules are stabilized by two calmodulin-regulated microtubule-associated proteins, E-STOP and N-STOP, which when suppressed in mice induce severe synaptic and behavioral deficits. Here we show that mature neurons also contain a 21-kDa STOP-like protein, SL21, which shares calmodulin-binding and microtubule-stabilizing homology domains with STOP proteins. Accordingly, in different biochemical or cellular assays, SL21 has calmodulin binding and microtubule stabilizing activity. However, in cultured hippocampal neurons, SL21 antibodies principally stain the somatic Golgi and punctate Golgi material in neurites. In cycling cells, transfected SL21 decorates microtubules when expressed at high levels but is otherwise principally visible at the Golgi. The Golgi targeting of SL21 depends on the presence of cysteine residues located within the SL21 N-terminal domain, suggesting that Golgi targeting may require SL21 palmitoylation. Accordingly we find that SL21 is palmitoylated in vivo. N-STOP and E-STOP, which contain the Golgi targeting sequences present in SL21, also display distinct Golgi staining when expressed at low level in cycling cells. Thus neuronal proteins of the STOP family have the capacity to associate with Golgi material, which could be important for STOP synaptic functions. Neuronal microtubules are stabilized by two calmodulin-regulated microtubule-associated proteins, E-STOP and N-STOP, which when suppressed in mice induce severe synaptic and behavioral deficits. Here we show that mature neurons also contain a 21-kDa STOP-like protein, SL21, which shares calmodulin-binding and microtubule-stabilizing homology domains with STOP proteins. Accordingly, in different biochemical or cellular assays, SL21 has calmodulin binding and microtubule stabilizing activity. However, in cultured hippocampal neurons, SL21 antibodies principally stain the somatic Golgi and punctate Golgi material in neurites. In cycling cells, transfected SL21 decorates microtubules when expressed at high levels but is otherwise principally visible at the Golgi. The Golgi targeting of SL21 depends on the presence of cysteine residues located within the SL21 N-terminal domain, suggesting that Golgi targeting may require SL21 palmitoylation. Accordingly we find that SL21 is palmitoylated in vivo. N-STOP and E-STOP, which contain the Golgi targeting sequences present in SL21, also display distinct Golgi staining when expressed at low level in cycling cells. Thus neuronal proteins of the STOP family have the capacity to associate with Golgi material, which could be important for STOP synaptic functions. Neurons contain abundant subpopulations of stable microtubules that resist depolymerizing conditions such as exposure to the cold. This property is due to microtubule association with E-and N-STOP 2The abbreviations used are: STOP, stable tubule only polypeptide; SL21, 21-kDa STOP-like protein; aa, amino acid(s); DIV, days (of differentiation) in vitro; mAb, monoclonal antibody; GST, glutathione S-transferase; PBS, phosphate-buffered saline; Pipes, 1,4-piperazinediethanesulfonic acid; DMEM, Dulbecco's modified Eagle's medium., two neuronal calmodulin-binding and calmodulin-regulated proteins (1Andrieux A. Salin P.A. Vernet M. Kujala P. Baratier J. Gory-Fauré S. Bosc C. Pointu H. Proietto D. Schweitzer A. Denarier E. Klumperman J. Job D. Genes Dev. 2002; 16: 2350-2364Crossref PubMed Scopus (146) Google Scholar, 2Guillaud L. Bosc C. Fourest-Lieuvin A. Denarier E. Pirollet F. Lafanechère L. Job D. J. Cell Biol. 1998; 142: 167-179Crossref PubMed Scopus (108) Google Scholar, 3Bosc C. Frank R. Denarier E. Ronjat M. Schweitzer A. Wehland J. Job D. J. Biol. Chem. 2001; 276: 30904-30913Abstract Full Text Full Text PDF PubMed Scopus (36) Google Scholar). STOP proteins are important for synaptic function and STOP null mice present defects in both short- and long-term synaptic plasticity, associated with severe behavioral and neurotransmitter deficits (1Andrieux A. Salin P.A. Vernet M. Kujala P. Baratier J. Gory-Fauré S. Bosc C. Pointu H. Proietto D. Schweitzer A. Denarier E. Klumperman J. Job D. Genes Dev. 2002; 16: 2350-2364Crossref PubMed Scopus (146) Google Scholar, 4Brun P. Bégou M. Andrieux A. Mouly-Badina L. Clerget M. Schweitzer A. Scarna H. Renaud B. Job D. Suaud-Chagny M.F. J. Neurochem. 