Homotypic fusion of immature secretory granules during maturation requires syntaxin 6

Abstract

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.

Figure 1

BotNT treatment does not inhibit ISG–ISG fusion. PC2 ISGs were treated with 20 nM BotNT A, C, D, or C and D, or A, C, and D for 30 min at 37°C in the presence of 0.5 mM ATP. (A) Pretreated or untreated ISGs were used in a fusion assay containing [³⁵S]-PC12 ISGs, as described in MATERIALS AND METHODS. Control ISGs were incubated in buffer alone. The extent of fusion is compared with controls and represents the signal obtained after subtraction of the background signal obtained in the absence of ISGs containing PC2. (B) Aliquots of untreated or treated ISG were solubilized and were subjected to SDS-PAGE and immunoblotting with the indicated antibodies. As expected, BotNT C cleaved syntaxin 1 (Synt 1) and BotNT D cleaved VAMP2. SNAP-25 was resistant to cleavage by BotNT A or C.

Figure 2

Codistribution of syntaxin 6 with a marker of ISGs but not MSGs. The distribution of endogenous SgII (A and E), p18 (B and H), and syntaxin 6 (D and G) in PC12/PC2 cells is shown by double labeling in confocal images, with the use of the anti-SgII antibody 175, the anti-p18 antibody 6B1/3, and anti-syntaxin 6 antibodies (D, mAb; G, polyclonal antibody), respectively. Superimposed images (C, F, and I) demonstrate the overlapping distribution of the syntaxin 6 only with SgII in TGN and ISGs. Full-length SgII, recognized by 175, appears mainly in the perinuclear region containing Golgi membranes and early ISGs (A), whereas p18 staining is found mainly distributed in maturing ISGs and MSGs distributed throughout the cytoplasm (B). Syntaxin 6 immunostaining can be found in the Golgi apparatus (D), where it colocalizes with SgII (E and F). In addition to the perinuclear Golgi staining, a punctate syntaxin 6 staining appears over the entire cell (D and G), which does not colocalize with p18 (H and I). Selected images from three independent experiments are shown.

Figure 3

Syntaxin 6 is present on ISGs from PC12 cells. PC12 ISGs and MSGs were isolated by subcellular fractionation involving two sequential sucrose gradients, as detailed in MATERIALS AND METHODS. (A) MSGs and ISGs were solubilized, subjected to SDS-PAGE, and immunoblotted with antibodies to SgII, syntaxin 6, and VAMP4. The number of MSGs and ISGs used were normalized to contain the same amount of the content protein SgII. (B) ISGs were subjected to immunoisolation with the use of empty beads (lane 1) or anti-syntaxin 6 beads (lane 2). The ISGs bound to the beads, and 1/10 the starting material (lane 3) was solubilized, subjected to SDS-PAGE, and subjected to immunoblotting with the antibodies indicated.

Figure 4

Antibodies to syntaxin 6, but not SNAP-25, SNAP-29, or VAMP4, inhibit ISG–ISG fusion. A standard fusion assay was performed with PC2 ISGs and [³⁵S]-sulfate-labeled ISGs. Both ISG populations were preincubated with (A) monoclonal anti-syntaxin 6 or syntaxin 1, or (B) SNAP-25 (line a), SNAP-29 (line b), or VAMP4 (line c) then were supplemented with cytosol and nucleotides. In (B) 5 μl and 10 μl (1× and 2×, respectively) of monoclonal SNAP-25, 10 μl and 20 μl of polyclonal SNAP-29 (1× and 2×, respectively), and 10 μl and 20 μl (1× and 2×, respectively) of affinity-purified polyclonal VAMP4 (0.2 μg/μl) antibodies were used. In (line b) the controls shown are done in the presence 20 μl of nonspecific preimmune sera. Fusion was assayed by determining the amount of p18 produced, as detailed in MATERIALS AND METHODS, and was quantitated as shown in (A) or as shown in (B). For quantitation, the signals used are those obtained after subtraction of the background obtained in the absence of PC2 ISGs. In (A), a representative experiment from a total of three independent experiments, performed in duplicate, is shown. The experiments in (B) were repeated two times (lines a and b) and three times (line c).

Figure 5

Syntaxin 6 is in a SNARE complex with SNAP-25 as well as SNAP-29/GS32, and both are regulated by NSF and α-SNAP. (A) ISG fractions (100 μg of protein) were subjected to immunoblotting with the use of SNAP-23, SNAP-25, or SNAP-29/GS32 antibodies. (B) Detergent-solubilized ISG membrane fractions were subjected to immunoprecipitation with the use of monoclonal antibodies against SNAP-25, syntaxin 6, or a nonspecific mAb as a control. Immunoprecipitates were analyzed by SDS-PAGE and immunoblotting with polyclonal antibodies to syntaxin 6 or SNAP-29/GS32, respectively. Immunoblotting with SNAP-29/GS32 antibody revealed that SNAP-29/GS32 was detected in the syntaxin 6 immunoprecipitates. An unspecific band was detected in both the control and syntaxin 6 immunoprecipitations (*). SUP corresponds to the supernatant left after the immunoprecipiatation reaction. (C) Detergent-solubilized ISG membrane fractions (100 μg) were incubated for 30 min at 4°C with recombinant NSF (2 μg) and α-SNAP (2 μg) and EDTA/ATP or Mg/ATP to provide assembly or disassembly conditions, respectively, followed by immunoprecipitation with syntaxin 6 antibodies. Immunoprecipitates were analyzed by SDS-PAGE and immunoblotting with the use of antibodies against SNAP-25 or SNAP-29/GS32, respectively. (D) Detergent solubilized ISGs (750 μg) were incubated with anti-syntaxin 6 magnetic beads, or magnetic beads alone. Immunoprecipitates were analyzed by SDS-PAGE and immunoblotting with polyclonal antibodies to VAMP4.

Figure 6

Anti-syntaxin 6 antibodies inhibit ISG–ISG fusion if added to only one population of ISGs. Anti-syntaxin 6 antibodies (concentration, 0.25 μg/μl) were added to the complete incubation on ice (complete + syn 6 ab). Alternatively, the PC2 ISGs, the PC12 ISGs, or both were preincubated with anti-syntaxin 6 antibodies, subjected to centrifugation, resuspended, and supplemented with the required components for fusion. A standard fusion assay was performed, and the amount of p18 produced was quantitated, as detailed in MATERIALS AND METHODS. A representative experiment performed in duplicate from three independent experiments is shown.

Figure 7

The schematic representation of gel filtration analysis of syntaxin 6, syntaxin 4, and SNAP-25. The soluble recombinant (A) syntaxin 6, (B) syntaxin 4, or (C) full-length SNAP-25 proteins were subjected to gel filtration with the use of a Superdex 200 column. The fractions shown correspond to fraction nos. 60 through 120, with dextran blue eluting at fraction no. 32. For calibration of the gel filtration column, ribonuclease A (13.7 kDa), ovalbumin (43 kDa), and aldolase (158 kDa) were used as size standards.