Role of a holo-insertase complex in the biogenesis of biophysically diverse ER membrane proteins.

Mammalian membrane proteins perform essential physiologic functions that rely on their accurate insertion and folding at the endoplasmic reticulum (ER). Using forward and arrayed genetic screens, we systematically studied the biogenesis of a panel of membrane proteins, including several G-protein-coupled receptors (GPCRs). We observed a central role for the insertase, the ER membrane protein complex (EMC), and developed a dual-guide approach to identify genetic modifiers of the EMC.

Role of a holo-insertase complex in the biogenesis of biophysically diverse ER membrane proteins.

Mammalian membrane proteins perform essential physiologic functions that rely on their accurate insertion and folding at the endoplasmic reticulum (ER). Using forward and arrayed genetic screens, we systematically studied the biogenesis of a panel of membrane proteins, including several G-protein coupled receptors (GPCRs). We observed a central role for the insertase, the ER membrane protein complex (EMC), and developed a dual-guide approach to identify genetic modifiers of the EMC.

A nanobody-based strategy for rapid and scalable purification of human protein complexes.

The isolation of proteins in high yield and purity is a major bottleneck for the analysis of their three-dimensional structure, function and interactome. Here, we present a streamlined workflow for the rapid production of proteins or protein complexes using lentiviral transduction of human suspension cells, combined with highly specific nanobody-mediated purification and proteolytic elution. Application of the method requires prior generation of a plasmid coding for a protein of interest (POI) fused to an N- or C-terminal GFP or ALFA peptide tag using a lentiviral plasmid toolkit we have designed.

A dual sgRNA library design to probe genetic modifiers using genome-wide CRISPRi screens.

Mapping genetic interactions is essential for determining gene function and defining novel biological pathways. We report a simple to use CRISPR interference (CRISPRi) based platform, compatible with Fluorescence Activated Cell Sorting (FACS)-based reporter screens, to query epistatic relationships at scale. This is enabled by a flexible dual-sgRNA library design that allows for the simultaneous delivery and selection of a fixed sgRNA and a second randomized guide, comprised of a genome-wide library, with a single transduction.

Macromolecular condensation buffers intracellular water potential.

Optimum protein function and biochemical activity critically depends on water availability because solvent thermodynamics drive protein folding and macromolecular interactions. Reciprocally, macromolecules restrict the movement of 'structured' water molecules within their hydration layers, reducing the available 'free' bulk solvent and therefore the total thermodynamic potential energy of water, or water potential. Here, within concentrated macromolecular solutions such as the cytosol, we found that modest changes in temperature greatly affect the water potential, and are counteracted by opposing changes in osmotic strength.

Coupled protein quality control during nonsense-mediated mRNA decay.

Translation of mRNAs containing premature termination codons (PTCs) results in truncated protein products with deleterious effects. Nonsense-mediated decay (NMD) is a surveillance pathway responsible for detecting PTC containing transcripts. Although the molecular mechanisms governing mRNA degradation have been extensively studied, the fate of the nascent protein product remains largely uncharacterized.

A selectivity filter in the ER membrane protein complex limits protein misinsertion at the ER.

Tail-anchored (TA) proteins play essential roles in mammalian cells, and their accurate localization is critical for proteostasis. Biophysical similarities lead to mistargeting of mitochondrial TA proteins to the ER, where they are delivered to the insertase, the ER membrane protein complex (EMC). Leveraging an improved structural model of the human EMC, we used mutagenesis and site-specific crosslinking to map the path of a TA protein from its cytosolic capture by methionine-rich loops to its membrane insertion through a hydrophilic vestibule.

A dual sgRNA library design to probe genetic modifiers using genome-wide CRISPRi screens.

Mapping genetic interactions is essential for determining gene function and defining novel biological pathways. We report a simple to use CRISPR interference (CRISPRi) based platform, compatible with Fluorescence Activated Cell Sorting (FACS)-based reporter screens, to query epistatic relationships at scale. This is enabled by a flexible dual-sgRNA library design that allows for the simultaneous delivery and selection of a fixed sgRNA and a second randomized guide, comprised of a genome-wide library, with a single transduction.

MTCH2 is a mitochondrial outer membrane protein insertase.

In the mitochondrial outer membrane, α-helical transmembrane proteins play critical roles in cytoplasmic-mitochondrial communication. Using genome-wide CRISPR screens, we identified mitochondrial carrier homolog 2 (MTCH2), and its paralog MTCH1, and showed that it is required for insertion of biophysically diverse tail-anchored (TA), signal-anchored, and multipass proteins, but not outer membrane β-barrel proteins. Purified MTCH2 was sufficient to mediate insertion into reconstituted proteoliposomes.

