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.

The exocyst complex in neurological disorders.

Exocytosis is the process by which secretory vesicles fuse with the plasma membrane to deliver materials to the cell surface or to release cargoes to the extracellular space. The exocyst-an evolutionarily conserved octameric protein complex-mediates spatiotemporal control of SNARE complex assembly for vesicle fusion and tethering the secretory vesicles to the plasma membrane. The exocyst participates in diverse cellular functions, including protein trafficking to the plasma membrane, membrane extension, cell polarity, neurite outgrowth, ciliogenesis, cytokinesis, cell migration, autophagy, host defense, and tumorigenesis.

Approaches to address bias in letters of recommendation.

Letters of recommendation are ubiquitous in the research enterprise. Requesting, writing, and reviewing letters of recommendation are all fraught with bias, especially for individuals from groups historically excluded from research environments. We detail how letter reviewers, requesters, and writers can make letters of recommendation a more equitable tool to evaluate scientists.

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.

Rab-E and its interaction with myosin XI are essential for polarised cell growth.

The fundamental process of polarised exocytosis requires the interconnected activity of molecular motors trafficking vesicular cargo within a dynamic cytoskeletal network. In plants, few mechanistic details are known about how molecular motors, such as myosin XI, associate with their secretory cargo to support the ubiquitous processes of polarised growth and cell division. Live-cell imaging coupled with targeted gene knockouts and a high-throughput RNAi assay enabled the first characterisation of the loss of Rab-E function.

Retro Is Cool: Structure of the Versatile Retromer Complex.

In this issue of Structure, Kendall et al. (2020) reveal the cryo-EM structure of the mammalian retromer complex, which is essential in sorting membrane proteins in endosomes. The retromer heterotrimer can oligomerize in multiple conformations; this versatility is promoted by a flexible interface of electrostatic residues on the VPS35 subunit.

Integrative structure and function of the yeast exocyst complex.

Exocyst is an evolutionarily conserved hetero-octameric tethering complex that plays a variety of roles in membrane trafficking, including exocytosis, endocytosis, autophagy, cell polarization, cytokinesis, pathogen invasion, and metastasis. Exocyst serves as a platform for interactions between the Rab, Rho, and Ral small GTPases, SNARE proteins, and Sec1/Munc18 regulators that coordinate spatial and temporal fidelity of membrane fusion. However, its mechanism is poorly described at the molecular level.

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.

SNARE complex assembly and disassembly.

A fundamental hallmark of eukaryotic cells is their compartmentalization into functionally distinct organelles, including those of the secretory and endocytic pathways. Transport of cargo between these compartments and to/from the cell surface is mediated by membrane-bound vesicles and tubules. Delivery of cargo is facilitated by SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor)-mediated membrane fusion of vesicles with their target compartments.

Capturing endosomal vesicles at the Golgi.

Membrane trafficking specificity between distinct compartments ensures that cargo proteins and lipids are delivered to their target organelle. However, accurate recognition of cargo carriers by tethering factors on target membranes is poorly understood. TBC1D23 is now identified as an adaptor that links endosome-derived vesicles with golgins at the trans-Golgi.

Getting mRNA-Containing Ribonucleoprotein Granules Out of a Nuclear Back Door.

A pivotal feature of long-lasting synaptic plasticity is the localization of RNAs and the protein synthesis machinery at synaptic sites. How and where ribonucleoprotein (RNP) transport granules that support this synthetic activity are formed is of fundamental importance. The prevailing model poses that the nuclear pore complex (NPC) is the sole gatekeeper for transit of cellular material in and out of the nucleus.

A novel homozygous VPS45 p.P468L mutation leading to severe congenital neutropenia with myelofibrosis.

VPS45-associated severe congenital neutropenia (SCN) is a rare disorder characterized by life-threating infections, neutropenia, neutrophil and platelet dysfunction, poor response to filgrastim, and myelofibrosis with extramedullary hematopoiesis. We present a patient with SCN due to a homozygous c.1403C>T (p.

The Trypanosome Exocyst: A Conserved Structure Revealing a New Role in Endocytosis.

Membrane transport is an essential component of pathogenesis for most infectious organisms. In African trypanosomes, transport to and from the plasma membrane is closely coupled to immune evasion and antigenic variation. In mammals and fungi an octameric exocyst complex mediates late steps in exocytosis, but comparative genomics suggested that trypanosomes retain only six canonical subunits, implying mechanistic divergence.

Subunit connectivity, assembly determinants and architecture of the yeast exocyst complex.

The exocyst is a hetero-octameric complex that has been proposed to serve as the tethering complex for exocytosis, although it remains poorly understood at the molecular level. Here, we purified endogenous exocyst complexes from Saccharomyces cerevisiae and showed that they are stable and consist of all eight subunits with equal stoichiometry. Using a combination of biochemical and auxin induced-degradation experiments in yeast, we mapped the subunit connectivity, identified two stable four-subunit modules within the octamer and demonstrated that several known exocyst-binding partners are not necessary for exocyst assembly and stability.

Three steps forward, two steps back: mechanistic insights into the assembly and disassembly of the SNARE complex.

Membrane fusion is a tightly controlled process in all eukaryotic cell types. The SNARE family of proteins is required for fusion throughout the exocytic and endocytic trafficking pathways. SNAREs on a transport vesicle interact with the cognate SNAREs on the target membrane, forming an incredibly stable SNARE complex that provides energy for the membranes to fuse, although many aspects of the mechanism remain elusive.

The Exocyst Subunit Sec6 Interacts with Assembled Exocytic SNARE Complexes.

In eukaryotic cells, membrane-bound vesicles carry cargo between intracellular compartments, to and from the cell surface, and into the extracellular environment. Many conserved families of proteins are required for properly localized vesicle fusion, including the multisubunit tethering complexes and the SNARE complexes. These protein complexes work together to promote proper vesicle fusion in intracellular trafficking pathways.

To protect or reject.

A protein known for its role in dismantling faulty SNARE complexes can also help to maintain complexes that have formed properly during membrane fusion.

Exorcising the exocyst complex.

The exocyst complex is an evolutionarily conserved multisubunit protein complex implicated in tethering secretory vesicles to the plasma membrane. Originally identified two decades ago in budding yeast, investigations using several different eukaryotic systems have since made great progress toward determination of the overall structure and organization of the eight exocyst subunits. Studies point to a critical role for the complex as a spatiotemporal regulator through the numerous protein and lipid interactions of its subunits, although a molecular understanding of exocyst function has been challenging to elucidate.