Lipid Droplets Big and Small: Basic Mechanisms That Make Them All.

Lipid droplets (LDs) are dynamic storage organelles with central roles in lipid and energy metabolism. They consist of a core of neutral lipids, such as triacylglycerol, which is surrounded by a monolayer of phospholipids and specialized surface proteins. The surface composition determines many of the LD properties, such as size, subcellular distribution, and interaction with partner organelles. Considering the diverse energetic and metabolic demands of various cell types, it is not surprising that LDs are highly heterogeneous within and between cell types. Despite their diversity, all LDs share a common biogenesis mechanism. However, adipocytes have evolved specific adaptations of these basic mechanisms, enabling the regulation of lipid and energy metabolism at both the cellular and organismal levels. Here, we discuss recent advances in the understanding of both the general mechanisms of LD biogenesis and the adipocyte-specific adaptations controlling these fascinating organelles.

Protein Quality Control and Lipid Droplet Metabolism.

Lipid droplets (LDs) are endoplasmic reticulum-derived organelles that consist of a core of neutral lipids encircled by a phospholipid monolayer decorated with proteins. As hubs of cellular lipid and energy metabolism, LDs are inherently involved in the etiology of prevalent metabolic diseases such as obesity and nonalcoholic fatty liver disease. The functions of LDs are regulated by a unique set of associated proteins, the LD proteome, which includes integral membrane and peripheral proteins. These proteins control key activities of LDs such as triacylglycerol synthesis and breakdown, nutrient sensing and signal integration, and interactions with other organelles. Here we review the mechanisms that regulate the composition of the LD proteome, such as pathways that mediate selective and bulk LD protein degradation and potential connections between LDs and cellular protein quality control.

Mechanisms of Tail-Anchored Membrane Protein Targeting and Insertion.

Proper localization of membrane proteins is essential for the function of biological membranes and for the establishment of organelle identity within a cell. Molecular machineries that mediate membrane protein biogenesis need to not only achieve a high degree of efficiency and accuracy, but also prevent off-pathway aggregation events that can be detrimental to cells. The posttranslational targeting of tail-anchored proteins (TAs) provides tractable model systems to probe these fundamental issues. Recent advances in understanding TA-targeting pathways reveal sophisticated molecular machineries that drive and regulate these processes. These findings also suggest how an interconnected network of targeting factors, cochaperones, and quality control machineries together ensures robust membrane protein biogenesis.

The CYTOLD and ERTOLD pathways for lipid droplet-protein targeting.

Lipid droplets (LDs) are the main organelles for lipid storage, and their surfaces contain unique proteins with diverse functions, including those that facilitate the deposition and mobilization of LD lipids. Among organelles, LDs have an unusual structure with an organic, hydrophobic oil phase covered by a phospholipid monolayer. The unique properties of LD monolayer surfaces require proteins to localize to LDs by distinct mechanisms. Here we review the two pathways known to mediate direct LD protein localization: the CYTOLD pathway mediates protein targeting from the cytosol toLDs, and the ERTOLD pathway functions in protein targeting from the endoplasmic reticulum toLDs. We describe the emerging principles for each targeting pathway in animal cells and highlight open questions in the field.

Antibody-guided proteases enable selective and catalytic degradation of challenging therapeutic targets.

The exquisite specificity, natural biological functions, and favorable development properties of antibodies make them highly effective agents as drugs. Monoclonal antibodies are particularly strong as inhibitors of systemically accessible targets where trough-level concentrations can sustain full target occupancy. Yet beyond this pharmacologic wheelhouse, antibodies perform suboptimally for targets of high abundance and those not easily accessible from circulation. Fundamentally, this restraint on broader application is due largely to the stoichiometric nature of their activity-one drug molecule is generally able to inhibit a maximum of two target molecules at a time. Enzymes in contrast are able to catalytically turnover multiple substrates, making them a natural sub-stoichiometric solution for targets of high abundance or in poorly accessible sites of action. However, enzymes have their own limitations as drugs, including, in particular, the polypharmacology and broad specificity often seen with native enzymes. In this study, we introduce antibody-guided proteolytic enzymes to enable selective sub-stoichiometric turnover of therapeutic targets. We demonstrate that antibody-mediated substrate targeting can enhance enzyme activity and specificity, with proof of concept for two challenging target proteins, amyloid-ß and immunoglobulin G. This work advances a new biotherapeutic platform that combines the favorable properties of antibodies and proteolytic enzymes to more effectively suppress high-bar therapeutic targets.

