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.

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.

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.

Activation of GCN2 by the ribosomal P-stalk.

Cells dynamically adjust their protein translation profile to maintain homeostasis in changing environments. During nutrient stress, the kinase general control nonderepressible 2 (GCN2) phosphorylates translation initiation factor eIF2α, initiating the integrated stress response (ISR). To examine the mechanism of GCN2 activation, we have reconstituted this process in vitro, using purified components. We find that recombinant human GCN2 is potently stimulated by ribosomes and, to a lesser extent, by tRNA. Hydrogen/deuterium exchange-mass spectrometry (HDX-MS) mapped GCN2-ribosome interactions to domain II of the uL10 subunit of the ribosomal P-stalk. Using recombinant, purified P-stalk, we showed that this domain of uL10 is the principal component of binding to GCN2; however, the conserved 14-residue C-terminal tails (CTTs) in the P1 and P2 P-stalk proteins are also essential for GCN2 activation. The HisRS-like and kinase domains of GCN2 show conformational changes upon binding recombinant P-stalk complex. Given that the ribosomal P-stalk stimulates the GTPase activity of elongation factors during translation, we propose that the P-stalk could link GCN2 activation to translational stress, leading to initiation of ISR.

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.

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.

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.

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.

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 3.4 angstroms by cryo-electron microscopy, permitting building of a nearly complete atomic model. We used structure-guided mutagenesis to demonstrate that substrate insertion requires a methionine-rich cytosolic loop and occurs via an enclosed hydrophilic vestibule within the membrane formed by the subunits EMC3 and EMC6. We propose that the EMC uses local membrane thinning and a positively charged patch to decrease the energetic barrier for insertion into the bilayer.

The ribosomal P-stalk couples amino acid starvation to GCN2 activation in mammalian cells.

The eukaryotic translation initiation factor 2α (eIF2α) kinase GCN2 is activated by amino acid starvation to elicit a rectifying physiological program known as the Integrated Stress Response (ISR). A role for uncharged tRNAs as activating ligands of yeast GCN2 is supported experimentally. However, mouse GCN2 activation has recently been observed in circumstances associated with ribosome stalling with no global increase in uncharged tRNAs. We report on a mammalian CHO cell-based CRISPR-Cas9 mutagenesis screen for genes that contribute to ISR activation by amino acid starvation. Disruption of genes encoding components of the ribosome P-stalk, uL10 and P1, selectively attenuated GCN2-mediated ISR activation by amino acid starvation or interference with tRNA charging without affecting the endoplasmic reticulum unfolded protein stress-induced ISR, mediated by the related eIF2α kinase PERK. Wildtype ribosomes isolated from CHO cells, but not those with P-stalk lesions, stimulated GCN2-dependent eIF2α phosphorylation in vitro. These observations support a model whereby lack of a cognate charged tRNA exposes a latent capacity of the ribosome P-stalk to activate GCN2 in cells and help explain the emerging link between ribosome stalling and ISR activation.

Disease Variants of FGFR3 Reveal Molecular Basis for the Recognition and Additional Roles for Cdc37 in Hsp90 Chaperone System.

Receptor tyrosine kinase FGFR3 is involved in many signaling networks and is frequently mutated in developmental disorders and cancer. The Hsp90/Cdc37 chaperone system is essential for function of normal and neoplastic cells. Here we uncover the mechanistic inter-relationships between these proteins by combining approaches including NMR, HDX-MS, and SAXS. We show that several disease-linked mutations convert FGFR3 to a stronger client, where the determinant underpinning client strength involves an allosteric network through the N-lobe and at the lobe interface. We determine the architecture of the client kinase/Cdc37 complex and demonstrate, together with site-specific information, that binding of Cdc37 to unrelated kinases induces a common, extensive conformational remodeling of the kinase N-lobe, beyond localized changes and interactions within the binary complex. As further shown for FGFR3, this processing by Cdc37 deactivates the kinase and presents it, in a specific orientation established in the complex, for direct recognition by Hsp90.

Deconvolution of Buparlisib's mechanism of action defines specific PI3K and tubulin inhibitors for therapeutic intervention.

BKM120 (Buparlisib) is one of the most advanced phosphoinositide 3-kinase (PI3K) inhibitors for the treatment of cancer, but it interferes as an off-target effect with microtubule polymerization. Here, we developed two chemical derivatives that differ from BKM120 by only one atom. We show that these minute changes separate the dual activity of BKM120 into discrete PI3K and tubulin inhibitors. Analysis of the compounds cellular growth arrest phenotypes and microtubule dynamics suggest that the antiproliferative activity of BKM120 is mainly due to microtubule-dependent cytotoxicity rather than through inhibition of PI3K. Crystal structures of BKM120 and derivatives in complex with tubulin and PI3K provide insights into the selective mode of action of this class of drugs. Our results raise concerns over BKM120's generally accepted mode of action, and provide a unique mechanistic basis for next-generation PI3K inhibitors with improved safety profiles and flexibility for use in combination therapies.

Structures of PI4KIIIß complexes show simultaneous recruitment of Rab11 and its effectors.

Phosphatidylinositol 4-kinases (PI4Ks) and small guanosine triphosphatases (GTPases) are essential for processes that require expansion and remodeling of phosphatidylinositol 4-phosphate (PI4P)-containing membranes, including cytokinesis, intracellular development of malarial pathogens, and replication of a wide range of RNA viruses. However, the structural basis for coordination of PI4K, GTPases, and their effectors is unknown. Here, we describe structures of PI4Kß (PI4KIIIß) bound to the small GTPase Rab11a without and with the Rab11 effector protein FIP3. The Rab11-PI4KIIIß interface is distinct compared with known structures of Rab complexes and does not involve switch regions used by GTPase effectors. Our data provide a mechanism for how PI4KIIIß coordinates Rab11 and its effectors on PI4P-enriched membranes and also provide strategies for the design of specific inhibitors that could potentially target plasmodial PI4KIIIß to combat malaria.