SOD1 integrates signals from oxygen and glucose to repress respiration.

Cu/Zn superoxide dismutase (SOD1) is an abundant enzyme that has been best studied as a regulator of antioxidant defense. Using the yeast Saccharomyces cerevisiae, we report that SOD1 transmits signals from oxygen and glucose to repress respiration. The mechanism involves SOD1-mediated stabilization of two casein kinase 1-gamma (CK1γ) homologs, Yck1p and Yck2p, required for respiratory repression. SOD1 binds a C-terminal degron we identified in Yck1p/Yck2p and promotes kinase stability by catalyzing superoxide conversion to peroxide. The effects of SOD1 on CK1γ stability are also observed with mammalian SOD1 and CK1γ and in a human cell line. Therefore, in a single circuit, oxygen, glucose, and reactive oxygen can repress respiration through SOD1/CK1γ signaling. Our data therefore may provide mechanistic insight into how rapidly proliferating cells and many cancers accomplish glucose-mediated repression of respiration in favor of aerobic glycolysis.

HRG-9 homologues regulate haem trafficking from haem-enriched compartments.

Haem is an iron-containing tetrapyrrole that is critical for a variety of cellular and physiological processes1-3. Haem binding proteins are present in almost all cellular compartments, but the molecular mechanisms that regulate the transport and use of haem within the cell remain poorly understood2,3. Here we show that haem-responsive gene 9 (HRG-9) (also known as transport and Golgi organization 2 (TANGO2)) is an evolutionarily conserved haem chaperone with a crucial role in trafficking haem out of haem storage or synthesis sites in eukaryotic cells. Loss of Caenorhabditis elegans hrg-9 and its paralogue hrg-10 results in the accumulation of haem in lysosome-related organelles, the haem storage site in worms. Similarly, deletion of the hrg-9 homologue TANGO2 in yeast and mammalian cells induces haem overload in mitochondria, the site of haem synthesis. We demonstrate that TANGO2 binds haem and transfers it from cellular membranes to apo-haemoproteins. Notably, homozygous tango2-/- zebrafish larvae develop pleiotropic symptoms including encephalopathy, cardiac arrhythmia and myopathy, and die during early development. These defects partially resemble the symptoms of human TANGO2-related metabolic encephalopathy and arrhythmias, a hereditary disease caused by mutations in TANGO24-8. Thus, the identification of HRG-9 as an intracellular haem chaperone provides a biological basis for exploring the aetiology and treatment of TANGO2-related disorders.

Sod1 integrates oxygen availability to redox regulate NADPH production and the thiol redoxome.

Cu/Zn superoxide dismutase (Sod1) is a highly conserved and abundant antioxidant enzyme that detoxifies superoxide (O2•-) by catalyzing its conversion to dioxygen (O2) and hydrogen peroxide (H2O2). Using Saccharomyces cerevisiae and mammalian cells, we discovered that a major aspect of the antioxidant function of Sod1 is to integrate O2 availability to promote NADPH production. The mechanism involves Sod1-derived H2O2 oxidatively inactivating the glycolytic enzyme, GAPDH, which in turn reroutes carbohydrate flux to the oxidative phase of the pentose phosphate pathway (oxPPP) to generate NADPH. The aerobic oxidation of GAPDH is dependent on and rate-limited by Sod1. Thus, Sod1 senses O2 via O2•- to balance glycolytic and oxPPP flux, through control of GAPDH activity, for adaptation to life in air. Importantly, this mechanism for Sod1 antioxidant activity requires the bulk of cellular Sod1, unlike for its role in protection against O2•- toxicity, which only requires <1% of total Sod1. Using mass spectrometry, we identified proteome-wide targets of Sod1-dependent redox signaling, including numerous metabolic enzymes. Altogether, Sod1-derived H2O2 is important for antioxidant defense and a master regulator of metabolism and the thiol redoxome.

Heme oxygenase-2 (HO-2) binds and buffers labile ferric heme in human embryonic kidney cells.

