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. We found that the back of Sec61 (BOS) complex, a component of the multipass translocon, was a physical and genetic interactor of the EMC. Functional and structural analysis of the EMC⋅BOS holocomplex showed that characteristics of a GPCR's soluble domain determine its biogenesis pathway. In contrast to prevailing models, no single insertase handles all substrates. We instead propose a unifying model for coordination between the EMC, the multipass translocon, and Sec61 for the biogenesis of diverse membrane proteins in human cells.

Triaging of α-helical proteins to the mitochondrial outer membrane by distinct chaperone machinery based on substrate topology.

Mitochondrial outer membrane ⍺-helical proteins play critical roles in mitochondrial-cytoplasmic communication, but the rules governing the targeting and insertion of these biophysically diverse proteins remain unknown. Here, we first defined the complement of required mammalian biogenesis machinery through genome-wide CRISPRi screens using topologically distinct membrane proteins. Systematic analysis of nine identified factors across 21 diverse ⍺-helical substrates reveals that these components are organized into distinct targeting pathways that act on substrates based on their topology. NAC is required for the efficient targeting of polytopic proteins, whereas signal-anchored proteins require TTC1, a cytosolic chaperone that physically engages substrates. Biochemical and mutational studies reveal that TTC1 employs a conserved TPR domain and a hydrophobic groove in its C-terminal domain to support substrate solubilization and insertion into mitochondria. Thus, the targeting of diverse mitochondrial membrane proteins is achieved through topological triaging in the cytosol using principles with similarities to ER membrane protein biogenesis systems.

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. We found that the back of sec61 (BOS) complex, a component of the 'multipass translocon', was a physical and genetic interactor of the EMC. Functional and structural analysis of the EMC•BOS holocomplex showed that characteristics of a GPCR's soluble domain determine its biogenesis pathway. In contrast to prevailing models, no single insertase handles all substrates. We instead propose a unifying model for coordination between the EMC, multipass translocon, and Sec61 for biogenesis of diverse membrane proteins in human cells.

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 interactions1. 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. This duality of temperature and osmotic strength enables simple manipulations of solvent thermodynamics to prevent cell death after extreme cold or heat shock. Physiologically, cells must sustain their activity against fluctuating temperature, pressure and osmotic strength, which impact water availability within seconds. Yet, established mechanisms of water homeostasis act over much slower timescales2,3; we therefore postulated the existence of a rapid compensatory response. We find that this function is performed by water potential-driven changes in macromolecular assembly, particularly biomolecular condensation of intrinsically disordered proteins. The formation and dissolution of biomolecular condensates liberates and captures free water, respectively, quickly counteracting thermal or osmotic perturbations of water potential, which is consequently robustly buffered in the cytoplasm. Our results indicate that biomolecular condensation constitutes an intrinsic biophysical feedback response that rapidly compensates for intracellular osmotic and thermal fluctuations. We suggest that preserving water availability within the concentrated cytosol is an overlooked evolutionary driver of protein (dis)order and function.

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. We use this approach to identify epistatic relationships for a defined biological pathway, showing both increased sensitivity and specificity than traditional growth screening approaches.

Triaging of α-helical proteins to the mitochondrial outer membrane by distinct chaperone machinery based on substrate topology.

Mitochondrial outer membrane α-helical proteins play critical roles in mitochondrial-cytoplasmic communication, but the rules governing the targeting and insertion of these biophysically diverse substrates remain unknown. Here, we first defined the complement of required mammalian biogenesis machinery through genome-wide CRISPRi screens using topologically distinct membrane proteins. Systematic analysis of nine identified factors across 21 diverse α-helical substrates reveals that these components are organized into distinct targeting pathways which act on substrates based on their topology. NAC is required for efficient targeting of polytopic proteins whereas signal-anchored proteins require TTC1, a novel cytosolic chaperone which physically engages substrates. Biochemical and mutational studies reveal that TTC1 employs a conserved TPR domain and a hydrophobic groove in its C-terminal domain to support substrate solubilization and insertion into mitochondria. Thus, targeting of diverse mitochondrial membrane proteins is achieved through topological triaging in the cytosol using principles with similarities to ER membrane protein biogenesis systems.

