Exocyst stimulates multiple steps of exocytic SNARE complex assembly and vesicle fusion.

Exocyst is a large multisubunit tethering complex essential for targeting and fusion of secretory vesicles in eukaryotic cells. Although the assembled exocyst complex has been proposed to tether vesicles to the plasma membrane and activate soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs) for membrane fusion, the key biochemical steps that exocyst stimulates in SNARE-mediated fusion are undetermined. Here we use a combination of single-molecule and bulk fluorescence assays to investigate the roles of purified octameric yeast exocyst complexes in a reconstituted yeast exocytic SNARE assembly and vesicle fusion system.

Dissecting the Structural Dynamics of the Nuclear Pore Complex.

Cellular processes are largely carried out by macromolecular assemblies, most of which are dynamic, having components that are in constant flux. One such assembly is the nuclear pore complex (NPC), an ∼50 MDa assembly comprised of ∼30 different proteins called Nups that mediates selective macromolecular transport between the nucleus and cytoplasm. We developed a proteomics method to provide a comprehensive picture of the yeast NPC component dynamics.

Exocyst structural changes associated with activation of tethering downstream of Rho/Cdc42 GTPases.

The exocyst complex plays a critical role in determining both temporal and spatial dynamics of exocytic vesicle tethering and fusion with the plasma membrane. However, the mechanism by which the exocyst functions and how it is regulated remain poorly understood. Here we describe a novel biochemical assay for the examination of exocyst function in vesicle tethering.

Exposing the Elusive Exocyst Structure.

A major challenge for a molecular understanding of membrane trafficking has been the elucidation of high-resolution structures of large, multisubunit tethering complexes that spatially and temporally control intracellular membrane fusion. Exocyst is a large hetero-octameric protein complex proposed to tether secretory vesicles at the plasma membrane to provide quality control of soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE)-mediated membrane fusion. Breakthroughs in methodologies, including sample preparation, biochemical characterization, fluorescence microscopy, and single-particle cryoelectron microscopy, are providing critical insights into the structure and function of the exocyst.

Cell cycle-dependent phosphorylation of Sec4p controls membrane deposition during cytokinesis.

Intracellular trafficking is an essential and conserved eukaryotic process. Rab GTPases are a family of proteins that regulate and provide specificity for discrete membrane trafficking steps by harnessing a nucleotide-bound cycle. Global proteomic screens have revealed many Rab GTPases as phosphoproteins, but the effects of this modification are not well understood.