Author Correction: Genome-wide SWAp-Tag yeast libraries for proteome exploration.

The version of Supplementary Table 1 originally published online with this article contained incorrect localization annotations for one plate. This error has been corrected in the online Supplementary Information.

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.

Genome-wide SWAp-Tag yeast libraries for proteome exploration.

Yeast libraries revolutionized the systematic study of cell biology. To extensively increase the number of such libraries, we used our previously devised SWAp-Tag (SWAT) approach to construct a genome-wide library of ~5,500 strains carrying the SWAT NOP1promoter-GFP module at the N terminus of proteins. In addition, we created six diverse libraries that restored the native regulation, created an overexpression library with a Cherry tag, or enabled protein complementation assays from two fragments of an enzyme or fluorophore. We developed methods utilizing these SWAT collections to systematically characterize the yeast proteome for protein abundance, localization, topology, and interactions.

Toolbox: Creating a systematic database of secretory pathway proteins uncovers new cargo for COPI.

A third of yeast genes encode for proteins that function in the endomembrane system. However, the precise localization for many of these proteins is still uncertain. Here, we visualized a collection of ~500 N-terminally, green fluorescent protein (GFP), tagged proteins of the yeast Saccharomyces cerevisiae. By co-localizing them with 7 known markers of endomembrane compartments we determined the localization for over 200 of them. Using this approach, we create a systematic database of the various secretory compartments and identify several new residents. Focusing in, we now suggest that Lam5 resides in contact sites between the endoplasmic reticulum and the late Golgi. Additionally, analysis of interactions between the COPI coat and co-localizing proteins from our screen identifies a subset of proteins that are COPI-cargo. In summary, our approach defines the protein roster within each compartment enabling characterization of the physical and functional organization of the endomembrane system and its components.

A pathway of targeted autophagy is induced by DNA damage in budding yeast.

Autophagy plays a central role in the DNA damage response (DDR) by controlling the levels of various DNA repair and checkpoint proteins; however, how the DDR communicates with the autophagy pathway remains unknown. Using budding yeast, we demonstrate that global genotoxic damage or even a single unrepaired double-strand break (DSB) initiates a previously undescribed and selective pathway of autophagy that we term genotoxin-induced targeted autophagy (GTA). GTA requires the action primarily of Mec1/ATR and Rad53/CHEK2 checkpoint kinases, in part via transcriptional up-regulation of central autophagy proteins. GTA is distinct from starvation-induced autophagy. GTA requires Atg11, a central component of the selective autophagy machinery, but is different from previously described autophagy pathways. By screening a collection of ∼6,000 yeast mutants, we identified genes that control GTA but do not significantly affect rapamycin-induced autophagy. Overall, our findings establish a pathway of autophagy specific to the DNA damage response.

Two novel effectors of trafficking and maturation of the yeast plasma membrane H+ -ATPase.

The endoplasmic reticulum (ER) is the entry site of proteins into the endomembrane system. Proteins exit the ER via coat protein II (COPII) vesicles in a selective manner, mediated either by direct interaction with the COPII coat or aided by cargo receptors. Despite the fundamental role of such receptors in protein sorting, only a few have been identified. To further define the machinery that packages secretory cargo and targets proteins from the ER to Golgi membranes, we used multiple systematic approaches, which revealed 2 uncharacterized proteins that mediate the trafficking and maturation of Pma1, the essential yeast plasma membrane proton ATPase. Ydl121c (Exp1) is an ER protein that binds Pma1, is packaged into COPII vesicles, and whose deletion causes ER retention of Pma1. Ykl077w (Psg1) physically interacts with Exp1 and can be found in the Golgi and coat protein I (COPI) vesicles but does not directly bind Pma1. Loss of Psg1 causes enhanced degradation of Pma1 in the vacuole. Our findings suggest that Exp1 is a Pma1 cargo receptor and that Psg1 aids Pma1 maturation in the Golgi or affects its retrieval. More generally our work shows the utility of high content screens in the identification of novel trafficking components.

