METTL17 is an Fe-S cluster checkpoint for mitochondrial translation.

Friedreich's ataxia (FA) is a debilitating, multisystemic disease caused by the depletion of frataxin (FXN), a mitochondrial iron-sulfur (Fe-S) cluster biogenesis factor. To understand the cellular pathogenesis of FA, we performed quantitative proteomics in FXN-deficient human cells. Nearly every annotated Fe-S cluster-containing protein was depleted, indicating that as a rule, cluster binding confers stability to Fe-S proteins. We also observed depletion of a small mitoribosomal assembly factor METTL17 and evidence of impaired mitochondrial translation. Using comparative sequence analysis, mutagenesis, biochemistry, and cryoelectron microscopy, we show that METTL17 binds to the mitoribosomal small subunit during late assembly and harbors a previously unrecognized [Fe4S4]2+ cluster required for its stability. METTL17 overexpression rescued the mitochondrial translation and bioenergetic defects, but not the cellular growth, of FXN-depleted cells. These findings suggest that METTL17 acts as an Fe-S cluster checkpoint, promoting translation of Fe-S cluster-rich oxidative phosphorylation (OXPHOS) proteins only when Fe-S cofactors are replete.

Continuous, but not intermittent, regimens of hypoxia prevent and reverse ataxia in a murine model of Friedreich's ataxia.

Friedreich's ataxia (FA) is a devastating, multi-systemic neurodegenerative disease affecting thousands of people worldwide. We previously reported that oxygen is a key environmental variable that can modify FA pathogenesis. In particular, we showed that chronic, continuous normobaric hypoxia (11% FIO2) prevents ataxia and neurological disease in a murine model of FA, although it did not improve cardiovascular pathology or lifespan. Here, we report the pre-clinical evaluation of seven 'hypoxia-inspired' regimens in the shFxn mouse model of FA, with the long-term goal of designing a safe, practical and effective regimen for clinical translation. We report three chief results. First, a daily, intermittent hypoxia regimen (16 h 11% O2/8 h 21% O2) conferred no benefit and was in fact harmful, resulting in elevated cardiac stress and accelerated mortality. The detrimental effect of this regimen is likely owing to transient tissue hyperoxia that results when daily exposure to 21% O2 combines with chronic polycythemia, as we could blunt this toxicity by pharmacologically inhibiting polycythemia. Second, we report that more mild regimens of chronic hypoxia (17% O2) confer a modest benefit by delaying the onset of ataxia. Third, excitingly, we show that initiating chronic, continuous 11% O2 breathing once advanced neurological disease has already started can rapidly reverse ataxia. Our studies showcase both the promise and limitations of candidate hypoxia-inspired regimens for FA and underscore the need for additional pre-clinical optimization before future translation into humans.

CYB561A3 is the key lysosomal iron reductase required for Burkitt B-cell growth and survival.

Epstein-Barr virus (EBV) causes endemic Burkitt lymphoma, the leading childhood cancer in sub-Saharan Africa. Burkitt cells retain aspects of germinal center B-cell physiology with MYC-driven B-cell hyperproliferation; however, little is presently known about their iron metabolism. CRISPR/Cas9 analysis highlighted the little-studied ferrireductase CYB561A3 as critical for Burkitt proliferation but not for that of the closely related EBV-transformed lymphoblastoid cells or nearly all other Cancer Dependency Map cell lines. Burkitt CYB561A3 knockout induced profound iron starvation, despite ferritinophagy ad plasma membrane transferrin upregulation. Elevated concentrations of ascorbic acid, a key CYB561 family electron donor, or the labile iron source ferrous citrate rescued Burkitt CYB561A3 deficiency. CYB561A3 knockout caused catastrophic lysosomal and mitochondrial damage and impaired mitochondrial respiration. Conversely, lymphoblastoid B cells with the transforming EBV latency III program were instead dependent on the STEAP3 ferrireductase. These results highlight CYB561A3 as an attractive therapeutic Burkitt lymphoma target.

