Structure of synaptophysin: a hexameric MARVEL-domain channel protein

Abstract

Synaptophysin I (SypI) is an archetypal member of the MARVEL-domain family of integral membrane proteins and one of the first synaptic vesicle proteins to be identified and cloned. Most all MARVEL-domain proteins are involved in membrane apposition and vesicle-trafficking events, but their precise role in these processes is unclear. We have purified mammalian SypI and determined its three-dimensional (3D) structure by using electron microscopy and single-particle 3D reconstruction. The hexameric structure resembles an open basket with a large pore and tenuous interactions within the cytosolic domain. The structure suggests a model for Synaptophysin's role in fusion and recycling that is regulated by known interactions with the SNARE machinery. This 3D structure of a MARVEL-domain protein provides a structural foundation for understanding the role of these important proteins in a variety of biological processes.

Figure 1. Comparison of transmembrane domains of synaptophysin family proteins and sequence alignment of Synaptophysin and Connexin

(A) Sequences are taken from the following: mouse synaptophysin I (Syph), mouse synaptoporin (Synpr), mouse synaptophysin-like protein 1 (Sypl1) and mouse synaptophysin-like protein 2 (Sypl2). These proteins are known to reside on synaptic vesicles and may account for the lack of phenotype seen when individual members of this family are removed. (B) Sequence alignment of the third transmembrane segment of Syp1 (SyphTM3) and the third transmembrane segment of connexin α 1 (ConxTM3).

Figure 2. Single particle electron microscopy of negatively stained synaptophysin I

(A) Low dose image of synaptophysin I complexes negatively stained with 2% ammonium molybdate on continuous carbon substrate. (Inset) magnified image of single synaptophysin I complex (scale bar = 100 nm). (B) Gallery of representative class averages displaying various views of the synaptophysin I complex. (C) Three views of the surface rendered density map of the synaptophysin I complex rendered at 20 Å resolution. The structure shows an overall diameter of 70 Å with an inner diameter of 30 Å. Density is contoured to 2.0 σ. (D) Mesh view of (C) with connexin docked into the density map. Highlighted area represents the lipid bilayer. (E) Stereo mesh view of (D) truncated to show the inner wall of the complex. The atomic model of connexin is docked into the density map with the third transmembrane domain represented in green. Highlighted area represents the lipid bilayer.

Figure 3. Chromatogram from gel filtration purification of synaptophysin I complex. Resolution analysis of synaptophysin I three-dimensional reconstruction and models of 5-fold and 7-fold imposed symmetry. Resolution analysis of synaptophysin I three-dimensional reconstruction and models of 5-fold and 7-fold imposed symmetry

(A) Chromatogram from gel filtration purification of synaptophysin I complex. Blue curve shows calibration standards for the purification column run under synaptophysin I isolation conditions. Arrows point to peaks representing 670 kDa and 160 kDa. Red curve shows gel filtration of solubilized synaptophysin I complex isolated from bovine brain tissue. Arrow points to the peak representing the 240 kDa synaptophysin I complex as verified by SDS-PAGE and estern blot analysis. (B) SDS-PAGE analysis of (L–R) purified synaptic vesicles, final purified synaptophysin I complex, and western blot analysis of the purified complex. Arrows show marked molecular weights. (C) Fourier shell correlation curve showing the calculated resolution of the reconstruction to be approximately 20 Å based on the FSC = 0.5 criterion. (D) Reconstruction of the same single particle data as Fig. 2 with 5-fold symmetry imposed. Density is contoured to 0.5 σ.

Figure 4. Comparison of the synaptophysin I complex with other known channel structures

(A) The synaptophysin I complex shows a similar structure and conductance to both connexin and mechanosensitive channels. Synaptophysin I has an outer diameter of 70 Å and a conductance of 0.4 nS. (B) Connexin (PDB-ID 1TXH) has an outer diameter of 70 Å and a conductance of 0.3 nS. (C) The small mechanosensitive channel MscS (PDB-ID 2OAU) has an outer diameter of 80 Å and a conductance of 1.0 nS. (D) The large mechanosensitive channel MscL (PDB-ID 2OAR) has an outer diameter of 50 Å and a conductance of 3.8 nS.

Figure 5. Model for Synaptophysin I (Syp1) complex involvement in vesicle fusion

Vesicles containing unassembled SNARE complexes approach the active zone of the pre-synaptic matrix. (A) Vesicles become “docked” through of t/v-SNARE interaction (step 1). Vesicle then proceed down one of two pathways: Full-fusion (FF) or Kiss-and-Run (KNR). Along the FF pathway SytI is the principal calcium sensor. Upon calcium influx, SytI binds to the SNARE complex and both C2 domains of SytI bind calcium and form a tight electrostatic interaction with the pre-synaptic membrane (E & F) which pulls the vesicle into pre-synaptic membrane. The SypI complex then binds with Syx on the plasma membrane and forms a fusion pore (step 2). The strong interaction of SytI with the pre-synaptic membrane and the tight formation of the SNARE complex disassociates Syb from Syp1 thus weaking the Syp1 complex and allowing the vesicle to fully fuse and dissociate the Syp1 hexamer (step 3). E shows a top view of the interaction of the SNARE complex and the dislodging of Syb from the Syp1 complex. In the KNR pathway SytIV is the principal calcium sensor and a weak interaction between SytIV and Syb occurs excluding SytI from interacting with Syb (B & C). Upon calcium influx only the C2B domain of SytIV binds calcium. This results in a weaker electrostatic interaction between SytIV and the pre-synaptic membrane (step 2*). The weak interaction of SytIV with the pre-synaptic membrane and the exclusion of Syb from forming a complete SNARE complex allows Syb to remain bound to the Syp1 complex and inhibits the vesicle from fully fusing (step 3*). B shows a top view of the interaction of Syb with Syx and how Syb remains bound to the Syp1 complex during transient fusion.