Coping with stress: How bacteria fine-tune protein synthesis and protein transport.

Maintaining a functional proteome under different environmental conditions is challenging for every organism, in particular for unicellular organisms, such as bacteria. In order to cope with changing environments and stress conditions, bacteria depend on strictly coordinated proteostasis networks that control protein production, folding, trafficking, and degradation. Regulation of ribosome biogenesis and protein synthesis are cornerstones of this cellular adaptation in all domains of life, which is rationalized by the high energy demand of both processes and the increased resistance of translationally silent cells against internal or external poisons. Reduced protein synthesis ultimately also reduces the substrate load for protein transport systems, which are required for maintaining the periplasmic, inner, and outer membrane subproteomes. Consequences of impaired protein transport have been analyzed in several studies and generally induce a multifaceted response that includes the upregulation of chaperones and proteases and the simultaneous downregulation of protein synthesis. In contrast, generally less is known on how bacteria adjust the protein targeting and transport machineries to reduced protein synthesis, e.g., when cells encounter stress conditions or face nutrient deprivation. In the current review, which is mainly focused on studies using Escherichia coli as a model organism, we summarize basic concepts on how ribosome biogenesis and activity are regulated under stress conditions. In addition, we highlight some recent developments on how stress conditions directly impair protein targeting to the bacterial membrane. Finally, we describe mechanisms that allow bacteria to maintain the transport of stress-responsive proteins under conditions when the canonical protein targeting pathways are impaired.

Structures of Get3d reveal a distinct architecture associated with the emergence of photosynthesis.

Homologs of the protein Get3 have been identified in all domains yet remain to be fully characterized. In the eukaryotic cytoplasm, Get3 delivers tail-anchored (TA) integral membrane proteins, defined by a single transmembrane helix at their C terminus, to the endoplasmic reticulum. While most eukaryotes have a single Get3 gene, plants are notable for having multiple Get3 paralogs. Get3d is conserved across land plants and photosynthetic bacteria and includes a distinctive C-terminal α-crystallin domain. After tracing the evolutionary origin of Get3d, we solve the Arabidopsis thaliana Get3d crystal structure, identify its localization to the chloroplast, and provide evidence for a role in TA protein binding. The structure is identical to that of a cyanobacterial Get3 homolog, which is further refined here. Distinct features of Get3d include an incomplete active site, a "closed" conformation in the apo-state, and a hydrophobic chamber. Both homologs have ATPase activity and are capable of binding TA proteins, supporting a potential role in TA protein targeting. Get3d is first found with the development of photosynthesis and conserved across 1.2 billion years into the chloroplasts of higher plants across the evolution of photosynthesis suggesting a role in the homeostasis of photosynthetic machinery.

Chlorotoxin binds to both matrix metalloproteinase 2 and neuropilin 1.

Chlorotoxin (CTX), a scorpion venom-derived 36-residue miniprotein, binds to and is taken up selectively by glioblastoma cells. Previous studies provided controversial results concerning target protein(s) of CTX. These included CLC3 chloride channel, matrix metalloproteinase 2 (MMP-2), regulators of MMP-2, annexin A2, and neuropilin 1 (NRP1). The present study aimed at clarifying which of the proposed binding partners can really interact with CTX using biochemical methods and recombinant proteins. For this purpose, we established two new binding assays based on anchoring the tested proteins to microbeads and quantifying the binding of CTX by flow cytometry. Screening of His-tagged proteins anchored to cobalt-coated beads indicated strong interaction of CTX with MMP-2 and NRP1, whereas binding to annexin A2 was not confirmed. Similar results were obtained with fluorophore-labeled CTX and CTX-displaying phages. Affinity of CTX to MMP-2 and NRP1 was assessed by the "immunoglobulin-coated bead" test, in which the proteins were anchored to beads by specific antibodies. This assay yielded highly reproducible data using both direct titration and displacement approach. The affinities of labeled and unlabeled CTX appeared to be similar for both MMP-2 and NRP1 with estimated KD values of 0.5 to 0.7 µM. Contrary to previous reports, we found that CTX does not inhibit the activity of MMP-2 and that CTX not only with free carboxyl end but also with carboxamide terminal end binds to NRP1. We conclude that the presented robust assays could also be applied for affinity-improving studies of CTX to its genuine targets using phage display libraries.

Mitochondrial antiviral-signalling protein is a client of the BAG6 protein quality control complex.