MitoCarta3.0: an updated mitochondrial proteome now with sub-organelle localization and pathway annotations.

The mammalian mitochondrial proteome is under dual genomic control, with 99% of proteins encoded by the nuclear genome and 13 originating from the mitochondrial DNA (mtDNA). We previously developed MitoCarta, a catalogue of over 1000 genes encoding the mammalian mitochondrial proteome. This catalogue was compiled using a Bayesian integration of multiple sequence features and experimental datasets, notably protein mass spectrometry of mitochondria isolated from fourteen murine tissues. Here, we introduce MitoCarta3.0. Beginning with the MitoCarta2.0 inventory, we performed manual review to remove 100 genes and introduce 78 additional genes, arriving at an updated inventory of 1136 human genes. We now include manually curated annotations of sub-mitochondrial localization (matrix, inner membrane, intermembrane space, outer membrane) as well as assignment to 149 hierarchical 'MitoPathways' spanning seven broad functional categories relevant to mitochondria. MitoCarta3.0, including sub-mitochondrial localization and MitoPathway annotations, is freely available at http://www.broadinstitute.org/mitocarta and should serve as a continued community resource for mitochondrial biology and medicine.

Genetic Screen for Cell Fitness in High or Low Oxygen Highlights Mitochondrial and Lipid Metabolism.

Human cells are able to sense and adapt to variations in oxygen levels. Historically, much research in this field has focused on hypoxia-inducible factor (HIF) signaling and reactive oxygen species (ROS). Here, we perform genome-wide CRISPR growth screens at 21%, 5%, and 1% oxygen to systematically identify gene knockouts with relative fitness defects in high oxygen (213 genes) or low oxygen (109 genes), most without known connection to HIF or ROS. Knockouts of many mitochondrial pathways thought to be essential, including complex I and enzymes in Fe-S biosynthesis, grow relatively well at low oxygen and thus are buffered by hypoxia. In contrast, in certain cell types, knockout of lipid biosynthetic and peroxisomal genes causes fitness defects only in low oxygen. Our resource nominates genetic diseases whose severity may be modulated by oxygen and links hundreds of genes to oxygen homeostasis.

Leigh Syndrome Mouse Model Can Be Rescued by Interventions that Normalize Brain Hyperoxia, but Not HIF Activation.

Leigh syndrome is a devastating mitochondrial disease for which there are no proven therapies. We previously showed that breathing chronic, continuous hypoxia can prevent and even reverse neurological disease in the Ndufs4 knockout (KO) mouse model of complex I (CI) deficiency and Leigh syndrome. Here, we show that genetic activation of the hypoxia-inducible factor transcriptional program via any of four different strategies is insufficient to rescue disease. Rather, we observe an age-dependent decline in whole-body oxygen consumption. These mice exhibit brain tissue hyperoxia, which is normalized by hypoxic breathing. Alternative experimental strategies to reduce oxygen delivery, including breathing carbon monoxide (600 ppm in air) or severe anemia, can reverse neurological disease. Therefore, unused oxygen is the most likely culprit in the pathology of this disease. While pharmacologic activation of the hypoxia response is unlikely to alleviate disease in vivo, interventions that safely normalize brain tissue hyperoxia may hold therapeutic potential.

Hypoxia Rescues Frataxin Loss by Restoring Iron Sulfur Cluster Biogenesis.

Friedreich's ataxia (FRDA) is a devastating, multisystemic disorder caused by recessive mutations in the mitochondrial protein frataxin (FXN). FXN participates in the biosynthesis of Fe-S clusters and is considered to be essential for viability. Here we report that when grown in 1% ambient O2, FXN null yeast, human cells, and nematodes are fully viable. In human cells, hypoxia restores steady-state levels of Fe-S clusters and normalizes ATF4, NRF2, and IRP2 signaling events associated with FRDA. Cellular studies and in vitro reconstitution indicate that hypoxia acts through HIF-independent mechanisms that increase bioavailable iron as well as directly activate Fe-S synthesis. In a mouse model of FRDA, breathing 11% O2 attenuates the progression of ataxia, whereas breathing 55% O2 hastens it. Our work identifies oxygen as a key environmental variable in the pathogenesis associated with FXN depletion, with important mechanistic and therapeutic implications.

