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

Sophie M Travis,Kevin Damico,I-Mei Yu,Conor Mcmahon,Safraz Hamid,Gabriel Ramirez-Arellano,Philip D Jeffrey,Frederick M Hughson

doi:10.1074/jbc.ra120.013654

Abstract

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.

Highlights

Eukaryotic cells use vesicles to transport cargo between organelles and to the plasma membrane for exocytosis
The S. cerevisiae Dsl1 complex subunit Tip20 was discovered as a cytoplasmic protein that interacts with the cytoplasmic domain of the endoplasmic reticulum (ER) Qb-sensitive factor attachment protein receptor (SNARE) Sec20 [40]
We initially conducted crystallization screens using both full-length S. cerevisiae Tip20 and an N-terminally truncated version in combination with various constructs representing the N-terminal region of Sec20

Summary

Introduction

Eukaryotic cells use vesicles to transport cargo between organelles and to the plasma membrane for exocytosis. Transmembrane anchors and adjacent SNARE motifs, most SNAREs contain N-terminal regions that can regulate SNARE assembly and/or interact with other proteins. The largest family of MTCs is the CATCHR (complexes associated with tethering containing helical rods) family, whose members function largely in trafficking to and from the Golgi [19, 26, 27]. The CATCHR-family complexes—GARP/EARP, exocyst, conserved oligomeric Golgi, and Dsl1—contain 3–8 subunits each Many of these subunits share a similar tertiary structure, the so-called CATCHR fold, comprising a series of helical bundles [28]. With the exception of the exocyst complex, the CATCHR complexes are multilegged and flexible [20, 30, 31] These attributes seem well-suited for roles in vesicle docking and SNARE complex assembly

Methods

Results

Conclusion