The heterotrimeric BAG6 complex coordinates the direct handover of newly synthesised tail-anchored (TA) membrane proteins from an SGTA-bound preloading complex to the endoplasmic reticulum (ER) delivery component TRC40. In contrast, defective precursors, including aberrant TA proteins, form a stable complex with this cytosolic protein quality control factor, enabling such clients to be either productively re-routed or selectively degraded. We identify the mitochondrial antiviral-signalling protein (MAVS) as an endogenous TA client of both SGTA and the BAG6 complex. Our data suggest that the BAG6 complex binds to a cytosolic pool of MAVS before its misinsertion into the ER membrane, from where it can subsequently be removed via ATP13A1-mediated dislocation. This BAG6-associated fraction of MAVS is dynamic and responds to the activation of an innate immune response, suggesting that BAG6 may modulate the pool of MAVS that is available for coordinating the cellular response to viral infection.

Co-translational biogenesis of lipid droplet integral membrane proteins.

Membrane proteins destined for lipid droplets (LDs), a major intracellular storage site for neutral lipids, are inserted into the endoplasmic reticulum (ER) and then trafficked to LDs where they reside in a hairpin loop conformation. Here, we show that LD membrane proteins can be delivered to the ER either co- or post-translationally and that their membrane-embedded region specifies pathway selection. The co-translational route for LD membrane protein biogenesis is insensitive to a small molecule inhibitor of the Sec61 translocon, Ipomoeassin F, and instead relies on the ER membrane protein complex (EMC) for membrane insertion. This route may even result in a transient exposure of the short N termini of some LD membrane proteins to the ER lumen, followed by putative topological rearrangements that would enable their transmembrane segment to form a hairpin loop and N termini to face the cytosol. Our study reveals an unexpected complexity to LD membrane protein biogenesis and identifies a role for the EMC during their co-translational insertion into the ER.

Lipid droplets form a network interconnected by the endoplasmic reticulum through which their proteins equilibrate.

Lipid droplets (LDs) are globular intracellular structures dedicated to the storage of neutral lipids. They are closely associated with the endoplasmic reticulum (ER) and are delineated by a monolayer of phospholipids that is continuous with the cytoplasmic leaflet of the ER membrane. LDs contain a specific set of proteins, but how these proteins are targeted to the LD surface is not fully understood. Here, we devised a yeast mating-based microscopic readout to monitor the transfer of LD proteins upon zygote formation. The results of this analysis indicate that ER fusion between mating partners is required for transfer of LD proteins and that this transfer is continuous, bidirectional and affects most LDs simultaneously. These observations suggest that LDs do not fuse upon mating of yeast cells, but that they form a network that is interconnected through the ER membrane. Consistent with this, ER-localized LD proteins rapidly move onto LDs of a mating partner and this protein transfer is affected by seipin, a protein important for proper LD biogenesis and the functional connection of LDs with the ER membrane.

Cathodoluminescent and Characteristic X-Ray-Emissive Rare-Earth-Doped Core/Shell Protein Labels for Spectromicroscopic Analysis of Cell Surface Receptors.

Understanding the localization and the interactions of biomolecules at the nanoscale and in the cellular context remains challenging. Electron microscopy (EM), unlike light-based microscopy, gives access to the cellular ultrastructure yet results in grey-scale images and averts unambiguous (co-)localization of biomolecules. Multimodal nanoparticle-based protein labels for correlative cathodoluminescence electron microscopy (CCLEM) and energy-dispersive X-ray spectromicroscopy (EDX-SM) are presented. The single-particle STEM-cathodoluminescence (CL) and characteristic X-ray emissivity of sub-20 nm lanthanide-doped nanoparticles are exploited as unique spectral fingerprints for precise label localization and identification. To maximize the nanoparticle brightness, lanthanides are incorporated in a low-phonon host lattice and separated from the environment using a passivating shell. The core/shell nanoparticles are then functionalized with either folic (terbium-doped) or caffeic acid (europium-doped). Their potential for (protein-)labeling is successfully demonstrated using HeLa cells expressing different surface receptors that bind to folic or caffeic acid, respectively. Both particle populations show single-particle CL emission along with a distinctive energy-dispersive X-ray signal, with the latter enabling color-based localization of receptors within swift imaging times well below 2 min per µ m $\umu\text{m}$ 2 while offering high resolution with a pixel size of 2.78 nm. Taken together, these results open a route to multi-color labeling based on electron spectromicroscopy.

Evolutionary cues of gene fusion and fission in plants.

KEY MESSAGE: Here, we systematically analyzed the potential fusion and fission events of neighboring genes in Arabidopsis genome and analyzed the influence on the protein targeting.