Heme oxygenases (HOs) detoxify heme by oxidatively degrading it into carbon monoxide, iron, and biliverdin, which is reduced to bilirubin and excreted. Humans express two isoforms of HO: the inducible HO-1, which is upregulated in response to excess heme and other stressors, and the constitutive HO-2. Much is known about the regulation and physiological function of HO-1, whereas comparatively little is known about the role of HO-2 in regulating heme homeostasis. The biochemical necessity for expressing constitutive HO-2 is dependent on whether heme is sufficiently abundant and accessible as a substrate under conditions in which HO-1 is not induced. By measuring labile heme, total heme, and bilirubin in human embryonic kidney HEK293 cells with silenced or overexpressed HO-2, as well as various HO-2 mutant alleles, we found that endogenous heme is too limiting a substrate to observe HO-2-dependent heme degradation. Rather, we discovered a novel role for HO-2 in the binding and buffering of heme. Taken together, in the absence of excess heme, we propose that HO-2 regulates heme homeostasis by acting as a heme buffering factor that controls heme bioavailability. When heme is in excess, HO-1 is induced, and both HO-2 and HO-1 can provide protection from heme toxicity via enzymatic degradation. Our results explain why catalytically inactive mutants of HO-2 are cytoprotective against oxidative stress. Moreover, the change in bioavailable heme due to HO-2 overexpression, which selectively binds ferric over ferrous heme, is consistent with labile heme being oxidized, thereby providing new insights into heme trafficking and signaling.

Paradoxical Roles of Antioxidant Enzymes: Basic Mechanisms and Health Implications.

Reactive oxygen species (ROS) and reactive nitrogen species (RNS) are generated from aerobic metabolism, as a result of accidental electron leakage as well as regulated enzymatic processes. Because ROS/RNS can induce oxidative injury and act in redox signaling, enzymes metabolizing them will inherently promote either health or disease, depending on the physiological context. It is thus misleading to consider conventionally called antioxidant enzymes to be largely, if not exclusively, health protective. Because such a notion is nonetheless common, we herein attempt to rationalize why this simplistic view should be avoided. First we give an updated summary of physiological phenotypes triggered in mouse models of overexpression or knockout of major antioxidant enzymes. Subsequently, we focus on a series of striking cases that demonstrate "paradoxical" outcomes, i.e., increased fitness upon deletion of antioxidant enzymes or disease triggered by their overexpression. We elaborate mechanisms by which these phenotypes are mediated via chemical, biological, and metabolic interactions of the antioxidant enzymes with their substrates, downstream events, and cellular context. Furthermore, we propose that novel treatments of antioxidant enzyme-related human diseases may be enabled by deliberate targeting of dual roles of the pertaining enzymes. We also discuss the potential of "antioxidant" nutrients and phytochemicals, via regulating the expression or function of antioxidant enzymes, in preventing, treating, or aggravating chronic diseases. We conclude that "paradoxical" roles of antioxidant enzymes in physiology, health, and disease derive from sophisticated molecular mechanisms of redox biology and metabolic homeostasis. Simply viewing antioxidant enzymes as always being beneficial is not only conceptually misleading but also clinically hazardous if such notions underpin medical treatment protocols based on modulation of redox pathways.

Mitochondrial-nuclear heme trafficking in budding yeast is regulated by GTPases that control mitochondrial dynamics and ER contact sites.

Heme is a cofactor and signaling molecule that is essential for much of aerobic life. All heme-dependent processes in eukaryotes require that heme is trafficked from its site of synthesis in the mitochondria to hemoproteins located throughout the cell. However, the mechanisms governing the mobilization of heme out of the mitochondria, and the spatio-temporal dynamics of these processes, are poorly understood. Here, using genetically encoded fluorescent heme sensors, we developed a live-cell assay to monitor heme distribution dynamics between the mitochondrial inner membrane, where heme is synthesized, and the mitochondrial matrix, cytosol and nucleus. Surprisingly, heme trafficking to the nucleus is ∼25% faster than to the cytosol or mitochondrial matrix, which have nearly identical heme trafficking dynamics, potentially supporting a role for heme as a mitochondrial-nuclear retrograde signal. Moreover, we discovered that the heme synthetic enzyme 5-aminolevulinic acid synthase (ALAS, also known as Hem1 in yeast), and GTPases in control of the mitochondrial dynamics machinery (Mgm1 and Dnm1) and ER contact sites (Gem1), regulate the flow of heme between the mitochondria and nucleus. Overall, our results indicate that there are parallel pathways for the distribution of bioavailable heme.This article has an associated First Person interview with the first author of the paper.

Using genetically encoded heme sensors to probe the mechanisms of heme uptake and homeostasis in Candida albicans.