2005; 94: 63-73Crossref PubMed Scopus (63) Google Scholar). At the ultrastructural level, the main signature of STOP suppression is a dramatic depletion of synaptic vesicle pools in glutamatergic neurons. E- and N-STOP arise from mRNA splicing of a single gene (5Denarier E. Aguezzoul M. Jolly C. Vourc'h C. Roure A. Andrieux A. Bosc C. Job D. Biochem. Biophys. Res. Commun. 1998; 243: 791-796Crossref PubMed Scopus (28) Google Scholar). These STOP proteins contain modular motifs (3Bosc C. Frank R. Denarier E. Ronjat M. Schweitzer A. Wehland J. Job D. J. Biol. Chem. 2001; 276: 30904-30913Abstract Full Text Full Text PDF PubMed Scopus (36) Google Scholar) that are either bifunctional (calmodulin-binding and microtubule-stabilizing sequences, Mc and Mn modules, respectively) or are comprised of calmodulin-binding sequences, unrelated to microtubule-stabilizing sequences. We have previously described a number of STOP splicing variants, which are principally present in non-neuronal cells (6Galiano M.R. Bosc C. Schweitzer A. Andrieux A. Job D. Hallak M.E. J. Neurosci. Res. 2004; 78: 329-337Crossref PubMed Scopus (33) Google Scholar), but for a long time, STOPs seemed to be unique with no STOP-related proteins. However, strong evidence for a 21-kDa STOP-like protein (SL21) has arisen from data base searches (3Bosc C. Frank R. Denarier E. Ronjat M. Schweitzer A. Wehland J. Job D. J. Biol. Chem. 2001; 276: 30904-30913Abstract Full Text Full Text PDF PubMed Scopus (36) Google Scholar). SL21 contains a 35-amino acid (aa) N-terminal stretch that shares 83% homology with the N-terminal part of E- or N-STOP and contains a calmodulin-binding motif. A second domain of SL21, spanning 24 aa, shares 71% homology with a microtubule-stabilizing Mn module of E- and N-STOP (3Bosc C. Frank R. Denarier E. Ronjat M. Schweitzer A. Wehland J. Job D. J. Biol. Chem. 2001; 276: 30904-30913Abstract Full Text Full Text PDF PubMed Scopus (36) Google Scholar, 7Bosc C. Andrieux A. Job D. Biochemistry. 2003; 42: 12125-12132Crossref PubMed Scopus (68) Google Scholar). Here, we demonstrate that SL21 is a neuronal protein, strictly expressed in the postnatal brain. We show that SL21 has microtubule stabilizing activity similar to E- and N-STOP. However, surprisingly, endogenous SL21 co-localizes with the somatic Golgi apparatus and with dendritic Golgi structures in cultured hippocampal neurons. We show that the N-terminal part of SL21 comprises a Golgi-targeting sequence with critical cysteine residues, which sustain SL21 palmitoylation. The SL21 Golgi targeting sequence is also present and functional in E-and N-STOP. Thus, in light of studies indicating that the presence of Golgi material in neurites and synapses may be important for synaptic plasticity (8Horton A.C. Ehlers M.D. Nat. Cell Biol. 2004; 6: 585-591Crossref PubMed Scopus (129) Google Scholar), Golgi interactions with STOP could be important for STOP function in synapses. Sequence Analysis—Mouse N-STOP protein sequence (GenBank™ accession number CAA75930) was submitted to the BLAST program to identify STOP-related proteins. GenBank™ accession numbers of these proteins are CAA63762 for rat N-STOP, NP_149052 for human N-STOP, XP_508647 for chimpanzee N-STOP, XP_596995 for cow N-STOP, NP_941001 for mouse SL21, XP_344040 for rat SL21, AAH06434 for human SL21, XP_516902 for chimpanzee SL21, and XP_608343 for cow SL21. Protein sequence alignments were performed using the multiple alignment software ClustalW (9Thompson J.D. Higgins D.G. Gibson T.J. Nucleic Acids Res. 1994; 22: 4673-4680Crossref PubMed Scopus (56590) Google Scholar). Plasmid Constructs—On the basis of the mouse SL21 cDNA sequence (GenBank™ accession number BY727771), the entire open reading frame of mouse SL21 was amplified by PCR. We used mouse brain Marathon Ready cDNA (BD Biosciences) and the Advantage-GC2 polymerase mix (BD Biosciences) with