A snapshot of the Physcomitrella N-terminome reveals N-terminal methylation of organellar proteins.

KEY MESSAGE: Analysis of the N-terminome of Physcomitrella reveals N-terminal monomethylation of nuclear-encoded, mitochondria-localized proteins. Post- or co-translational N-terminal modifications of proteins influence their half-life as well as mediating protein sorting to organelles via cleavable N-terminal sequences that are recognized by the respective translocation machinery. Here, we provide an overview on the current modification state of the N-termini of over 4500 proteins from the model moss Physcomitrella (Physcomitrium patens) using a compilation of 24 N-terminomics datasets. Our data reveal distinct proteoforms and modification states and confirm predicted targeting peptide cleavage sites of 1,144 proteins localized to plastids and the thylakoid lumen, to mitochondria, and to the secretory pathway. In addition, we uncover extended N-terminal methylation of mitochondrial proteins. Moreover, we identified PpNTM1 (P. patens alpha N-terminal protein methyltransferase 1) as a candidate for protein methylation in plastids, mitochondria, and the cytosol. These data can now be used to optimize computational targeting predictors, for customized protein fusions and their targeted localization in biotechnology, and offer novel insights into potential dual targeting of proteins.

Sequence-based features that are determinant for tail-anchored membrane protein sorting in eukaryotes.

The correct targeting and insertion of tail-anchored (TA) integral membrane proteins is critical for cellular homeostasis. TA proteins are defined by a hydrophobic transmembrane domain (TMD) at their C-terminus and are targeted to either the ER or mitochondria. Derived from experimental measurements of a few TA proteins, there has been little examination of the TMD features that determine localization. As a result, the localization of many TA proteins are misclassified by the simple heuristic of overall hydrophobicity. Because ER-directed TMDs favor arrangement of hydrophobic residues to one side, we sought to explore the role of geometric hydrophobic properties. By curating TA proteins with experimentally determined localizations and assessing hypotheses for recognition, we bioinformatically and experimentally verify that a hydrophobic face is the most accurate singular metric for separating ER and mitochondria-destined yeast TA proteins. A metric focusing on an 11 residue segment of the TMD performs well when classifying human TA proteins. The most inclusive predictor uses both hydrophobicity and C-terminal charge in tandem. This work provides context for previous observations and opens the door for more detailed mechanistic experiments to determine the molecular factors driving this recognition.

Enhanced membrane binding of oncogenic G protein αqQ209L confers resistance to inhibitor YM-254890.

Heterotrimeric G proteins couple activated G protein-coupled receptors (GPCRs) to intracellular signaling pathways. They can also function independently of GPCR activation upon acquiring mutations that prevent GTPase activity and result in constitutive signaling, as occurs with the αqQ209L mutation in uveal melanoma. YM-254890 (YM) can inhibit signaling by both GPCR-activated WT αq and GPCR-independent αqQ209L. Although YM inhibits WT αq by binding to αq-GDP and preventing GDP/GTP exchange, the mechanism of YM inhibition of cellular αqQ209L remains to be fully understood. Here, we show that YM promotes a subcellular redistribution of αqQ209L from the plasma membrane (PM) to the cytoplasm. To test if this loss of PM localization could contribute to the mechanism of inhibition of αqQ209L by YM, we developed and examined N-terminal mutants of αqQ209L, termed PM-restricted αqQ209L, in which the addition of membrane-binding motifs enhanced PM localization and prevented YM-promoted redistribution. Treatment of cells with YM failed to inhibit signaling by these PM-restricted αqQ209L. Additionally, pull-down experiments demonstrated that YM promotes similar conformational changes in both αqQ209L and PM-restricted αqQ209L, resulting in increased binding to ßγ and decreased binding to regulator RGS2, and effectors p63RhoGEF-DH/PH and phospholipase C-ß. GPCR-dependent signaling by PM-restricted WT αq is strongly inhibited by YM, demonstrating that resistance to YM inhibition by membrane-binding mutants is specific to constitutively active αqQ209L. Together, these results indicate that changes in membrane binding impact the ability of YM to inhibit αqQ209L and suggest that YM contributes to inhibition of αqQ209L by promoting its relocalization.

Determining the targeting specificity of the selective peroxisomal targeting factor Pex9.