Candida albicans is a major fungal pathogen that can utilise hemin and haemoglobin as iron sources in the iron-scarce host environment. While C. albicans is a heme prototroph, we show here that it can also efficiently utilise external heme as a cellular heme source. Using genetically encoded ratiometric fluorescent heme sensors, we show that heme extracted from haemoglobin and free hemin enter the cells with different kinetics. Heme supplied as haemoglobin is taken up via the Common in Fungal Extracellular Membrane (CFEM) hemophore cascade, and reaches the cytoplasm over several hours, whereas entry of free hemin via CFEM-dependent and independent pathways is much faster, less than an hour. To prevent an influx of extracellular heme from reaching toxic levels in the cytoplasm, the cells deploy Hmx1, a heme oxygenase. Hmx1 was previously suggested to be involved in utilisation of haemoglobin and hemin as iron sources, but we find that it is primarily required to prevent heme toxicity. Taken together, the combination of novel heme sensors with genetic analysis revealed new details of the fungal mechanisms of heme import and homeostasis, necessary to balance the uses of heme as essential cofactor and potential iron source against its toxicity.

Cutting in-line with iron: ribosomal function and non-oxidative RNA cleavage.

Divalent metal cations are essential to the structure and function of the ribosome. Previous characterizations of the ribosome performed under standard laboratory conditions have implicated Mg2+ as a primary mediator of ribosomal structure and function. Possible contributions of Fe2+ as a ribosomal cofactor have been largely overlooked, despite the ribosome's early evolution in a high Fe2+ environment, and the continued use of Fe2+ by obligate anaerobes inhabiting high Fe2+ niches. Here, we show that (i) Fe2+ cleaves RNA by in-line cleavage, a non-oxidative mechanism that has not previously been shown experimentally for this metal, (ii) the first-order in-line rate constant with respect to divalent cations is >200 times greater with Fe2+ than with Mg2+, (iii) functional ribosomes are associated with Fe2+ after purification from cells grown under low O2 and high Fe2+ and (iv) a small fraction of Fe2+ that is associated with the ribosome is not exchangeable with surrounding divalent cations, presumably because those ions are tightly coordinated by rRNA and deeply buried in the ribosome. In total, these results expand the ancient role of iron in biochemistry and highlight a possible new mechanism of iron toxicity.

Human ribosomal G-quadruplexes regulate heme bioavailability.

The in vitro formation of stable G-quadruplexes (G4s) in human rRNA was recently reported. However, their formation in cells and their cellular roles were not resolved. Here, by taking a chemical biology approach that integrates results from immunofluorescence, G4 ligands, heme-affinity reagents, and a genetically encoded fluorescent heme sensor, we report that human ribosomes can form G4s in vivo that regulate heme bioavailability. Immunofluorescence experiments indicate that the vast majority of extra-nuclear G4s are associated with rRNA. Moreover, titrating human cells with a G4 ligand alters the ability of ribosomes to bind heme and disrupts cellular heme bioavailability as measured by a genetically encoded fluorescent heme sensor. Overall, these results suggest that ribosomes play a role in regulating heme homeostasis.

Cu/Zn Superoxide Dismutase (Sod1) regulates the canonical Wnt signaling pathway.

Cu/Zn Superoxide Dismutase (Sod1) catalyzes the disproportionation of cytotoxic superoxide radicals (O2•-) into oxygen (O2) and hydrogen peroxide (H2O2), a key signaling molecule. In Saccharomyces cerevisiae, we previously discovered that Sod1 participates in an H2O2-mediated redox signaling circuit that links nutrient availability to the control of energy metabolism. In response to glucose and O2, Sod1-derived H2O2 stabilizes a pair of conserved plasma membrane kinases - yeast casein kinase 1 and 2 (Yck1/2) - that signal glycolytic growth and the repression of respiration. The Yck1/2 homolog in humans, casein kinase 1-γ (CK1γ), is an integral component of the Wingless and Int-1 (Wnt) signaling pathway, which is essential for regulating cell fate and proliferation in early development and adult tissue and is dysregulated in many cancers. Herein, we establish the conservation of the SOD1/YCK1 redox signaling axis in humans by finding that SOD1 regulates CK1γ expression in human embryonic kidney 293 (HEK293) cells and is required for canonical Wnt signaling and Wnt-dependent cell proliferation.