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. Here, we use a fluorescent reporter system in mammalian cells to reveal a selective degradation pathway specifically targeting the protein product of an NMD mRNA. We show that this process is post-translational and dependent on the ubiquitin proteasome system. To systematically uncover factors involved in NMD-linked protein quality control, we conducted genome-wide flow cytometry-based screens. Our screens recovered known NMD factors but suggested that protein degradation did not depend on the canonical ribosome-quality control (RQC) pathway. A subsequent arrayed screen demonstrated that protein and mRNA branches of NMD rely on a shared recognition event. Our results establish the existence of a targeted pathway for nascent protein degradation from PTC containing mRNAs, and provide a reference for the field to identify and characterize required factors.

A genome-wide optical pooled screen reveals regulators of cellular antiviral responses.

The infection of mammalian cells by viruses and innate immune responses to infection are spatiotemporally organized processes. Cytosolic RNA sensors trigger nuclear translocation of the transcription factor interferon regulatory factor 3 (IRF3) and consequent induction of host immune responses to RNA viruses. Previous genetic screens for factors involved in viral sensing did not resolve changes in the subcellular localization of host or viral proteins. Here, we increased the throughput of our optical pooled screening technology by over fourfold. This allowed us to carry out a genome-wide CRISPR knockout screen using high-resolution multiparameter imaging of cellular responses to Sendai virus infection coupled with in situ cDNA sequencing by synthesis (SBS) to identify 80,408 single guide RNAs (sgRNAs) in 10,366,390 cells-over an order of magnitude more genomic perturbations than demonstrated previously using an in situ SBS readout. By ranking perturbations using human-designed and deep learning image feature scores, we identified regulators of IRF3 translocation, Sendai virus localization, and peroxisomal biogenesis. Among the hits, we found that ATP13A1, an ER-localized P5A-type ATPase, is essential for viral sensing and is required for targeting of mitochondrial antiviral signaling protein (MAVS) to mitochondrial membranes where MAVS must be localized for effective signaling through retinoic acid-inducible gene I (RIG-I). The ability to carry out genome-wide pooled screens with complex high-resolution image-based phenotyping dramatically expands the scope of functional genomics approaches.

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. We use this approach to identify epistatic relationships for a defined biological pathway, showing both increased sensitivity and specificity than traditional growth screening approaches.

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. Here we focus on how these two sets of machinery target, transfer, and insert TAs into the lipid bilayer in close collaboration with quality control machinery. Additionally, we highlight the unifying features of the insertion process as revealed by recent structures of the GET and EMC membrane protein complexes.

Maximizing CRISPRi efficacy and accessibility with dual-sgRNA libraries and optimal effectors.

CRISPR interference (CRISPRi) enables programmable, reversible, and titratable repression of gene expression (knockdown) in mammalian cells. Initial CRISPRi-mediated genetic screens have showcased the potential to address basic questions in cell biology, genetics, and biotechnology, but wider deployment of CRISPRi screening has been constrained by the large size of single guide RNA (sgRNA) libraries and challenges in generating cell models with consistent CRISPRi-mediated knockdown. Here, we present next-generation CRISPRi sgRNA libraries and effector expression constructs that enable strong and consistent knockdown across mammalian cell models. First, we combine empirical sgRNA selection with a dual-sgRNA library design to generate an ultra-compact (1-3 elements per gene), highly active CRISPRi sgRNA library. Next, we compare CRISPRi effectors to show that the recently published Zim3-dCas9 provides an excellent balance between strong on-target knockdown and minimal non-specific effects on cell growth or the transcriptome. Finally, we engineer a suite of cell lines with stable expression of Zim3-dCas9 and robust on-target knockdown. Our results and publicly available reagents establish best practices for CRISPRi genetic screening.

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 ß-barrel proteins. Purified MTCH2 was sufficient to mediate insertion into reconstituted proteoliposomes. Functional and mutational studies suggested that MTCH2 has evolved from a solute carrier transporter. MTCH2 uses membrane-embedded hydrophilic residues to function as a gatekeeper for the outer membrane, controlling mislocalization of TAs into the endoplasmic reticulum and modulating the sensitivity of leukemia cells to apoptosis. Our identification of MTCH2 as an insertase provides a mechanistic explanation for the diverse phenotypes and disease states associated with MTCH2 dysfunction.

Mapping information-rich genotype-phenotype landscapes with genome-scale Perturb-seq.