Accurate and regulated protein targeting is crucial for cellular function and proteostasis. In the yeast Saccharomyces cerevisiae, peroxisomal matrix proteins, which harboring a Peroxisomal Targeting Signal 1 (PTS1), can utilize two paralog targeting factors, Pex5 and Pex9, to target correctly. While both proteins are similar and recognize PTS1 signals, Pex9 targets only a subset of Pex5 cargo proteins. However, what defines this substrate selectivity remains uncovered. Here, we used unbiased screens alongside directed experiments to identify the properties underlying Pex9 targeting specificity. We find that the specificity of Pex9 is largely determined by the hydrophobic nature of the amino acid preceding the PTS1 tripeptide of its cargos. This is explained by structural modeling of the PTS1-binding cavities of the two factors showing differences in their surface hydrophobicity. Our work outlines the mechanism by which targeting specificity is achieved, enabling dynamic rewiring of the peroxisomal proteome in changing metabolic needs.

Systematic multi-level analysis of an organelle proteome reveals new peroxisomal functions.

Seventy years following the discovery of peroxisomes, their complete proteome, the peroxi-ome, remains undefined. Uncovering the peroxi-ome is crucial for understanding peroxisomal activities and cellular metabolism. We used high-content microscopy to uncover peroxisomal proteins in the model eukaryote - Saccharomyces cerevisiae. This strategy enabled us to expand the known peroxi-ome by ~40% and paved the way for performing systematic, whole-organellar proteome assays. By characterizing the sub-organellar localization and protein targeting dependencies into the organelle, we unveiled non-canonical targeting routes. Metabolomic analysis of the peroxi-ome revealed the role of several newly identified resident enzymes. Importantly, we found a regulatory role of peroxisomes during gluconeogenesis, which is fundamental for understanding cellular metabolism. With the current recognition that peroxisomes play a crucial part in organismal physiology, our approach lays the foundation for deep characterization of peroxisome function in health and disease.

Proteomic Identification of Coxiella burnetii Effector Proteins Targeted to the Host Cell Mitochondria During Infection.

Modulation of the host cell is integral to the survival and replication of microbial pathogens. Several intracellular bacterial pathogens deliver bacterial proteins, termed "effector proteins" into the host cell during infection by sophisticated protein translocation systems, which manipulate cellular processes and functions. The functional contribution of individual effectors is poorly characterized, particularly in intracellular bacterial pathogens with large effector protein repertoires. Technical caveats have limited the capacity to study these proteins during a native infection, with many effector proteins having only been demonstrated to be translocated during over-expression of tagged versions. Here, we developed a novel strategy to examine effector proteins in the context of infection. We coupled a broad, unbiased proteomics-based screen with organelle purification to study the host-pathogen interactions occurring between the host cell mitochondrion and the Gram-negative, Q fever pathogen Coxiella burnetii. We identify four novel mitochondrially-targeted C. burnetii effector proteins, renamed Mitochondrial Coxiella effector protein (Mce) B to E. Examination of the subcellular localization of ectopically expressed proteins confirmed their mitochondrial localization, demonstrating the robustness of our approach. Subsequent biochemical analysis and affinity enrichment proteomics of one of these effector proteins, MceC, revealed the protein localizes to the inner membrane and can interact with components of the mitochondrial quality control machinery. Our study adapts high-sensitivity proteomics to study intracellular host-pathogen interactions, providing a robust strategy to examine the subcellular localization of effector proteins during native infection. This approach could be applied to a range of pathogens and host cell compartments to provide a rich map of effector dynamics throughout infection.

Uncovering targeting priority to yeast peroxisomes using an in-cell competition assay.

Approximately half of eukaryotic proteins reside in organelles. To reach their correct destination, such proteins harbor targeting signals recognized by dedicated targeting pathways. It has been shown that differences in targeting signals alter the efficiency in which proteins are recognized and targeted. Since multiple proteins compete for any single pathway, such differences can affect the priority for which a protein is catered. However, to date the entire repertoire of proteins with targeting priority, and the mechanisms underlying it, have not been explored for any pathway. Here we developed a systematic tool to study targeting priority and used the Pex5-mediated targeting to yeast peroxisomes as a model. We titrated Pex5 out by expressing high levels of a Pex5-cargo protein and examined how the localization of each peroxisomal protein is affected. We found that while most known Pex5 cargo proteins were outcompeted, several cargo proteins were not affected, implying that they have high targeting priority. This priority group was dependent on metabolic conditions. We dissected the mechanism of priority for these proteins and suggest that targeting priority is governed by different parameters, including binding affinity of the targeting signal to the cargo factor, the number of binding interfaces to the cargo factor, and more. This approach can be modified to study targeting priority in various organelles, cell types, and organisms.