Sec24p and Sec16p cooperate to regulate the GTP cycle of the COPII coat

Vesicle budding from the endoplasmic reticulum (ER) employs a cycle of GTP binding and hydrolysis to regulate assembly of the COPII coat. We have identified a novel mutation (sec24‐m11) in the cargo‐binding subunit, Sec24p, that specifically impacts the GTP‐dependent generation of vesicles in vitro. Using a high‐throughput approach, we defined genetic interactions between sec24‐m11 and a variety of trafficking components of the early secretory pathway, including the candidate COPII regulators, Sed4p and Sec16p. We defined a fragment of Sec16p that markedly inhibits the Sec23p‐ and Sec31p‐stimulated GTPase activity of Sar1p, and demonstrated that the Sec24p‐m11 mutation diminished this inhibitory activity, likely by perturbing the interaction of Sec24p with Sec16p. The consequence of the heightened GTPase activity when Sec24p‐m11 is present is the generation of smaller vesicles, leading to accumulation of ER membranes and more stable ER exit sites. We propose that association of Sec24p with Sec16p creates a novel regulatory complex that retards the GTPase activity of the COPII coat to prevent premature vesicle scission, pointing to a fundamental role for GTP hydrolysis in vesicle release rather than in coat assembly/disassembly

Sec24p and Sec16p cooperate to regulate the GTP cycle of the COPII coat

Vesicle budding from the endoplasmic reticulum (ER) employs a cycle of GTP binding and hydrolysis to regulate assembly of the COPII coat. We have identified a novel mutation (sec24-m11) in the cargo-binding subunit, Sec24p, that specifically impacts the GTP-dependent generation of vesicles in vitro. Using a high-throughput approach, we defined genetic interactions between sec24-m11 and a variety of trafficking components of the early secretory pathway, including the candidate COPII regulators, Sed4p and Sec16p. We defined a fragment of Sec16p that markedly inhibits the Sec23p- and Sec31p-stimulated GTPase activity of Sar1p, and demonstrated that the Sec24p-m11 mutation diminished this inhibitory activity, likely by perturbing the interaction of Sec24p with Sec16p. The consequence of the heightened GTPase activity when Sec24p-m11 is present is the generation of smaller vesicles, leading to accumulation of ER membranes and more stable ER exit sites. We propose that association of Sec24p with Sec16p creates a novel regulatory complex that retards the GTPase activity of the COPII coat to prevent premature vesicle scission, pointing to a fundamental role for GTP hydrolysis in vesicle release rather than in coat assembly/disassembly

Deciphering the Role of p24 Proteins in COPII-mediated Protein Secretion

In eukaryotic cells, proteins continuously flux through the organelles of the secretory pathway in an essential cellular process called protein secretion. This dynamic process originates at the endoplasmic reticulum (ER), where translating ribosomes push linear peptides into the ER membrane and lumen. ER chaperones assist in folding nascent peptides into three-dimensional conformations and proteins are concentrated into membrane-encapsulated vesicles bound for the Golgi apparatus. ER to Golgi transport is mediated by a set of cytosolic coat proteins called COPII. The COPII coat polymerizes into a lattice on the ER membrane that is able to bend the membrane around secretory cargo and bud off a spherical vesicle. Protein secretion is subject to rapid changes as a cell responds to its environment and requirements for viability alter. In addition to accommodating short-term demands, such as translational up-regulation, evolved complexity of secretory proteins over time, has also required that secretory components adapt. In both cases changes in secretory demands require that the COPII proteins have an inherent flexibility to navigate these changes without disrupting secretory flux. In this work I have examined a family of quintessential secretory cargo, p24 proteins, that challenge protein secretion. This family of proteins forms a hetero-tetrameric complex that cycles between the ER and the Golgi and mediates transport of glycosylphosphatidylinositol-anchored proteins (GPI-APs). Here I present evidence that suggests, when present in vesicles, both p24 proteins and their GPI-AP cargo present a challenge to vesicle formation. I posit that three attributes of these proteins present a local barrier to membrane bending: Lumenal asymmetric distribution across the membrane, high cellular abundance and affinity for ceramide rich membranes. I have also elucidated mechanisms that the coat has evolved to accommodate troublesome cargo such as p24 proteins, which enhance structural scaffolding and increase average vesicle size. Finally I present preliminary findings indicating that p24s also contribute to ER homeostasis by preventing aberrant incorporation of proteins into vesicles. Comprehensively, these findings have shed light on the role of p24 proteins in vesicles. Traditionally thought to be canonical ER cargo receptors, these proteins also appear capable of contributing to the composition of the vesicles in which they reside, and impacting trafficking efficiency in two ways: First by directly mediating transport of GPI-APs and second by uniformly packing vesicles to avoid wasteful secretion. My work has contributed to a growing notion in the field that secretory cargo are not inert passengers but active participants in vesicle mediated secretion

ER Cargo Properties Specify a Requirement for COPII Coat Rigidity Mediated by Sec13p