A central goal of genetics is to define the relationships between genotypes and phenotypes. High-content phenotypic screens such as Perturb-seq (CRISPR-based screens with single-cell RNA-sequencing readouts) enable massively parallel functional genomic mapping but, to date, have been used at limited scales. Here, we perform genome-scale Perturb-seq targeting all expressed genes with CRISPR interference (CRISPRi) across >2.5 million human cells. We use transcriptional phenotypes to predict the function of poorly characterized genes, uncovering new regulators of ribosome biogenesis (including CCDC86, ZNF236, and SPATA5L1), transcription (C7orf26), and mitochondrial respiration (TMEM242). In addition to assigning gene function, single-cell transcriptional phenotypes allow for in-depth dissection of complex cellular phenomena-from RNA processing to differentiation. We leverage this ability to systematically identify genetic drivers and consequences of aneuploidy and to discover an unanticipated layer of stress-specific regulation of the mitochondrial genome. Our information-rich genotype-phenotype map reveals a multidimensional portrait of gene and cellular function.

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. In cultured cells and in tissue we find that compensation involves electroneutral active transport of Na+, K+, and Cl- through differential activity of SLC12A family cotransporters. In cardiomyocytes ex vivo and in vivo, compensatory ion fluxes confer daily variation in electrical activity. Perturbation of soluble protein abundance has commensurate effects on ion composition and cellular function across the circadian cycle. Thus, circadian regulation of the proteome impacts ion homeostasis with substantial consequences for the physiology of electrically active cells such as cardiomyocytes.

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. WNK1 uses a conserved amphipathic helix to stabilize the soluble subunit, EMC2, by binding to the EMC2-8 interface. Shielding this hydrophobic surface prevents promiscuous interactions of unassembled EMC2 and directly competes for binding of E3 ubiquitin ligases, permitting assembly. Depletion of WNK1 thus destabilizes both the EMC and its membrane protein clients. This work describes an unexpected role for WNK1 in protein biogenesis and defines the general requirements of an assembly factor that will apply across the proteome.

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. Our data suggest that the stability of each subunit is differentially regulated. In WRB's absence, CAML folds incorrectly, causing aberrant exposure of a hydrophobic transmembrane domain to the ER lumen. When present, WRB can correct the topology of CAML both in vitro and in cells. In contrast, WRB can independently fold correctly but is still degraded in the absence of CAML. We therefore propose that there are at least two distinct regulatory pathways for the surveillance of orphan subunits in the mammalian ER.

EMC Is Required to Initiate Accurate Membrane Protein Topogenesis.

Mammals encode ∼5,000 integral membrane proteins that need to be inserted in a defined topology at the endoplasmic reticulum (ER) membrane by mechanisms that are incompletely understood. Here, we found that efficient biogenesis of ß1-adrenergic receptor (ß1AR) and other G protein-coupled receptors (GPCRs) requires the conserved ER membrane protein complex (EMC). Reconstitution studies of ß1AR biogenesis narrowed the EMC requirement to the co-translational insertion of the first transmembrane domain (TMD). Without EMC, a proportion of TMD1 inserted in an inverted orientation or failed altogether. Purified EMC and SRP receptor were sufficient for correctly oriented TMD1 insertion, while the Sec61 translocon was necessary for insertion of the next TMD. Enforcing TMD1 topology with an N-terminal signal peptide bypassed the EMC requirement for insertion in vitro and restored efficient biogenesis of multiple GPCRs in EMC-knockout cells. Thus, EMC inserts TMDs co-translationally and cooperates with the Sec61 translocon to ensure accurate topogenesis of many membrane proteins.

Transmembrane Domain Recognition during Membrane Protein Biogenesis and Quality Control.

One-fourth of eukaryotic genes code for integral membrane proteins, nearly all of which are inserted and assembled at the endoplasmic reticulum (ER). The defining feature of membrane proteins is one or more transmembrane domains (TMDs). During membrane protein biogenesis, TMDs are selectively recognized, shielded, and chaperoned into the lipid bilayer, where they often assemble with other TMDs. If maturation fails, exposed TMDs serve as a cue for engagement of degradation pathways. Thus, TMD-recognition factors in the cytosol and ER are essential for membrane protein biogenesis and quality control. Here, we discuss the growing assortment of cytosolic and membrane-embedded TMD-recognition factors, the pathways within which they operate, and mechanistic principles of recognition.