Cooperative Functions of the Retrograde Vesicle Fusion Machinery

Abstract

N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs) mediate most intracellular membrane fusion events. SNAREs from apposed membranes interact to form membrane-bridging complexes that pull the membranes together, leading to lipid mixing and fusion. While SNAREs alone can drive membrane fusion, they are insufficient to fuse membranes at physiologically relevant speeds. To accelerate the process, membrane tethering complexes (MTC) mediate an initial attachment between a vesicle and a target membrane, and Sec1/Munc18 (SM) proteins accelerate the SNARE assembly step. How these families of proteins cooperate in vesicle capture and fusion, however, is poorly understood. Here, we present two structures of the Golgi-endoplasmic reticulum (ER) retrograde pathway fusion machinery. The first structure, the Saccharomyces cerevisiae Dsl1 MTC bound to the ER SNAREs Sec20 and Use1, is the first reported structure of a tethering complex engaged with other fusion machinery. Together, the trimeric Dsl1 complex and SNAREs form a rigid heteropentamer, the assembly of which is essential for yeast viability. This complex also bears unexpected similarities to the much-larger MTC exocyst, suggesting possible principles of MTC function. The second structure, the SM protein Sly1 bound to the ER SNARE Ufe1, constitutes the remainder of the ER-associated fusion machinery. The remaining Golgi-ER SNARE, Sec22, is embedded in and contributed by the Golgi-derived vesicle. Both the Dsl1 complex and Sly1 remain stably associated with SNAREs during SNARE assembly, resulting in an octameric supercomplex. Our data support a model in which the Dsl1 complex and Sly1 collaborate to localize SNAREs at the site of fusion and drive SNARE assembly and membrane fusion.