Pex35 is a regulator of peroxisome abundance.

Peroxisomes are cellular organelles with vital functions in lipid, amino acid and redox metabolism. The cellular formation and dynamics of peroxisomes are governed by PEX genes; however, the regulation of peroxisome abundance is still poorly understood. Here, we use a high-content microscopy screen in Saccharomyces cerevisiae to identify new regulators of peroxisome size and abundance. Our screen led to the identification of a previously uncharacterized gene, which we term PEX35, which affects peroxisome abundance. PEX35 encodes a peroxisomal membrane protein, a remote homolog to several curvature-generating human proteins. We systematically characterized the genetic and physical interactome as well as the metabolome of mutants in PEX35, and we found that Pex35 functionally interacts with the vesicle-budding-inducer Arf1. Our results highlight the functional interaction between peroxisomes and the secretory pathway.

Iron affects Ire1 clustering propensity and the amplitude of endoplasmic reticulum stress signaling.

The unfolded protein response (UPR) allows cells to adjust secretory pathway capacity according to need. Ire1, the endoplasmic reticulum (ER) stress sensor and central activator of the UPR is conserved from the budding yeast Saccharomyces cerevisiae to humans. Under ER stress conditions, Ire1 clusters into foci that enable optimal UPR activation. To discover factors that affect Ire1 clustering, we performed a high-content screen using a whole-genome yeast mutant library expressing Ire1-mCherry. We imaged the strains following UPR induction and found 154 strains that displayed alterations in Ire1 clustering. The hits were enriched for iron and heme effectors and binding proteins. By performing pharmacological depletion and repletion, we confirmed that iron (Fe3+) affects UPR activation in both yeast and human cells. We suggest that Ire1 clustering propensity depends on membrane composition, which is governed by heme-dependent biosynthesis of sterols. Our findings highlight the diverse cellular functions that feed into the UPR and emphasize the cross-talk between organelles required to concertedly maintain homeostasis.

One library to make them all: streamlining the creation of yeast libraries via a SWAp-Tag strategy.

The yeast Saccharomyces cerevisiae is ideal for systematic studies relying on collections of modified strains (libraries). Despite the significance of yeast libraries and the immense variety of available tags and regulatory elements, only a few such libraries exist, as their construction is extremely expensive and laborious. To overcome these limitations, we developed a SWAp-Tag (SWAT) method that enables one parental library to be modified easily and efficiently to give rise to an endless variety of libraries of choice. To showcase the versatility of the SWAT approach, we constructed and investigated a library of ∼1,800 strains carrying SWAT-GFP modules at the amino termini of endomembrane proteins and then used it to create two new libraries (mCherry and seamless GFP). Our work demonstrates how the SWAT method allows fast and effortless creation of yeast libraries, opening the door to new ways of systematically studying cell biology.

Saccharomyces cerevisiae cells lacking Pex3 contain membrane vesicles that harbor a subset of peroxisomal membrane proteins.

Pex3 has been proposed to be important for the exit of peroxisomal membrane proteins (PMPs) from the ER, based on the observation that PMPs accumulate at the ER in Saccharomyces cerevisiae pex3 mutant cells. Using a combination of microscopy and biochemical approaches, we show that a subset of the PMPs, including the receptor docking protein Pex14, localizes to membrane vesicles in S. cerevisiae pex3 cells. These vesicles are morphologically distinct from the ER and do not co-sediment with ER markers in cell fractionation experiments. At the vesicles, Pex14 assembles with other peroxins (Pex13, Pex17, and Pex5) to form a complex with a composition similar to the PTS1 import pore in wild-type cells. Fluorescence microscopy studies revealed that also the PTS2 receptor Pex7, the importomer organizing peroxin Pex8, the ubiquitin conjugating enzyme Pex4 with its recruiting PMP Pex22, as well as Pex15 and Pex25 co-localize with Pex14. Other peroxins (including the RING finger complex and Pex27) did not accumulate at these structures, of which Pex11 localized to mitochondria. In line with these observations, proteomic analysis showed that in addition to the docking proteins and Pex5, also Pex7, Pex4/Pex22 and Pex25 were present in Pex14 complexes isolated from pex3 cells. However, formation of the entire importomer was not observed, most likely because Pex8 and the RING proteins were absent in the Pex14 protein complexes. Our data suggest that peroxisomal membrane vesicles can form in the absence of Pex3 and that several PMPs can insert in these vesicles in a Pex3 independent manner.