primers containing BglII extensions. The resulting PCR product was first cloned into pCR2.1-TOPO (Invitrogen). For expression in mammalian cells, the complete coding sequence was cloned in pSG5 vector (Stratagene) and in pcDNA3.1(-)/Myc-His-A vector (Invitrogen) to be fused with the myc epitope DNA. For production and purification in the bacterial system, the complete coding sequence was cloned into pGex-4T3 vector (Amersham Biosciences) to fuse GST after the last amino acids of SL21. The two SL21 deletion mutants, obtained by PCR, correspond to Met + aa 35-191 (SL21Δ2-34) and to aa 1-122 + aa 146-191 (SL21ΔMn3) according to the numbering of mouse SL21 protein. The replacement of Cys5, Cys10, and Cys11 by glycine residues (SL21-C(5/10/11)G) was obtained by PCR, with a degenerate 5′-oligonucleotide (5′-AgA TCT ATg gCg Tgg CCC ggC ATC AgC Cgg CTA ggC ggC CTg gCC-3′). The STOP coding sequence of LNtΔMn1Mn2, corresponding to aa 1-123 + Ala + aa 139-161 + Ile-Gln + aa 175-225 according to the numbering of rat N-STOP, was amplified by PCR and then subcloned into the pcDNA3.1(-)/Myc-His-A vector to be fused with the myc epitope DNA. N-STOP plasmid was described previously (2Guillaud L. Bosc C. Fourest-Lieuvin A. Denarier E. Pirollet F. Lafanechère L. Job D. J. Cell Biol. 1998; 142: 167-179Crossref PubMed Scopus (108) Google Scholar, 10Bosc C. Cronk J.D. Pirollet F. Watterson D.M. Haiech J. Job D. Margolis R.L. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 2125-2130Crossref PubMed Scopus (96) Google Scholar). The STOP coding sequence of N-STOPΔ2-19, corresponding to Met + aa 20-952 according to the numbering of rat N-STOP, was amplified by PCR and then subcloned into the pSG5 vector. All of the SL21 and STOP constructs were confirmed by DNA sequencing. Production of SL21-GST—Plasmid pGex-4T3 containing SL21 cDNA was transformed in One Shot BL21 (DEA3) Star cells (Invitrogen) according to the manufacturer's instructions. Transformed cells expressing SL21-GST were grown at 37 °C to an A600 of 0.6, and induced with 0.5 mm isopropyl 1-thio-β-d-galactopyranoside for 3 h at 37 °C. Cells were pelleted, resuspended in PBS containing 1% Triton X-100, protease inhibitors (Complete Mixture tablets, Roche Applied Science), and sonicated for 1.5 min before centrifugation (17,000 × g for 10 min). The supernatant was incubated with glutathione-Sepharose beads. These beads were placed in columns and successively washed with PBS containing 1% Triton X-100, then with PBS, and finally with 50 mm Tris, pH 8.0. The GST fusion proteins were eluted with 10 mm glutathione in 50 mm Tris, pH 8.0. Western Blot Analysis—Tissues from mice were homogenized in a 100 mm K-Pipes, 1 mm EGTA, and 1 mm MgCl2 buffer, pH 6.65. The homogenates were centrifuged at 200,000 × g for 30 min at 4 °C. Supernatants were then harvested and analyzed by SDS-PAGE (11Laemmli U.K. Nature. 1970; 227: 680-685Crossref PubMed Scopus (208375) Google Scholar). For immunoblotting, proteins were subsequently transferred onto nitrocellulose membranes and processed as described previously (12Lieuvin A. Labbé J.C. Dorée M. Job D. J. Cell Biol. 1994; 124: 985-996Crossref PubMed Scopus (101) Google Scholar). Sedimentation of Proteins with Microtubules—All proteins were preclarified at 150,000 × g for 15 min in a TL-100 Ultracentrifuge at 4 °C prior to the start of experiments. STOP proteins were purified as described in Pirollet et al. (13Pirollet F. Derancourt J. Haiech J. Job D. Margolis R.L. Biochemistry. 1992; 31: 8849-8855Crossref PubMed Scopus (52) Google Scholar). A microtubule-binding assay was performed as in Masson and Kreis (14Masson D. Kreis T.E. J. Cell Biol. 1993; 123: 357-371Crossref PubMed Scopus (89) Google Scholar) using taxol-stabilized microtubules (4 μm) as substrates and a 60% glycerol cushion. Assay of Calmodulin Binding to Immobilized Peptide—Immobilized peptide arrays corresponding to the human SL21 were produced according to Frank (15Frank R. Tetrahedron. 