Promiscuous interaction of SNAP-25 with all plasma membrane syntaxins in a neuroendocrine cell

Abstract

SNAP-25 (25 kDa synaptosome-associated protein) is found in cells that release neurotransmitters and hormones, and plays a central role in the fusion of secretory vesicles with the plasma membrane. SNAP-25 has been shown to interact specifically with syntaxin 1, a 35 kDa membrane protein, to mediate the fusion process. Here, we investigated whether other known syntaxin isoforms found at the plasma membrane can serve as binding partners for SNAP-25 in vivo. In our analysis, we employed rat phaeochromocytoma PC12 cells that are often used as a model of neuronal functions. We now show that these cells contain large amounts of SNAP-25, which interacts not only with syntaxin 1, but also with ubiquitous syntaxins 2, 3 and 4. The plasma membrane syntaxins appear to occupy complementary domains at the plasma membrane. In defined reactions, the ubiquitous plasma membrane syntaxin isoforms, when in binary complexes with SNAP-25, readily bound vesicular synaptobrevin to form SDS-resistant SNARE (soluble N-ethylmaleimide-sensitive fusion protein attachment protein receptor) complexes implicated in membrane fusion. However, vesicular synaptotagmin and cytosolic complexin, both implicated in the fusion process, exhibited differential ability to interact with the SNARE complexes formed by syntaxins 1-4, suggesting that the plasma membrane syntaxins may mediate vesicle fusion events with different properties.

Figure 1. Neuroendocrine PC12 cells display the full complement of plasma membrane syntaxins as well as enrichment of SNAP-25, in comparison with mature chromaffin cells from the adrenal gland

A Western immunoblot using anti-SNAP-25 and anti-syntaxin (Syx) antibodies of chromaffin cells (CC), and six tumour cell lines (PC12, HeLa, Raji, MCF, COS7 and Jurkat) is shown. Normalization of total cell protein (30 μg per lane) was verified by Coomassie staining (CS). Positions of molecular-mass markers (in kDa) are shown on the right of the gels.

Figure 2. Distribution of endogenous syntaxins 1–4, SNAP-25 and SNAP-23 in PC12 cells

(A) Cells were co-immunostained using mouse anti-SNAP-25 and rabbit anti-syntaxin-1–4 (Syx1–Syx4) (B) Co-immunostaining of PC12 cells using mouse anti-SNAP-25 and rabbit SNAP-23. (C) Cells were co-immunostained using HPC-1 monoclonal anti-syntaxin-1 antibody and either anti-SNAP-23 or anti-SNAP-25 antibodies. Scale bar, 10 μm.

Figure 3. Syntaxins 1–4 can occupy distinct patches of the plasma membrane of PC12 cells

Cells were co-immunostained using mouse anti-syntaxin-1 (Syx1) and rabbit anti-syntaxin-2–4 (Syx2–Syx4). The rectangular areas shown in the bottom panels in (A) are magnified in the respective panels in (B) [scale bar in (A), upper left panel, 10 μm; scale bars in (B), 1 μm].

Figure 4. SNAP-25 interacts in vivo with all four plasma membrane syntaxin isoforms

(A) Immunoprecipitation experiments of PC12 cell extracts using either control Sepharose beads or Sepharose beads with a covalently attached monoclonal anti-SNAP-25 antibody are shown. A fraction of the load (one-hundredth) and of the pull-down material (one-tenth) was probed using rabbit antibodies against the indicated proteins. (B) MS analysis of the anti-SNAP-25 pull-down material reveals partial peptide coverage for all four plasma membrane syntaxin isoforms.

Figure 5. Syntaxins 3 and 4 are present in the brain, and are capable of in vitro interaction with both SNAP-25 and synaptobrevin 2

(A) Western immunoblot analysis of rat internal organs and different parts of brain. Protein normalization (30 μg per lane) was verified by Coomassie staining (CS). (B) Recombinant syntaxins 3 and 4 form SDS-resistant SNARE complexes upon addition of SNAP-25 and synaptobrevin 2 (syb2) in a 30 min reaction at 24 °C. Coomassie stained gel. (C) Syntaxins 3 and 4 (Syx3 and Syx4) are as capable of direct binding to SNAP-25 as syntaxin 1. Immobilized GST fusion proteins of syntaxins were incubated with SNAP-25 for 30 min and, after extensive washing, were analysed by SDS/PAGE and Sypro Orange staining. A control reaction was performed using glutathione–Sepharose beads without GST–syntaxin, but in the presence of SNAP-25.

Figure 6. SNARE complexes formed by syntaxins 1–4, SNAP-25 and synaptobrevin 2 differentially interact with complexin and synaptotagmin 1

SNAP-25 and synaptobrevin 2 were pre-bound to GST–syntaxin (GST-Syx) beads (upper panel; Coomassie-stained gel). The four SNARE complexes were incubated with rat brain Triton X-100 extract for 1 h at 4 °C. Following washing, the SNARE complexes were immunoblotted using anti-complexin (Cxn) or anti-synaptotagmin-1 (Syt1) antibodies (lower two panels). Syb2, synaptobrevin 2.