Characterization of proteome dynamics during growth in oleate reveals a new peroxisome-targeting receptor.

To optimally perform the diversity of metabolic functions that occur within peroxisomes, cells must dynamically regulate peroxisome size, number and content in response to the cell state and the environment. Except for transcriptional regulation little is known about the mechanisms used to perform this complicated feat. Focusing on the yeast Saccharomyces cerevisiae, we used complementary high-content screens to follow changes in localization of most proteins during growth in oleate. We found extensive changes in cellular architecture and identified several proteins that colocalized with peroxisomes that had not previously been considered peroxisomal proteins. One of the newly identified peroxisomal proteins, Ymr018w, is a protein with an unknown function that is similar to the yeast and human peroxisomal targeting receptor Pex5. We demonstrate that Ymr018w is a new peroxisomal-targeting receptor that targets a subset of matrix proteins to peroxisomes. We, therefore, renamed Ymr018w, Pex9, and suggest that Pex9 is a condition-specific targeting receptor that enables the dynamic rewiring of peroxisomes in response to metabolic needs. Moreover, we suggest that Pex5-like receptors might also exist in vertebrates.

Database for High Throughput Screening Hits (dHITS): a simple tool to retrieve gene specific phenotypes from systematic screens done in yeast.

In the last decade several collections of Saccharomyces cerevisiae yeast strains have been created. In these collections every gene is modified in a similar manner such as by a deletion or the addition of a protein tag. Such libraries have enabled a diversity of systematic screens, giving rise to large amounts of information regarding gene functions. However, often papers describing such screens focus on a single gene or a small set of genes and all other loci affecting the phenotype of choice ('hits') are only mentioned in tables that are provided as supplementary material and are often hard to retrieve or search. To help unify and make such data accessible, we have created a Database of High Throughput Screening Hits (dHITS). The dHITS database enables information to be obtained about screens in which genes of interest were found as well as the other genes that came up in that screen - all in a readily accessible and downloadable format. The ability to query large lists of genes at the same time provides a platform to easily analyse hits obtained from transcriptional analyses or other screens. We hope that this platform will serve as a tool to facilitate investigation of protein functions to the yeast community.

Combining Deep Sequencing, Proteomics, Phosphoproteomics, and Functional Screens To Discover Novel Regulators of Sphingolipid Homeostasis.

Sphingolipids (SLs) are essential components of cell membranes and are broad-range bioactive signaling molecules. SL levels must be tightly regulated as imbalances affect cellular function and contribute to pathologies ranging from neurodegenerative and metabolic disorders to cancer and aging. Deciphering how SL homeostasis is maintained and uncovering new regulators is required for understanding lipid biology and for identifying new targets for therapeutic interventions. Here we combine omics technologies to identify the changes of the transcriptome, proteome, and phosphoproteome in the yeast Saccharomyces cerevisiae upon SL depletion induced by myriocin. Surprisingly, while SL depletion triggers important changes in the expression of regulatory proteins involved in SL homeostasis, the most dramatic regulation occurs at the level of the phosphoproteome, suggesting that maintaining SL homeostasis demands rapid responses. To discover which of the phosphoproteomic changes are required for the cell's first-line response to SL depletion, we overlaid our omics results with systematic growth screens for genes required during growth in myriocin. By following the rate of SL biosynthesis in those candidates that are both affecting growth and are phosphorylated in response to the drug, we uncovered Atg9, Stp4, and Gvp36 as putative new regulators of SL homeostasis.