1992; 48: 9217-9232Crossref Scopus (937) Google Scholar) with an ABIMED ASP 222 automated SPOT robot. The protein sequence was subdivided into 15-mer peptides with an overlap of 12 aa. The peptides were as an on a The peptides are to the aa a The that of Frank and R. H. Biol. 1996; Google Scholar). binding assay were performed as described (3Bosc C. Frank R. Denarier E. Ronjat M. Schweitzer A. Wehland J. Job D. J. Biol. Chem. 2001; 276: 30904-30913Abstract Full Text Full Text PDF PubMed Scopus (36) Google Scholar), using Cell were as described previously J. Neurosci. PubMed Google Scholar). brain from mice were and in in at 37 °C for 15 cells were at a of on in One after the was to containing and (Invitrogen). and cells were cultured in or with and 1% (Invitrogen). and of or cells were transfected with the described using according to the manufacturer's instructions. cells were either processed for or to min on or to for 30 min). Cells were then in mm Pipes, 1 mm EGTA, 1 mm Triton X-100, pH for 1 min and processed for antibodies to rat to J. J. Cell Biol. PubMed Scopus Google to and to (BD to STOP (2Guillaud L. Bosc C. Fourest-Lieuvin A. Denarier E. Pirollet F. Lafanechère L. Job D. J. Cell Biol. 1998; 142: 167-179Crossref PubMed Scopus (108) Google Scholar) to antibodies were peptide corresponding to aa of mouse SL21 was with the corresponding peptide and used to and to for Western and antibodies GST or and neurons were then either in mm Pipes, pH 1 mm EGTA, 1 mm Triton X-100, at °C for min before or were for 10 min in at °C. Cells were then incubated with antibodies for min in and with antibodies for Cells were analyzed with a or with an 50 by were using a and cells were either or transfected with or SL21 One after cells were for 3 h with of acid (Amersham The acid was resuspended in of and with 1 of containing Cells were washed and with centrifugation at × g for cells were in mm Tris, mm 1% Triton X-100, pH in the presence of protease inhibitors (Complete Mixture tablets, Roche Applied and mm centrifugation of the at × g for min at 4 the supernatant was with mm and incubated for h at with of myc for h at with 30 of protein (Amersham centrifugation at × g for the were washed in for 10 min at 4 °C. were by and were for 30 min in acid and then for 30 min with (Amersham Biosciences) to the The were and on for 10 days at °C. STOP in and N-STOP contains two of bifunctional calmodulin-binding and E- and N-STOP also contain calmodulin-binding sequences unrelated to Mn or Mc The first aa of SL21 83% homology with the N-terminal aa of E- and N-STOP, which in the functional calmodulin-binding (3Bosc C. Frank R. Denarier E. Ronjat M. Schweitzer A. Wehland J. Job D. J. Biol. Chem. 2001; 276: 30904-30913Abstract Full Text Full Text PDF PubMed Scopus (36) Google Scholar). A second domain of SL21, spanning 24 aa, shares 71% homology with the E- and N-STOP bifunctional calmodulin-binding and microtubule-stabilizing module (3Bosc C. Frank R. Denarier E. Ronjat M. Schweitzer A. Wehland J. Job D. J. Biol. Chem. 2001; 276: 30904-30913Abstract Full Text Full Text PDF PubMed Scopus (36) Google Scholar). The domains of N-STOP and SL21 are of of the STOP calmodulin-binding and were as calmodulin-binding motifs on N-STOP peptide arrays (3Bosc C. Frank R. Denarier E. Ronjat M. Schweitzer A. Wehland J. Job D. J. Biol. Chem. 2001; 276: 30904-30913Abstract Full Text Full Text PDF PubMed Scopus (36) Google Scholar). A similar assay with SL21 peptides A and that the STOP and motifs of SL21 and also and 1 and with two calmodulin-binding and and 4 and motifs in N-STOP have to to in (3Bosc C. Frank R. Denarier E. Ronjat M. Schweitzer A. Wehland J. Job D. J. Biol. Chem. 2001; 276: 30904-30913Abstract Full Text Full Text PDF PubMed Scopus (36) Google Scholar). Accordingly, that SL21-GST to calmodulin as in The binding was strictly on the presence of as by the of SL21 from the calmodulin by and by the of SL21 to calmodulin in the of The binding of SL21-GST to calmodulin is by SL21, as GST was to to calmodulin Thus, SL21 as a calmodulin-binding protein in Binding and of E- and N-STOP, Mn STOP association with microtubules with resulting of microtubule exposure to the or to (3Bosc C. Frank