Structure of a membrane tethering complex incorporating multiple SNAREs.

Most membrane fusion reactions in eukaryotic cells are mediated by multisubunit tethering complexes (MTCs) and SNARE proteins. MTCs are much larger than SNAREs and are thought to mediate the initial attachment of two membranes. Complementary SNAREs then form membrane-bridging complexes whose assembly draws the membranes together for fusion. Here we present a cryo-electron microscopy structure of the simplest known MTC, the 255-kDa Dsl1 complex of Saccharomyces cerevisiae, bound to the two SNAREs that anchor it to the endoplasmic reticulum. N-terminal domains of the SNAREs form an integral part of the structure, stabilizing a Dsl1 complex configuration with unexpected similarities to the 850-kDa exocyst MTC. The structure of the SNARE-anchored Dsl1 complex and its comparison with exocyst reveal what are likely to be common principles underlying MTC function. Our structure also implies that tethers and SNAREs can work together as a single integrated machine.

Structure of a Membrane Tethering Complex Incorporating Multiple SNAREs.

Most membrane fusion reactions in eukaryotic cells are mediated by membrane tethering complexes (MTCs) and SNARE proteins. MTCs are much larger than SNAREs and are thought to mediate the initial attachment of two membranes. Complementary SNAREs then form membrane-bridging complexes whose assembly draws the membranes together for fusion. Here, we present a cryo-EM structure of the simplest known MTC, the 255-kDa Dsl1 complex, bound to the two SNAREs that anchor it to the endoplasmic reticulum. N-terminal domains of the SNAREs form an integral part of the structure, stabilizing a Dsl1 complex configuration with remarkable and unexpected similarities to the 850-kDa exocyst MTC. The structure of the SNARE-anchored Dsl1 complex and its comparison with exocyst reveal what are likely to be common principles underlying MTC function. Our structure also implies that tethers and SNAREs can work together as a single integrated machine.

Structural basis for the binding of SNAREs to the multisubunit tethering complex Dsl1.

Multisubunit-tethering complexes (MTCs) are large (250 to >750 kDa), conserved macromolecular machines that are essential for soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE)-mediated membrane fusion in all eukaryotes. MTCs are thought to organize membrane trafficking by mediating the initial long-range interaction between a vesicle and its target membrane and promoting the formation of membrane-bridging SNARE complexes. Previously, we reported the structure of the yeast Dsl1 complex, the simplest known MTC, which is essential for coat protein I (COPI) mediated transport from the Golgi to the endoplasmic reticulum (ER). This structure suggests how the Dsl1 complex might tether a vesicle to its target membrane by binding at one end to the COPI coat and at the other to ER-associated SNAREs. Here, we used X-ray crystallography to investigate these Dsl1-SNARE interactions in greater detail. The Dsl1 complex comprises three subunits that together form a two-legged structure with a central hinge. We found that distal regions of each leg bind N-terminal Habc domains of the ER SNAREs Sec20 (a Qb-SNARE) and Use1 (a Qc-SNARE). The observed binding modes appear to anchor the Dsl1 complex to the ER target membrane while simultaneously ensuring that both SNAREs are in open conformations, with their SNARE motifs available for assembly. The proximity of the two SNARE motifs, and therefore their ability to enter the same SNARE complex, will depend on the relative orientation of the two Dsl1 legs. These results underscore the critical roles of SNARE N-terminal domains in mediating interactions with other elements of the vesicle docking and fusion machinery.

CTCF-Induced Circular DNA Complexes Observed by Atomic Force Microscopy.

The CTCF protein has emerged as a key architectural protein involved in genome organization. Although hypothesized to initiate DNA looping, direct evidence of CTCF-induced DNA loop formation is still missing. Several studies have shown that the 11 zinc finger (11 ZF) domain of CTCF is actively involved in DNA binding. We here use atomic force microscopy to examine the effect of the 11 ZF domain comprising residues 266-579 (11 ZF CTCF) and the 3 ZF domain comprising residues 402-494 (6-8 ZF CTCF) of human CTCF on the DNA morphology. Our results show that both domains alter the DNA architecture from the relaxed morphology observed in control DNA samples to compact circular complexes, meshes, and networks, offering important insights into the multivalent character of the 11 ZF CTCF domain. Atomic force microscopy images reveal quasi-circular DNA/CTCF complexes, which are destabilized upon replacing the 11 ZF CTCF by the 6-8 ZF CTCF domain, highlighting the role of the 11 ZF motif in loop formation. Intriguingly, the formation of circular DNA/CTCF complexes is dominated by non-specific binding, whereby contour length and height profiles suggest a single DNA molecule twice wrapped around the protein.