Natural genetic variation for expression of a SWEET transporter among wild species of Solanum lycopersicum (tomato) determines the hexose composition of ripening tomato fruit.

The sugar content of Solanum lycopersicum (tomato) fruit is a primary determinant of taste and quality. Cultivated tomato fruit are characterized by near-equimolar levels of the hexoses glucose and fructose, derived from the hydrolysis of translocated sucrose. As fructose is perceived as approximately twice as sweet as glucose, increasing its concentration at the expense of glucose can improve tomato fruit taste. Introgressions of the FgrH allele from the wild species Solanum habrochaites (LA1777) into cultivated tomato increased the fructose-to-glucose ratio of the ripe fruit by reducing glucose levels and concomitantly increasing fructose levels. In order to identify the function of the Fgr gene, we combined a fine-mapping strategy with RNAseq differential expression analysis of near-isogenic tomato lines. The results indicated that a SWEET protein was strongly upregulated in the lines with a high fructose-to-glucose ratio. Overexpressing the SWEET protein in transgenic tomato plants dramatically reduced the glucose levels and increased the fructose : glucose ratio in the developing fruit, thereby proving the function of the protein. The SWEET protein was localized to the plasma membrane and expression of the SlFgr gene in a yeast line lacking native hexose transporters complemented growth with glucose, but not with fructose. These results indicate that the SlFgr gene encodes a plasma membrane-localized glucose efflux transporter of the SWEET family, the overexpression of which reduces glucose levels and may allow for increased fructose levels. This article identifies the function of the tomato Fgr gene as a SWEET transporter, the upregulation of which leads to a modified sugar accumulation pattern in the fleshy fruit. The results point to the potential of the inedible wild species to improve fruit sugar accumulation via sugar transport mechanisms.

Antibodies to biotin enable large-scale detection of biotinylation sites on proteins.

Although purification of biotinylated molecules is highly efficient, identifying specific sites of biotinylation remains challenging. We show that anti-biotin antibodies enable unprecedented enrichment of biotinylated peptides from complex peptide mixtures. Live-cell proximity labeling using APEX peroxidase followed by anti-biotin enrichment and mass spectrometry yielded over 1,600 biotinylation sites on hundreds of proteins, an increase of more than 30-fold in the number of biotinylation sites identified compared to streptavidin-based enrichment of proteins.

The SND proteins constitute an alternative targeting route to the endoplasmic reticulum.

In eukaryotes, up to one-third of cellular proteins are targeted to the endoplasmic reticulum, where they undergo folding, processing, sorting and trafficking to subsequent endomembrane compartments. Targeting to the endoplasmic reticulum has been shown to occur co-translationally by the signal recognition particle (SRP) pathway or post-translationally by the mammalian transmembrane recognition complex of 40 kDa (TRC40) and homologous yeast guided entry of tail-anchored proteins (GET) pathways. Despite the range of proteins that can be catered for by these two pathways, many proteins are still known to be independent of both SRP and GET, so there seems to be a critical need for an additional dedicated pathway for endoplasmic reticulum relay. We set out to uncover additional targeting proteins using unbiased high-content screening approaches. To this end, we performed a systematic visual screen using the yeast Saccharomyces cerevisiae, and uncovered three uncharacterized proteins whose loss affected targeting. We suggest that these proteins work together and demonstrate that they function in parallel with SRP and GET to target a broad range of substrates to the endoplasmic reticulum. The three proteins, which we name Snd1, Snd2 and Snd3 (for SRP-independent targeting), can synthetically compensate for the loss of both the SRP and GET pathways, and act as a backup targeting system. This explains why it has previously been difficult to demonstrate complete loss of targeting for some substrates. Our discovery thus puts in place an essential piece of the endoplasmic reticulum targeting puzzle, highlighting how the targeting apparatus of the eukaryotic cell is robust, interlinked and flexible.