Multiple prebiotic metals mediate translation.

Today, Mg2+ is an essential cofactor with diverse structural and functional roles in life's oldest macromolecular machine, the translation system. We tested whether ancient Earth conditions (low O2, high Fe2+, and high Mn2+) can revert the ribosome to a functional ancestral state. First, SHAPE (selective 2'-hydroxyl acylation analyzed by primer extension) was used to compare the effect of Mg2+, Fe2+, and Mn2+ on the tertiary structure of rRNA. Then, we used in vitro translation reactions to test whether Fe2+ or Mn2+ could mediate protein production, and quantified ribosomal metal content. We found that (i) Mg2+, Fe2+, and Mn2+ had strikingly similar effects on rRNA folding; (ii) Fe2+ and Mn2+ can replace Mg2+ as the dominant divalent cation during translation of mRNA to functional protein; and (iii) Fe and Mn associate extensively with the ribosome. Given that the translation system originated and matured when Fe2+ and Mn2+ were abundant, these findings suggest that Fe2+ and Mn2+ played a role in early ribosomal evolution.

Heme and hemoglobin suppress amyloid ß-mediated inflammatory activation of mouse astrocytes.

Glial immune activity is a key feature of Alzheimer's disease (AD). Given that the blood factors heme and hemoglobin (Hb) are both elevated in AD tissues and have immunomodulatory roles, here we sought to interrogate their roles in modulating ß-amyloid (Aß)-mediated inflammatory activation of astrocytes. We discovered that heme and Hb suppress immune activity of primary mouse astrocytes by reducing expression of several proinflammatory cytokines (e.g. RANTES (regulated on activation normal T cell expressed and secreted)) and the scavenger receptor CD36 and reducing internalization of Aß(1-42) by astrocytes. Moreover, we found that certain soluble (>75-kDa) Aß(1-42) oligomers are primarily responsible for astrocyte activation and that heme or Hb association with these oligomers reverses inflammation. We further found that heme up-regulates phosphoprotein signaling in the phosphoinositide 3-kinase (PI3K)/Akt pathway, which regulates a number of immune functions, including cytokine expression and phagocytosis. The findings in this work suggest that dysregulation of Hb and heme levels in AD brains may contribute to impaired amyloid clearance and that targeting heme homeostasis may reduce amyloid pathogenesis. Altogether, we propose heme as a critical molecular link between amyloid pathology and AD risk factors, such as aging, brain injury, and stroke, which increase Hb and heme levels in the brain.

Heme bioavailability and signaling in response to stress in yeast cells.

Protoheme (hereafter referred to as heme) is an essential cellular cofactor and signaling molecule that is also potentially cytotoxic. To mitigate heme toxicity, heme synthesis and degradation are tightly coupled to heme utilization in order to limit the intracellular concentration of "free" heme. Such a model, however, would suggest that a readily accessible steady-state, bioavailable labile heme (LH) pool is not required for supporting heme-dependent processes. Using the yeast Saccharomyces cerevisiae as a model and fluorescent heme sensors, site-specific heme chelators, and molecular genetic approaches, we found here that 1) yeast cells preferentially use LH in heme-depleted conditions; 2) sequestration of cytosolic LH suppresses heme signaling; and 3) lead (Pb2+) stress contributes to a decrease in total heme, but an increase in LH, which correlates with increased heme signaling. We also observed that the proteasome is involved in the regulation of the LH pool and that loss of proteasomal activity sensitizes cells to Pb2+ effects on heme homeostasis. Overall, these findings suggest an important role for LH in supporting heme-dependent functions in yeast physiology.

Glyceraldehyde-3-phosphate dehydrogenase is a chaperone that allocates labile heme in cells.

Cellular heme is thought to be distributed between a pool of sequestered heme that is tightly bound within hemeproteins and a labile heme pool required for signaling and transfer into proteins. A heme chaperone that can hold and allocate labile heme within cells has long been proposed but never been identified. Here, we show that the glycolytic protein glyceraldehyde-3-phosphate dehydrogenase (GAPDH) fulfills this role by acting as an essential repository and allocator of bioavailable heme to downstream protein targets. We identified a conserved histidine in GAPDH that is needed for its robust heme binding both in vitro and in mammalian cells. Substitution of this histidine, and the consequent decreases in GAPDH heme binding, antagonized heme delivery to both cytosolic and nuclear hemeprotein targets, including inducible nitric-oxide synthase (iNOS) in murine macrophages and the nuclear transcription factor Hap1 in yeast, even though this GAPDH variant caused cellular levels of labile heme to rise dramatically. We conclude that by virtue of its heme-binding property, GAPDH binds and chaperones labile heme to create a heme pool that is bioavailable to downstream proteins. Our finding solves a fundamental question in cell biology and provides a new foundation for exploring heme homeostasis in health and disease.

