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Roles of the cytoplasmic and transmembrane domains of syntaxins in intracellular localization and trafficking

¹ Department of Physiology, Kyorin University, Mitaka, Tokyo 181-8611, Japan
² Toyama Chemical Co. Ltd, Sinjuku, Tokyo 160-0023, Japan

View larger version (64K): [in a new window]   Fig. 1. Intracellular localization of wild-type syntaxins. Clone 9 cells were transfected with HA-tagged wild-type syntaxins 5, 6, 7 and 8 as described in Materials and Methods. After incubation for 12 hours, the cells were fixed and stained with anti-HA antibody and antibodies against indicated marker proteins. Syntaxins 5 and 6 overlapped with ß-COP and TGN38, respectively. Syntaxin 7 was colocalized with EEA1 but not with lamp-1, and syntaxin 8 was partially colocalized with both EEA1 and lamp-1.

View larger version (41K): [in a new window]   Fig. 2. Immunofluorescence analysis of wild-type and chimeric syntaxins. PC12h cells were electroporated with the indicated HA-tagged wild-type syntaxins (a,c,e,g,i) or HA-tagged syntaxin 1A chimeras containing the transmembrane domain of syntaxin 5, 6, 7 or 8 (b,d,f,h, respectively), as described Materials and Methods. After incubation for 24 hours, the cells were treated for an additional 24 hours with 20 mM sodium butyrate. The cells were fixed and stained with anti-HA antibody. Syn1-5 localized to the CGN, as did wild-type syntaxin 5. Other chimeras were localized at the plasma membrane, but did not localize to the same compartments as did their wild-type counterparts containing the same transmembrane domains.

View larger version (66K): [in a new window]   Fig. 3. The cytoplasmic domains are important for the intracellular localization of syntaxins. Clone 9 cells were transfected with HA-tagged chimeras composed of the transmembrane domain of syntaxin 1A and the cytoplasmic domain of either syntaxin 5, 6, 7 or 8. After incubation for 12 hours, the cells were fixed and stained with anti-HA antibody and antibodies against indicated marker proteins. Each chimera localized to the same compartment(s) as did its wild-type counterpart with the same cytoplasmic domain.

View larger version (34K): [in a new window]   Fig. 4. Immunofluorescence analysis of chimeric syntaxins in BFA-treated cells. Clone 9 cells were transfected with HA-tagged syntaxin 5 (a,a'), syn5-1 (b,b'), syn6-5 (d,d'), syntaxin 6 (e,e'), and syn6-1 (f,f') and PC12h cells were transfected with HA-tagged syn1-5 (c,c') in the absence (a-f) or presence (a'-f') of BFA. After incubation for expression, the cells were treated with 5 µg/ml BFA for 30 minutes before fixing. The cells were stained with anti-HA antibody. In BFA-treated cells, chimeras containing either the cytoplasmic domain or the transmembrane domain of syntaxin 5 were redistributed from the CGN into the ER, as was syntaxin 5 (a'-d'). By contrast, in BFA-treated cells, syntaxin 6 and syn6-1 were redistributed from the TGN to the MTOC (e',f').

View larger version (54K): [in a new window]   Fig. 5. Syntaxin 8 is present at the plasma membrane. Clone 9 cells were transfected with HA-tagged wild-type syntaxins and incubated for 12 hours for low-level expression (a-d) or 24 hours for overexpression (a'-d') before fixing. The cells were stained with anti-HA antibody. In low-expression cells, syntaxins 6, 7 and 8 were faintly labeled at the plasma membrane (b-d; arrows) besides their specific intracellular localizations. In overexpressing cells, syntaxins 6, 7 and 8, but not syntaxin 5, were present at the plasma membrane.

View larger version (75K): [in a new window]   Fig. 6. The cytoplasmic domains of syntaxins 7 and 8 direct their internalization from the plasma membrane. PC12h cells were electroporated with c-myc-tagged wild-type syntaxins (a,d,g), transmembrane domain chimeras (b,e,h), or cytoplasmic domain chimeras (c,f,i), and were processed for antibody uptake experiments. After incubation for 24 hours, the cells were treated for an additional 48 hours with 20 mM sodium butyrate. The cells were incubated for 3 hours in the presence of mouse anti-c-myc antibody before fixing. The cells were fixed and stained with FITC-conjugated anti-mouse IgG. Incubation of wild-type syntaxin-expressing cells with anti-c-myc antibody resulted in efficient labeling of intracellular compartments. Similar results were obtained in cells expressing syn6-1, syn7-1 or syn8-1. However, incubation of cells expressing syn1-6, syn1-7 or syn1-8 with anti-c-myc antibody resulted in labeling only at the plasma membrane.

View larger version (51K): [in a new window]   Fig. 7. Di-leucine-based motifs are important for the intracellular localization and trafficking of syntaxins 7 and 8. A putative di-leucine-based motif is present in the cytoplasmic domains of syntaxin 7 (amino acids 162-168) and syntaxin 8 (amino acids 77-83). Mutations in these motifs were made as indicated by underlining. Clone 9 cells were transfected with syn7-mut (a,b) or syn8-mut (d,e), each of which was fused to an N-terminal HA tag. For intracellular localization analysis, the cells were incubated for 12 hours (a,d) or 24 hours (b,e) and stained with anti-HA antibody. For antibody uptake experiments, Clone 9 cells were transfected with mutants in which the C-termini were fused to three c-myc tags (c,f). After 24 hours incubation, the cells were incubated for 3 hours in the presence of mouse anti-c-myc antibody before fixing. The cells were fixed and stained with FITC-conjugated anti-mouse IgG. (a-c) Syn7-mut was localized to endosomes and at the plasma membrane. In antibody uptake experiments, labeling was found only at the plasma membrane. (d-f) Syn8-mut was localized to the perinuclear Golgi region. No staining was observed in antibody uptake experiments.

View larger version (22K): [in a new window]   Fig. 8. Schematic representation of vesicular transport of syntaxins between intracellular compartments. The 35 kDa isoform of syntaxin 5 used in this study is retained at the CGN by virtue of either the cytoplasmic domain (CD) or the transmembrane domain (TMD). Substitution of the cytoplasmic domain (CD-substitution), but not the transmembrane domain, of syntaxins 6, 7 and 8 with that of syntaxin 1A prevents their internalization. The di-leucine-based motifs in the cytoplasmic domains of syntaxins 7 and 8 have important, but distinct, roles in intracellular localization. Mutation of the di-leucine-based motif of syntaxin 7 (amino acids 162-168) inhibited internalization of syntaxin 7. By contrast, mutation of the di-leucine-based motif of syntaxin 8 (amino acids 77-83) resulted in accumulation of syntaxin 8 at the perinuclear Golgi region. The transport pathways of syntaxins 5, 6, 7 and 8 are denoted by gray, green, blue and red, respectively.