Homotypic fusion of immature secretory granules during maturation requires syntaxin 6.
Homotypic fusion of immature secretory granules (ISGs) gives rise to mature secretory granules (MSGs), the storage compartment in endocrine and neuroendocrine cells for hormones and neuropeptides. With the use of a cell-free fusion assay, we investigated which soluble N-ethylmaleimide-sensitive fusion protein attachment receptor (SNARE) molecules are involved in the homotypic fusion of ISGs. Interestingly, the SNARE molecules mediating the exocytosis of MSGs in neuroendocrine cells, syntaxin 1, SNAP-25, and VAMP2, were not involved in homotypic ISG fusion. Instead, we have identified syntaxin 6 as a component of the core machinery responsible for homotypic ISG fusion. Subcellular fractionation studies and indirect immunofluorescence microscopy show that syntaxin 6 is sorted away during the maturation of ISGs to MSGs. Although, syntaxin 6 on ISG membranes is associated with SNAP-25 and SNAP-29/GS32, we could not find evidence that these target (t)-SNARE molecules are involved in homotypic ISG fusion. Nor could we find any involvement for the vesicle (v)-SNARE VAMP4, which is known to be associated with syntaxin 6. Importantly, we have shown that homotypic fusion requires the function of syntaxin 6 on both donor as well as acceptor membranes, which suggests that t-t-SNARE interactions, either direct or indirect, may be required during fusion of ISG membranes.
Wendler
F
F
Secretory Pathway Laboratory, Imperial Cancer Research Fund, London WC2A 3PX, UK.
Page
L
L
Urbé
S
S
Tooze
S A
SA
D000818:Animals; D000954:Antigens, Surface / Q000378:metabolism; D002462:Cell Membrane / Q000378:metabolism; D002474:Cell-Free System; D002850:Chromatography, Gel; D004305:Dose-Response Relationship, Drug; D004703:Endocrine System / Q000378:metabolism; D019084:Fluorescent Antibody Technique, Indirect; D008565:Membrane Proteins / Q000737:chemistry / Q000378:metabolism / Q000502:physiology*; D008856:Microscopy, Fluorescence; D009419:Nerve Tissue Proteins / Q000737:chemistry / Q000378:metabolism; D016716:PC12 Cells; D011233:Precipitin Tests; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D050765:Qa-SNARE Proteins; D050683:R-SNARE Proteins; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D011994:Recombinant Proteins / Q000378:metabolism; D050600:SNARE Proteins; D022142:Secretory Vesicles / Q000378:metabolism*; D013347:Subcellular Fractions; D050825:Synaptosomal-Associated Protein 25; D050827:Syntaxin 1; D033921:Vesicular Transport Proteins
D000954:Antigens, Surface; D008565:Membrane Proteins; D009419:Nerve Tissue Proteins; D050765:Qa-SNARE Proteins; D050683:R-SNARE Proteins; D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins; D050600:SNARE Proteins; C494210:Snap25 protein, rat; C494213:Stx1a protein, rat; D050825:Synaptosomal-Associated Protein 25; D050827:Syntaxin 1; D033921:Vesicular Transport Proteins