Eukaryotic secretory proteins exit the endoplasmic reticulum (ER) via transport vesicles generated bythe essential coat protein complex II (COPII) proteins. The outer coat complex, Sec13-Sec31,forms a scaffold that is thought to enforce curvature. By exploiting yeast bypass-of-sec-thirteen (bst)mutants, where Sec13p is dispensable, we probed the relationship between a compromised COPIIcoat and the cellular context in which it could still function. Genetic and biochemical analysessuggested that Sec13p was required to generate vesicles from membranes that contained asymmetricallydistributed cargoes that were likely to confer opposing curvature. Thus, Sec13p may rigidify theCOPII cage and increase its membrane-bending capacity; this function could be bypassed when a bstmutation renders the membrane more deformable

Traffic of p24 Proteins and COPII Coat Composition Mutually Influence Membrane Scaffolding

Eukaryotic protein secretion requires efficient and accurate delivery of diverse secretory and membrane proteins. This process initiates in the ER, where vesicles are sculpted by the essential COPII coat. The Sec13p subunit of the COPII coat contributes to membrane scaffolding, which enforces curvature on the nascent vesicle. A requirement for Sec13p can be bypassed when traffic of lumenally oriented membrane proteins is abrogated. Here we sought to further explore the impact of cargo proteins on vesicle formation. We show that efficient ER export of the p24 family of proteins is a major driver of the requirement for Sec13p. The scaffolding burden presented by the p24 complex is met in part by the cargo adaptor Lst1p, which binds to a subset of cargo, including the p24 proteins. We propose that the scaffolding function of Lst1p is required to generate vesicles that can accommodate difficult cargo proteins that include large oligomeric assemblies and asymmetrically distributed membrane proteins. Vesicles that contain such cargoes are also more dependent on scaffolding by Sec13p, and may serve as a model for large carrier formation in other systems

Performance characteristics of five immunoassays for SARS-CoV-2: a head-to-head benchmark comparison

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has caused a global pandemic in 2020. Testing is crucial for mitigating public health and economic effects. Serology is considered key to population-level surveillance and potentially individual-level risk assessment. However, immunoassay performance has not been compared on large, identical sample sets. We aimed to investigate the performance of four high-throughput commercial SARS-CoV-2 antibody immunoassays and a novel 384-well ELISA.We did a head-to-head assessment of SARS-CoV-2 IgG assay (Abbott, Chicago, IL, USA), LIAISON SARS-CoV-2 S1/S2 IgG assay (DiaSorin, Saluggia, Italy), Elecsys Anti-SARS-CoV-2 assay (Roche, Basel, Switzerland), SARS-CoV-2 Total assay (Siemens, Munich, Germany), and a novel 384-well ELISA (the Oxford immunoassay). We derived sensitivity and specificity from 976 pre-pandemic blood samples (collected between Sept 4, 2014, and Oct 4, 2016) and 536 blood samples from patients with laboratory-confirmed SARS-CoV-2 infection, collected at least 20 days post symptom onset (collected between Feb 1, 2020, and May 31, 2020). Receiver operating characteristic (ROC) curves were used to assess assay thresholds.At the manufacturers' thresholds, for the Abbott assay sensitivity was 92·7% (95% CI 90·2–94·8) and specificity was 99·9% (99·4–100%); for the DiaSorin assay sensitivity was 96·2% (94·2–97·7) and specificity was 98·9% (98·0–99·4); for the Oxford immunoassay sensitivity was 99·1% (97·8–99·7) and specificity was 99·0% (98·1–99·5); for the Roche assay sensitivity was 97·2% (95·4–98·4) and specificity was 99·8% (99·3–100); and for the Siemens assay sensitivity was 98·1% (96·6–99·1) and specificity was 99·9% (99·4–100%). All assays achieved a sensitivity of at least 98% with thresholds optimised to achieve a specificity of at least 98% on samples taken 30 days or more post symptom onset.Four commercial, widely available assays and a scalable 384-well ELISA can be used for SARS-CoV-2 serological testing to achieve sensitivity and specificity of at least 98%. The Siemens assay and Oxford immunoassay achieved these metrics without further optimisation. This benchmark study in immunoassay assessment should enable refinements of testing strategies and the best use of serological testing resource to benefit individuals and population health.Public Health England and UK National Institute for Health Research

Guidelines for the use and interpretation of assays for monitoring autophagy (4th edition)

In 2008, we published the first set of guidelines for standardizing research in autophagy. Since then, this topic has received increasing attention, and many scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Thus, it is important to formulate on a regular basis updated guidelines for monitoring autophagy in different organisms. Despite numerous reviews, there continues to be confusion regarding acceptable methods to evaluate autophagy, especially in multicellular eukaryotes. Here, we present a set of guidelines for investigators to select and interpret methods to examine autophagy and related processes, and for reviewers to provide realistic and reasonable critiques of reports that are focused on these processes. These guidelines are not meant to be a dogmatic set of rules, because the appropriateness of any assay largely depends on the question being asked and the system being used. Moreover, no individual assay is perfect for every situation, calling for the use of multiple techniques to properly monitor autophagy in each experimental setting. Finally, several core components of the autophagy machinery have been implicated in distinct autophagic processes (canonical and noncanonical autophagy), implying that genetic approaches to block autophagy should rely on targeting two or more autophagy-related genes that ideally participate in distinct steps of the pathway. Along similar lines, because multiple proteins involved in autophagy also regulate other cellular pathways including apoptosis, not all of them can be used as a specific marker for bona fide autophagic responses. Here, we critically discuss current methods of assessing autophagy and the information they can, or cannot, provide. Our ultimate goal is to encourage intellectual and technical innovation in the field