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. Exocyst subunits are essential for cell viability; and mutations or variants in several exocyst subunits have been implicated in human diseases, mostly neurodevelopmental disorders and ciliopathies. These conditions often share common features such as developmental delay, intellectual disability, and brain abnormalities. In this review, we summarize the mutations and variants in exocyst subunits that have been linked to disease and discuss the implications of exocyst dysfunction in other disorders.

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. We discovered that, although all Nups display uniformly slow turnover, their exchange rates vary considerably. Surprisingly, this exchange rate was relatively unrelated to each Nup's position, accessibility, or role in transport but correlated with its structural role; scaffold-forming Nups exchange slowly, whereas flexible connector Nups threading throughout the NPC architecture exchange more rapidly. Targeted perturbations in the NPC structure revealed a dynamic resilience to damage. Our approach opens a new window into macromolecular assembly 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. Yeast two-hybrid and subsequent in silico structural prediction uncovered a specific interaction between Rab-E and myosin XI that is conserved between P. patens and A. thaliana. Rab-E co-localises with myosin XI at sites of active exocytosis, and at the growing tip both proteins are spatiotemporally coupled. Rab-E is required for normal plant growth in P. patens and the rab-E and myosin XI phenotypes are rescued by A. thaliana's Rab-E1c and myosin XI-K/E, respectively. Both PpMyoXI and AtMyoXI-K interact with PpRabE14, and the interaction is specifically mediated by PpMyoXI residue V1422. This interaction is required for polarised growth. Our results suggest that the interaction of Rab-E and myosin XI is a conserved feature of polarised growth in plants.

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. Here, we determine the molecular architecture of the yeast exocyst complex by an integrative approach, based on a 3D density map from negative-stain electron microscopy (EM) at ~16 Å resolution, 434 disuccinimidyl suberate and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride cross-links from chemical-crosslinking mass spectrometry, and partial atomic models of the eight subunits. The integrative structure is validated by a previously determined cryo-EM structure, cross-links, and distances from in vivo fluorescence microscopy. Our subunit configuration is consistent with the cryo-EM structure, except for Sec5. While not observed in the cryo-EM map, the integrative model localizes the N-terminal half of Sec3 near the Sec6 subunit. Limited proteolysis experiments suggest that the conformation of Exo70 is dynamic, which may have functional implications for SNARE and membrane interactions. This study illustrates how integrative modeling based on varied low-resolution structural data can inform biologically relevant hypotheses, even in the absence of high-resolution data.

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. Importantly, the assay is stimulated by gain-of-function mutations in the Exo70 component of the exocyst, selected for their ability to bypass Rho/Cdc42 activation in vivo. Single-particle electron microscopy and 3D reconstructions of negatively stained exocyst complexes reveal a structural change in the mutant exocyst that exposes a binding site for the v-SNARE. We demonstrate a v-SNARE requirement in our tethering assay and increased v-SNARE binding to exocyst gain-of-function complexes. Together, these data suggest an allosteric mechanism for activation involving a conformational change in one subunit of the complex, which is relayed through the complex to regulate its biochemical activity in vitro, as well as overall function in vivo.

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. These studies now pose more questions than answers for understanding fundamental functional mechanisms, and they open wide the door for future studies to elucidate interactions with protein and membrane partners, potential conformational changes, and molecular insights into tethering reactions.

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. Vesicles contain a variety of cargos, including lipids, membrane proteins, signaling molecules, biosynthetic and hydrolytic enzymes, and the trafficking machinery itself. Proper function of membrane trafficking is required for cellular growth, division, movement, and cell-cell communication. Defects in these processes have been implicated in a variety of human diseases, such as cancer, diabetes, neurodegenerative disorders, ciliopathies, and infections. The elucidation of the mechanisms of SNARE assembly and disassembly is key to understanding how membrane fusion is regulated throughout eukaryotes. Here, we introduce the SNARE proteins, their structures and functions in eukaryotic cells, and discuss recent breakthroughs in elucidating the regulation of SNARE assembly and disassembly through the use of high-resolution structural biology and biophysical techniques.

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. However, insights from the nuclear assembly of large viral capsids highlight a back door route for nuclear escape, a process referred to nuclear envelope (NE) budding. Recent studies indicate that NE budding might be an endogenous cellular process for the nuclear export of very large RNPs and protein aggregates. In Drosophila, this mechanism is required for synaptic plasticity, but its role may extend beyond the nervous system, in tissues where local changes in translation are required. Here we discuss these recent findings and a potential relationship between NE budding and the NPC.

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.P468L) mutation in VPS45, critical regulator of SNARE-dependent membrane fusion. Structural modeling indicates that P468, like the T224 and E238 residues affected by previously reported mutations, cluster in a VPS45 "hinge" region, indicating its critical role in membrane fusion and VPS45-associated SCN. Bone marrow transplantation, complicated by early graft failure rescued with stem cell boost, led to resolution of the hematopoietic phenotype.

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. We directly determined the composition of the Trypanosoma brucei exocyst by affinity isolation and demonstrate that the parasite complex is nonameric, retaining all eight canonical subunits (albeit highly divergent at the sequence level) plus a novel essential subunit, Exo99. Exo99 and Sec15 knockdowns have remarkably similar phenotypes in terms of viability and impact on morphology and trafficking pathways. Significantly, both Sec15 and Exo99 have a clear function in endocytosis, and global proteomic analysis indicates an important role in maintaining the surface proteome. Taken together these data indicate additional exocyst functions in trypanosomes, which likely include endocytosis, recycling and control of surface composition. Knockdowns in HeLa cells suggest that the role in endocytosis is shared with metazoan cells. We conclude that, whilst the trypanosome exocyst has novel components, overall functionality appears conserved, and suggest that the unique subunit may provide therapeutic opportunities.

The Secret Life of Tethers: The Role of Tethering Factors in SNARE Complex Regulation.

Trafficking in eukaryotic cells is a tightly regulated process to ensure correct cargo delivery to the proper destination organelle or plasma membrane. In this review, we focus on how the vesicle fusion machinery, the SNARE complex, is regulated by the interplay of the multisubunit tethering complexes (MTC) with the SNAREs and Sec1/Munc18 (SM) proteins. Although these factors are used in different stages of membrane trafficking, e.g., Golgi to plasma membrane transport vs. vacuolar fusion, and in a variety of diverse eukaryotic cell types, many commonalities between their functions are being revealed. We explore the various protein-protein interactions and findings from functional reconstitution studies in order to highlight both their common features and the differences in their modes of regulation. These studies serve as a starting point for mechanistic explorations in other systems.

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. Furthermore, we visualized the structure of the yeast complex by using negative-stain electron microscopy; our results indicate that the exocyst exists predominantly as a stable, octameric complex with an elongated architecture that suggests that the subunits are contiguous helical bundles packed together into a bundle of long rods.

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. Recent advances in single-molecule and high-resolution structural methods provide exciting new insights into how SNARE complexes assemble, including measurements of assembly energetics and identification of intermediates in the assembly pathway. These techniques were also key in elucidating mechanistic details into how the SNARE complex is disassembled, including details of the energetics required for ATP-dependent α-SNAP/NSF-mediated SNARE complex disassembly, and the structural changes that accompany ATP hydrolysis by the disassembly machinery. Additionally, SNARE complex formation and disassembly are tightly regulated processes; innovative biochemical and biophysical characterization has deepened our understanding of how these regulators work to control membrane fusion and exocytosis.