A TAle of Two Pathways: Tail-Anchored Protein Insertion at the Endoplasmic Reticulum.

Tail-anchored (TA) proteins are an essential class of integral membrane proteins required for many aspects of cellular physiology. TA proteins contain a single carboxy-terminal transmembrane domain that must be post-translationally recognized, guided to, and ultimately inserted into the correct cellular compartment. The majority of TA proteins begin their biogenesis in the endoplasmic reticulum (ER) and utilize two parallel strategies for targeting and insertion: the guided-entry of tail-anchored proteins (GET) and ER-membrane protein complex (EMC) pathways.

Compensatory ion transport buffers daily protein rhythms to regulate osmotic balance and cellular physiology.

Between 6-20% of the cellular proteome is under circadian control and tunes mammalian cell function with daily environmental cycles. For cell viability, and to maintain volume within narrow limits, the daily variation in osmotic potential exerted by changes in the soluble proteome must be counterbalanced. The mechanisms and consequences of this osmotic compensation have not been investigated before.

WNK1 is an assembly factor for the human ER membrane protein complex.

The assembly of nascent proteins into multi-subunit complexes is a tightly regulated process that must occur at high fidelity to maintain cellular homeostasis. The ER membrane protein complex (EMC) is an essential insertase that requires seven membrane-spanning and two soluble cytosolic subunits to function. Here, we show that the kinase with no lysine 1 (WNK1), known for its role in hypertension and neuropathy, functions as an assembly factor for the human EMC.

SARS-CoV-2 Disrupts Splicing, Translation, and Protein Trafficking to Suppress Host Defenses.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a recently identified coronavirus that causes the respiratory disease known as coronavirus disease 2019 (COVID-19). Despite the urgent need, we still do not fully understand the molecular basis of SARS-CoV-2 pathogenesis. Here, we comprehensively define the interactions between SARS-CoV-2 proteins and human RNAs.

Structural basis for membrane insertion by the human ER membrane protein complex.

A defining step in the biogenesis of a membrane protein is the insertion of its hydrophobic transmembrane helices into the lipid bilayer. The nine-subunit endoplasmic reticulum (ER) membrane protein complex (EMC) is a conserved co- and posttranslational insertase at the ER. We determined the structure of the human EMC in a lipid nanodisc to an overall resolution of 3.

Differential Modes of Orphan Subunit Recognition for the WRB/CAML Complex.

A large proportion of membrane proteins must be assembled into oligomeric complexes for function. How this process occurs is poorly understood, but it is clear that complex assembly must be tightly regulated to avoid accumulation of orphan subunits with potential cytotoxic effects. We interrogated assembly in mammalian cells by using the WRB/CAML complex, an essential insertase for tail-anchored proteins in the endoplasmic reticulum (ER), as a model system.

White spot lesions: Does etching really matter?

Aim: The clinical significance of acid etching prior to orthodontic bonding is controversial. In the present study, we evaluated the effect of 15 seconds of acid etching on enamel demineralization.

Methods: Twenty-seven human molars were sectioned and assigned to two groups.

Toward a structural understanding of co-translational protein translocation.

The translocation of most eukaryotic secreted and integral membrane proteins occurs co-translationally at the endoplasmic reticulum (ER). These nascent polypeptides are recognized on the ribosome by the signal recognition particle (SRP), targeted to the ER, and translocated across or inserted into the membrane by the Sec61 translocation channel. Structural analysis of these co-translational processes has been challenging due to the size, complexity, and flexibility of the targeting and translocation machinery.

Development of the chemical exposure monitor with indoor positioning (CEMWIP) for workplace VOC surveys.

The purpose of this article was to research and develop a direct-reading exposure assessment method that combined a real-time location system with a wireless direct-reading personal chemical sensor. The personal chemical sensor was a photoionization device for detecting volatile organic compounds. The combined system was calibrated and tested against the same four standard gas concentrations and calibrated at one standard location and tested at four locations that included the standard locations.

Structure of the Sec61 channel opened by a signal sequence.

Secreted and integral membrane proteins compose up to one-third of the biological proteome. These proteins contain hydrophobic signals that direct their translocation across or insertion into the lipid bilayer by the Sec61 protein-conducting channel. The molecular basis of how hydrophobic signals within a nascent polypeptide trigger channel opening is not understood.