R. Denarier E. Ronjat M. Schweitzer A. Wehland J. Job D. J. Biol. Chem. 2001; 276: 30904-30913Abstract Full Text Full Text PDF PubMed Scopus (36) Google Scholar). In with the presence of an Mn module sequence in SL21, SL21 with taxol-stabilized microtubules in microtubule binding in the in the of microtubules In the microtubule binding N-STOP with GST in the In of were and then incubated with SL21-GST at was pelleted, and SL21 was analyzed on in both and The of at which of SL21 with microtubules was In SL21-GST at was with microtubules were pelleted, and SL21 was analyzed on in both and In SL21 at 4 was both in the supernatant and in the at of SL21, from to 0.5 SL21 was only associated with these that SL21 as a microtubule-binding protein in We used cells for microtubule-binding and in vivo. cells are of STOP, of SL21, and of or microtubules transfected in cells, a SL21 (SL21Δ2-34) the N-terminal domain and containing the Mn module microtubules and induced both microtubule and microtubule to with the of the Mn module of SL21 in vivo. the of the SL21 in cells was the the N-terminal domain (SL21Δ2-34) due to the of the N-terminal and of used Western of mouse with SL21 to the of SL21. SL21 was only in brain In from SL21 expression was low at and after We then the presence of SL21 in neuronal using both and SL21 was in neuronal and cultured in the presence or of In SL21 was present in neurons in but from cells in hippocampal cultured neurons, SL21 antibodies principally a corresponding to the somatic Golgi as by with the At of neuronal in in to the somatic both SL21 and antibodies a staining of corresponding to staining of Golgi A.C. Ehlers M.D. J. Neurosci. 2003; PubMed Google Scholar). A distinct microtubule staining was also visible after protein of the somatic Golgi with A in neurons induced a dramatic of SL21 and both of which These that SL21 is expressed in neuronal cells and that to to the somatic and to Golgi material neurites. of SL21 in Cells from and Golgi-targeting in used cells, which have a somatic and SL21 for of SL21 and domain expressed in cells, the of SL21 as a function of expression At low levels of SL21 was principally on the Golgi At levels of was also distinct SL21 staining of microtubules The deletion of the Mn module suppressed the microtubule association of SL21 at expression Golgi at levels of expression in cells, a deletion of SL21 N-terminal amino acids (SL21Δ2-34) associated with microtubules as in the of cells with no Golgi In microtubule in cells, we that SL21 expressed at high levels and the deletion microtubule stabilizing the SL21 which Golgi no on microtubule these that the targeting of SL21 to microtubules depends on the presence of the Mn module and that SL21 comprises a Golgi-targeting sequence located in the N-terminal domain of the protein. of is for protein association with membranes and has previously in neuronal proteins associated with and with the such as R. S. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). at cysteine residues with residues M.D. Biophys. PubMed Scopus Google Scholar). SL21 contains cysteine residues, located in the domain 10 and and by residues these were in the of SL21 to the Golgi we produced SL21 in which Cys5, Cys10, and Cys11 were by glycine These constructs were then transfected in cells, and the Golgi of the was In to SL21, which to the the SL21 the cysteine residues were to the Golgi but with microtubules These a of SL21 N-terminal Cys5, Cys10, and Cys11 for of SL21 to Golgi SL21 cells were incubated with with or prior with or and were from and the proteins were analyzed both by Western using SL21 and by in SL21 that SL21 is palmitoylated in cells and the of Cys5, Cys10, and Cys11 in These were obtained for and similar were when using of Golgi of N-terminal Golgi-targeting sequence of SL21, which is is also present at the of N-STOP Accordingly, a N-terminal of N-STOP containing the sequence and for microtubule-binding and was to the Golgi when transfected in cells the presence of such a functional Golgi-targeting sequence at E- and N-STOP have at the Golgi in which