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doi: 10.1242/10.1242/jcs.00139

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Drosophila syntaxin 16 is a Q-SNARE implicated in Golgi dynamics

¹ Programme in Cell Biology The Hospital for Sick Children, University of Toronto, 555 University Avenue, Toronto, Ontario M5G 1x8 Canada
² Programme in Developmental Biology, The Hospital for Sick Children, University of Toronto, 555 University Avenue, Toronto, Ontario M5G 1x8 Canada
³ Department of Biochemistry, University of Toronto, 555 University Avenue, Toronto, Ontario M5G 1x8 Canada
⁴ Department of Molecular and Medical Genetics, University of Toronto, 555 University Avenue, Toronto, Ontario M5G 1x8 Canada

View larger version (47K): [in a new window]   Fig. 1. Alignment of dSyx16 with hSyx16. The sequences are numbered on the right. Identical amino acids are shaded black. Conserved amino acids are shaded gray. A potential transmembrane domain at the C-terminal end is underlined. The star below the residue Q indicates the central residue of the predicted coiled-coil. The heptad repeats are numbered above the sequence.

View larger version (38K): [in a new window]   Fig. 2. dSyx16 binds dSNAP in a concentration-dependent fashion. From lane 1 to 5, immobilized GST-dSyx16 or GST were incubated with 10, 4, 2, 0 and 10 µg of recombinant dSNAP, respectively. Proteins on the glutathione beads were then eluted and subjected to SDS-PAGE and western blot analysis.

View larger version (147K): [in a new window]   Fig. 3. dSyx16 interacts genetically with NSF2 during wing margin development. Wings from wild-type fly (A) and flies overexpressing dominant-negative NSF2 alone (B) or together with soluble dSyx1670 to 329 (C) or together with dSyx1670 to 352 (D) are shown. Several independent transgenic lines inserted with either the soluble dSyx16 or dSyx1670 to 352 were tested and the phenotypes shown in C and D have been consistently observed.

View larger version (54K): [in a new window]   Fig. 4. dSyx16 is a membrane protein expressed ubiquitously in Drosophila. Embryos after deposition (AED) were collected and allowed to develop for indicated period of time (A). Larvae, pupae, adults were collected and imaginal discs were dissected from third instar larvae (B). All samples were lysed in SDS-homogenization buffer and subject to SDS-PAGE and western blot analysis. In C, crude membrane fraction from homogenized Oregon R adults was treated with H2O, KCl, Na2CO3, Urea, Triton X-100 or SDS and then centrifuged to separate the soluble from the insoluble. All samples were re-suspended to the same volume before subjecting them to SDS-PAGE and western blot analysis.

View larger version (46K): [in a new window]   Fig. 5. dSyx16 distribution in salivary gland cells. Salivary glands were dissected from third instar lava, fixed in 4% paraformaldehyde and stained with anti-dSyx16 (A) and anti-p120 (B). The merged image is shown in C. Overlapping of large puncta is indicated by filled arrow. Open arrows point to a small punctate structure. Arrowheads indicate nuclei. Scale bar, 10 µM.

View larger version (36K): [in a new window]   Fig. 6. dSyx16 localization in S2 cells. Cells treated with DMSO (A,C,E,G) or brefeldin A (B,D,F,H) were fixed and co-stained with antibodies against p120 (A,B) and dSyx16 (C,D). Note the small but distinct punctate Golgi pattern in the absence of BFA and the big aggregates with BFA treatment. Arrows point to the ring structure, which can be observed at different focal planes in other cells. Scale bar, 10 µM.

View larger version (61K): [in a new window]   Fig. 7. dSyx16 distribution during cell division in Drosophila testis. Testes from Oregan R were dissected and stained with anti-dSyx16 (A,E), anti-p120 (B) or propidium iodide (F). A-D are interphase cells with intact nuclei arrows. dSyx16 is localized in distinct puncta (arrowhead). E-H are anaphase cells with dSyx16 much more dispersed (open arrowhead). Occasionally, larger puncta can be observed (open arrows), but they are not comparable with those in interphase cells. Scale bars, 10 µM.

View larger version (71K): [in a new window]   Fig. 8. Overexpression of dSyx16 in S2 cells. S2 cells transfected with myc-dSyx1670 to 329 (A-C) or myc-dSyx1670 to 352 (D-I) were fixed and co-stained with anti-myc (red channel) and anti-p120 (green channel, B,C,E,F) or anti-lva (green channel, H,I) antibodies. The arrows point to transfected cells. Open arrowheads point to non-transfected cells. Scale bar, 10 µM.