Heme dynamics and trafficking factors revealed by genetically encoded fluorescent heme sensors.

Heme is an essential cofactor and signaling molecule. Heme acquisition by proteins and heme signaling are ultimately reliant on the ability to mobilize labile heme (LH). However, the properties of LH pools, including concentration, oxidation state, distribution, speciation, and dynamics, are poorly understood. Herein, we elucidate the nature and dynamics of LH using genetically encoded ratiometric fluorescent heme sensors in the unicellular eukaryote Saccharomyces cerevisiae We find that the subcellular distribution of LH is heterogeneous; the cytosol maintains LH at ∼20-40 nM, whereas the mitochondria and nucleus maintain it at concentrations below 2.5 nM. Further, we find that the signaling molecule nitric oxide can initiate the rapid mobilization of heme in the cytosol and nucleus from certain thiol-containing factors. We also find that the glycolytic enzyme glyceraldehyde phosphate dehydrogenase constitutes a major cellular heme buffer, and is responsible for maintaining the activity of the heme-dependent nuclear transcription factor heme activator protein (Hap1p). Altogether, we demonstrate that the heme sensors can be used to reveal fundamental aspects of heme trafficking and dynamics and can be used across multiple organisms, including Escherichia coli, yeast, and human cell lines.

Regulation of intracellular heme trafficking revealed by subcellular reporters.

Heme is an essential prosthetic group in proteins that reside in virtually every subcellular compartment performing diverse biological functions. Irrespective of whether heme is synthesized in the mitochondria or imported from the environment, this hydrophobic and potentially toxic metalloporphyrin has to be trafficked across membrane barriers, a concept heretofore poorly understood. Here we show, using subcellular-targeted, genetically encoded hemoprotein peroxidase reporters, that both extracellular and endogenous heme contribute to cellular labile heme and that extracellular heme can be transported and used in toto by hemoproteins in all six subcellular compartments examined. The reporters are robust, show large signal-to-background ratio, and provide sufficient range to detect changes in intracellular labile heme. Restoration of reporter activity by heme is organelle-specific, with the Golgi and endoplasmic reticulum being important sites for both exogenous and endogenous heme trafficking. Expression of peroxidase reporters in Caenorhabditis elegans shows that environmental heme influences labile heme in a tissue-dependent manner; reporter activity in the intestine shows a linear increase compared with muscle or hypodermis, with the lowest heme threshold in neurons. Our results demonstrate that the trafficking pathways for exogenous and endogenous heme are distinct, with intrinsic preference for specific subcellular compartments. We anticipate our results will serve as a heuristic paradigm for more sophisticated studies on heme trafficking in cellular and whole-animal models.

Heme Gazing: Illuminating Eukaryotic Heme Trafficking, Dynamics, and Signaling with Fluorescent Heme Sensors.

Heme (iron protoporphyrin IX) is an essential protein prosthetic group and signaling molecule required for most life on Earth. All heme-dependent processes require the dynamic and rapid mobilization of heme from sites of synthesis or uptake to hemoproteins present in virtually every subcellular compartment. The cytotoxicity and hydrophobicity of heme necessitate that heme mobilization be carefully controlled to mitigate the deleterious effects of this essential toxin. Indeed, a number of disorders, including certain cancers, cardiovascular diseases, and aging and age-related neurodegenerative diseases, are tied to defects in heme homeostasis. However, the molecules and mechanisms that mediate heme transport and trafficking, and the dynamics of these processes, are poorly understood. This is in large part due to the lack of physical tools for probing cellular heme. Herein, we discuss the recent development of fluorescent probes that can monitor and image kinetically labile heme with respect to its mobilization and role in signaling. In particular, we will highlight how heme gazing with these tools can uncover new heme trafficking factors upon being integrated with genetic screens and illuminate the concentration, subcellular distribution, and dynamics of labile heme in various physiological contexts. Altogether, the monitoring of labile heme, along with recent biochemical and cell biological studies demonstrating the reversible regulation of certain cellular processes by heme, is challenging us to reconceptualize heme from being a static cofactor buried in protein active sites to a dynamic and mobile signaling molecule.