The Protease Ste24 Clears Clogged Translocons.

Translocation into the endoplasmic reticulum (ER) is the first step in the biogenesis of thousands of eukaryotic endomembrane proteins. Although functional ER translocation has been avidly studied, little is known about the quality control mechanisms that resolve faulty translocational states. One such faulty state is translocon clogging, in which the substrate fails to properly translocate and obstructs the translocon pore. To shed light on the machinery required to resolve clogging, we carried out a systematic screen in Saccharomyces cerevisiae that highlighted a role for the ER metalloprotease Ste24. We could demonstrate that Ste24 approaches the translocon upon clogging, and it interacts with and generates cleavage fragments of the clogged protein. Importantly, these functions are conserved in the human homolog, ZMPSTE24, although disease-associated mutant forms of ZMPSTE24 fail to clear the translocon. These results shed light on a new and critical task of Ste24, which safeguards the essential process of translocation.

The PH gene determines fruit acidity and contributes to the evolution of sweet melons.

Taste has been the subject of human selection in the evolution of agricultural crops, and acidity is one of the three major components of fleshy fruit taste, together with sugars and volatile flavour compounds. We identify a family of plant-specific genes with a major effect on fruit acidity by map-based cloning of C. melo PH gene (CmPH) from melon, Cucumis melo taking advantage of the novel natural genetic variation for both high and low fruit acidity in this species. Functional silencing of orthologous PH genes in two distantly related plant families, cucumber and tomato, produced low-acid, bland tasting fruit, showing that PH genes control fruit acidity across plant families. A four amino-acid duplication in CmPH distinguishes between primitive acidic varieties and modern dessert melons. This fortuitous mutation served as a preadaptive antecedent to the development of sweet melon cultigens in Central Asia over 1,000 years ago.

A cytosolic degradation pathway, prERAD, monitors pre-inserted secretory pathway proteins.

The endoplasmic reticulum (ER) identifies and disposes of misfolded secretory pathway proteins through the actions of ER-associated degradation (ERAD) pathways. It is becoming evident that a substantial fraction of the secretome transiently resides in the cytosol before translocating into the ER, both in yeast and in higher eukaryotes. To uncover factors that monitor this transient cytosolic protein pool, we carried out a genetic screen in Saccharomyces cerevisiae. Our findings highlighted a pre-insertional degradation mechanism at the cytosolic leaflet of the ER, which we term prERAD. prERAD relies on the concurrent action of the ER-localized ubiquitylation and deubiquitylation machineries Doa10 and Ubp1. By recognizing C-terminal hydrophobic motifs, prERAD tags for degradation pre-inserted proteins that have remained on the cytosolic leaflet of the ER for too long. Our discoveries delineate a new cellular safeguard, which ensures that every stage of secretory pathway protein biogenesis is scrutinized and regulated.

Characterization of an M28 metalloprotease family member residing in the yeast vacuole.

The systematic and complete characterization of the Saccharomyces cerevisiae genome and proteome has been stalled in some cases by misannotated genes. One such gene is YBR074W, which was initially annotated as two independent open reading frames (ORFs). We now report on Ybr074, a metalloprotease family member that was initially predicted to reside in the endoplasmic reticulum (ER). Therefore, we tested the hypothesis that Ybr074 may be an ER quality control protease. Instead, indirect immunofluorescence images indicate that Ybr074 is a vacuolar protein, and by employing protease protection assays, we demonstrate that a conserved M28 metalloprotease domain is oriented within the lumen. Involvement of Ybr074 in ER protein quality control was ruled out by examining the stabilities of several well-characterized substrates in strains lacking Ybr074. Finally, using a proteomic approach, we show that disrupting Ybr074 function affects the levels of select factors implicated in vacuolar trafficking and osmoregulation. Together, our data indicate that Ybr074 is the only multispanning vacuolar membrane protease found in the yeast Saccharomyces cerevisiae.