could be due either to a of the STOP sequences and Mc or to these we N-STOP in and In neurons, STOP a staining of the a STOP at the Golgi to (2Guillaud L. Bosc C. Fourest-Lieuvin A. Denarier E. Pirollet F. Lafanechère L. Job D. J. Cell Biol. 1998; 142: 167-179Crossref PubMed Scopus (108) Google Scholar). in cells, transfected N-STOP staining was principally at the Golgi at low levels of expression with both Golgi and microtubule staining present at high expression levels a transfected N-STOP the Golgi targeting sequence to associate with the Golgi these that the to to Golgi material is a property of STOP and STOP-like proteins, of the presence of a Golgi-targeting sequence in N-terminal In SL21 and neuronal STOPs and as a of and calmodulin-binding proteins, both a Golgi-targeting sequence and microtubule-stabilizing We that the STOP Golgi-targeting sequence is only present in E- and N-STOP and is from of the STOP (6Galiano M.R. Bosc C. Schweitzer A. Andrieux A. Job D. Hallak M.E. J. Neurosci. Res. 2004; 78: 329-337Crossref PubMed Scopus (33) Google Scholar). are E- and N-STOP, SL21 is to neurons, and both SL21 and N-STOP are expressed only in the postnatal brain. such in and is an in the cellular of endogenous SL21 and N-STOP in neurons, with SL21 associated with the somatic Golgi and N-STOP with both SL21 and N-STOP are principally at the Golgi when expressed at low levels in cells, are also visible on microtubules at high levels of The endogenous SL21 and N-STOP in neurons may in expression with N-STOP abundant SL21. may also SL21 or STOP The Golgi targeting of SL21 and of STOP depends on which is a and protein Biol. Full Text Full Text PDF PubMed Scopus Google Scholar, Cell Biol. PubMed Scopus Google Scholar). the microtubule binding activity of Mn be by calmodulin D. Margolis R.L. Proc. Natl. Acad. Sci. U. S. A. 78: PubMed Scopus (108) Google Scholar) in the of STOP, the of a protein from microtubules to J. L. J. S. F. Bosc C. Fourest-Lieuvin A. L. Salin P. Job D. Andrieux A. J. Biol. Chem. Full Text Full Text PDF Scopus Google Scholar). neurons contain microtubule-associated proteins with Golgi binding The somatic Golgi in neurons has an similar to that in cells, the Golgi the and Golgi the A.C. B. Ehlers M.D. 2005; 48: Full Text Full Text PDF PubMed Scopus Google Scholar). is evidence for the presence of Golgi material neurites. ultrastructural studies have membranes to Golgi within the of Nat. Neurosci. PubMed Scopus Google Scholar, Biol. 2001; Full Text Full Text PDF PubMed Scopus Google Scholar). neurons both somatic Golgi and structures located into the These Golgi are structures and are to or of may be important for synaptic plasticity (8Horton A.C. Ehlers M.D. Nat. Cell Biol. 2004; 6: 585-591Crossref PubMed Scopus (129) Google Scholar, A.C. Ehlers M.D. J. Neurosci. 2003; PubMed Google Scholar). The of Golgi binding may be important for the function of N-STOP in synaptic plasticity (1Andrieux A. Salin P.A. Vernet M. Kujala P. Baratier J. Gory-Fauré S. Bosc C. Pointu H. Proietto D. Schweitzer A. Denarier E. Klumperman J. Job D. Genes Dev. 2002; 16: 2350-2364Crossref PubMed Scopus (146) Google Scholar). may STOP with or material the Golgi D. Nat. Neurosci. 2002; PubMed Scopus Google Scholar) and may be to the dramatic of STOP suppression on the synaptic vesicle (1Andrieux A. Salin P.A. Vernet M. Kujala P. Baratier J. Gory-Fauré S. Bosc C. Pointu H. Proietto D. Schweitzer A. Denarier E. Klumperman J. Job D. Genes Dev. 2002; 16: 2350-2364Crossref PubMed Scopus (146) Google Scholar). has to a in neuronal protein and in the of and the associated proteins at synapses D. Nat. Neurosci. 2002; PubMed Scopus Google Scholar, A. 2005; PubMed Scopus Google Scholar). to the present SL21 and STOP have STOP suppression is by SL21 in and we have in SL21 expression is the of SL21 in the presence of We have performed SL21 in hippocampal neurons, with is as for N-STOP, SL21 function be by suppression in of synaptic function be STOP or SL21 null may in which to the of the Golgi in brain functions. We A. Schweitzer and for for in and and J. C. for