Heme Mobilization in Animals: A Metallolipid's Journey.

Heme is universally recognized as an essential and ubiquitous prosthetic group that enables proteins to carry out a diverse array of functions. All heme-dependent processes, from protein hemylation to heme signaling, require the dynamic and rapid mobilization of heme to hemoproteins present in virtually every subcellular compartment. The cytotoxicity and hydrophobicity of heme necessitates that heme mobilization is carefully controlled at the cellular and systemic level. However, the molecules and mechanisms that mediate heme homeostasis are poorly understood. In this Account, we provide a heuristic paradigm with which to conceptualize heme trafficking and highlight the most recent developments in the mechanisms underlying heme trafficking. As an iron-containing tetrapyrrole, heme exhibits properties of both transition metals and lipids. Accordingly, we propose its transport and trafficking will reflect principles gleaned from the trafficking of both metals and lipids. Using this conceptual framework, we follow the flow of heme from the final step of heme synthesis in the mitochondria to hemoproteins present in various subcellular organelles. Further, given that many cells and animals that cannot make heme can assimilate it intact from nutritional sources, we propose that intercellular heme trafficking pathways must exist. This necessitates that heme be able to be imported and exported from cells, escorted between cells and organs, and regulated at the organismal level via a coordinated systemic process. In this Account, we highlight recently discovered heme transport and trafficking factors and provide the biochemical foundation for the cell and systems biology of heme. Altogether, we seek to reconceptualize heme from an exchange inert cofactor buried in hemoprotein active sites to an exchange labile and mobile metallonutrient.

Evaluation of the Intrinsic Zn(II) Affinity of a Cys3His1 Site in the Absence of Protein Folding Effects.

Zinc finger transcription factors are the largest class of metalloproteins in the human genome. Binding of Zn(II) to their canonical Cys2His2, Cys3His1, or Cys4 sites results in metal-induced protein folding events required to achieve their biologically active structures. However, the coupled nature of metal binding and protein folding obscures the individual free energy contributions of each process toward overall zinc finger stabilization. Herein, we separate the energetic contributions of metal-ligand interactions from those of protein-protein interactions using a natural protein scaffold that retains essentially identical structures with and without Zn(II) bound, the 59 amino acid zinc binding domain of human transcription factor IIB (ZBD-TFIIB). The formation constant of Zn(II)-ZBD-TFIIB, which contains a single Cys3His1 site, was determined to be 1.5 × 10(15) M(-1) via fluorimetry and isothermal titration calorimetry. Isothermal titration calorimetry showed that Zn(II) binding is entropically favored at pH 5.5, 7.0, and 8.0 and enthalpically favored at pH 8.0 but slightly enthalpically disfavored at pH 5.5 and 7.0. The conditional dissociation constants of Zn(II)-ZBD-TFIIB and natural Cys3His1 zinc finger proteins were compared to determine the free energy cost of protein folding in the latter. Our analysis reveals that the energetic cost to fold zinc finger proteins is minimal relative to the contribution of Zn(II) binding and suggests that the true role of Zn(II) binding may be to modulate protein dynamics and/or kinetically template the protein folding process.

The molecular mechanism of on-demand sterol biosynthesis at organelle contact sites.

Contact-sites are specialized zones of proximity between two organelles, essential for organelle communication and coordination. The formation of contacts between the Endoplasmic Reticulum (ER), and other organelles, relies on a unique membrane environment enriched in sterols. However, how these sterol-rich domains are formed and maintained had not been understood. We found that the yeast membrane protein Yet3, the homolog of human BAP31, is localized to multiple ER contact sites. We show that Yet3 interacts with all the enzymes of the post-squalene ergosterol biosynthesis pathway and recruits them to create sterol-rich domains. Increasing sterol levels at ER contacts causes its depletion from the plasma membrane leading to a compensatory reaction and altered cell metabolism. Our data shows that Yet3 provides on-demand sterols at contacts thus shaping organellar structure and function. A molecular understanding of this protein's functions gives new insights into the role of BAP31 in development and pathology.