A network of cytosolic factors targets SRP-independent proteins to the endoplasmic reticulum.

Translocation into the endoplasmic reticulum (ER) is an initial and crucial biogenesis step for all secreted and endomembrane proteins in eukaryotes. ER insertion can take place through the well-characterized signal recognition particle (SRP)-dependent pathway or the less-studied route of SRP-independent translocation. To better understand the prevalence of the SRP-independent pathway, we systematically defined the translocational dependence of the yeast secretome. By combining hydropathy-based analysis and microscopy, we uncovered that a previously unappreciated fraction of the yeast secretome translocates without the aid of the SRP. Furthermore, we validated a family of SRP-independent substrates-the glycosylphosphatidylinositol (GPI)-anchored proteins. Studying this family, we identified a determinant for ER targeting and uncovered a network of cytosolic proteins that facilitate SRP-independent targeting and translocation. These findings highlight the underappreciated complexity of SRP-independent translocation, which enables this pathway to efficiently cope with its extensive substrate flux.

All roads lead to Rome (but some may be harder to travel): SRP-independent translocation into the endoplasmic reticulum.

Translocation into the endoplasmic reticulum (ER) is the first biogenesis step for hundreds of eukaryotic secretome proteins. Over the past 30 years, groundbreaking biochemical, structural and genetic studies have delineated one conserved pathway that enables ER translocation- the signal recognition particle (SRP) pathway. However, it is clear that this is not the only pathway which can mediate ER targeting and insertion. In fact, over the past decade, several SRP-independent pathways have been uncovered, which recognize proteins that cannot engage the SRP and ensure their subsequent translocation into the ER. These SRP-independent pathways face the same challenges that the SRP pathway overcomes: chaperoning the preinserted protein while in the cytosol, targeting it rapidly to the ER surface and generating vectorial movement that inserts the protein into the ER. This review strives to summarize the various mechanisms and machineries which mediate these stages of SRP-independent translocation, as well as examine why SRP-independent translocation is utilized by the cell. This emerging understanding of the various pathways utilized by secretory proteins to insert into the ER draws light to the complexity of the translocational task, and underlines that insertion into the ER might be more varied and tailored than previously appreciated.

Protein degradation: BAGging up the trash.

Cells efficiently uncover and degrade proteins that are misfolded. However, we know very little about what cells do to protect themselves from mislocalized proteins. A new study reveals a novel quality control pathway that recognizes and degrades secretory pathway proteins that have failed to target to the endoplasmic reticulum.

COP9 signalosome subunit 7 from Arabidopsis interacts with and regulates the small subunit of ribonucleotide reductase (RNR2).

The COP9 Signalosome protein complex (CSN) is a pleiotropic regulator of plant development and contains eight-subunits. Six of these subunits contain the PCI motif which mediates specific protein interactions necessary for the integrity of the complex. COP9 complex subunit 7 (CSN7) contains an N-terminal PCI motif followed by a C-terminal extension which is also necessary for CSN function. A yeast-interaction trap assay identified the small subunit of ribonucelotide reductase (RNR2) from Arabidopsis as interacting with the C-terminal section of CSN7. This interaction was confirmed in planta by both bimolecular fluorescence complementation and immuoprecipitation assays with endogenous proteins. The subcellular localization of RNR2 was primarily nuclear in meristematic regions, and cytoplasmic in adult cells. RNR2 was constitutively nuclear in csn7 mutant seedlings, and was also primarily nuclear in wild type seedlings following exposure to UV-C. These two results correlate with constitutive expression of several DNA-damage response genes in csn7 mutants, and to increased tolerance of csn7 seedlings to UV-C treatment. We propose that the CSN is a negative regulator of RNR activity in Arabidopsis.