A defect in the RNA-processing protein HNRPDL causes limb-girdle muscular dystrophy 1G (LGMD1G).

Limb-girdle muscular dystrophies (LGMD) are a heterogeneous group of genetically determined muscle disorders with a primary or predominant involvement of the pelvic or shoulder girdle musculature. More than 20 genes with autosomal recessive (LGMD2A to LGMD2Q) and autosomal dominant inheritance (LGMD1A to LGMD1H) have been mapped/identified to date. Mutations are known for six among the eight mapped autosomal dominant forms: LGMD1A (myotilin), LGMD1B (lamin A/C), LGMD1C (caveolin-3), LGMD1D (desmin), LGMD1E (DNAJB6), and more recently for LGMD1F (transportin-3). Our group previously mapped the LGMD1G gene at 4q21 in a Caucasian-Brazilian family. We now mapped a Uruguayan family with patients displaying a similar LGMD1G phenotype at the same locus. Whole genome sequencing identified, in both families, mutations in the HNRPDL gene. HNRPDL is a heterogeneous ribonucleoprotein family member, which participates in mRNA biogenesis and metabolism. Functional studies performed in S. cerevisiae showed that the loss of HRP1 (yeast orthologue) had pronounced effects on both protein levels and cell localizations, and yeast proteome revealed dramatic reorganization of proteins involved in RNA-processing pathways. In vivo analysis showed that hnrpdl is important for muscle development in zebrafish, causing a myopathic phenotype when knocked down. The present study presents a novel association between a muscular disorder and a RNA-related gene and reinforces the importance of RNA binding/processing proteins in muscle development and muscle disease. Understanding the role of these proteins in muscle might open new therapeutic approaches for muscular dystrophies.

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.

Sexually dimorphic oxytocin circuits drive intragroup social conflict and aggression in wild house mice.

In nature, both males and females engage in competitive aggressive interactions to resolve social conflicts, yet the behavioral principles guiding such interactions and their underlying neural mechanisms remain poorly understood. Through circuit manipulations in wild mice, we unveil oxytocin-expressing (OT+) neurons in the hypothalamic paraventricular nucleus (PVN) as a neural hub governing behavior in dyadic and intragroup social conflicts, influencing the degree of behavioral sexual dimorphism. We demonstrate that OT+ PVN neurons are essential and sufficient in promoting aggression and dominance hierarchies, predominantly in females. Furthermore, pharmacogenetic activation of these neurons induces a change in the 'personality' traits of the mice within groups, in a sex-dependent manner. Finally, we identify an innervation from these OT neurons to the ventral tegmental area that drives dyadic aggression, in a sex-specific manner. Our data suggest that competitive aggression in naturalistic settings is mediated by a sexually dimorphic OT network connected with reward-related circuitry.

Functional MRI of murine olfactory bulbs at 15.2T reveals characteristic activation patters when stimulated by different odors.

Thanks to its increased sensitivity, single-shot ultrahigh field functional MRI (UHF fMRI) could lead to valuable insight about subtle brain functions such as olfaction. However, UHF fMRI experiments targeting small organs next to air voids, such as the olfactory bulb, are severely affected by field inhomogeneity problems. Spatiotemporal Encoding (SPEN) is an emerging single-shot MRI technique that could provide a route for bypassing these complications. This is here explored with single-shot fMRI studies on the olfactory bulbs of male and female mice performed at 15.2T. SPEN images collected on these organs at a 108 µm in-plane resolution yielded remarkably large and well-defined responses to olfactory cues. Under suitable T2* weightings these activation-driven changes exceeded 5% of the overall signal intensity, becoming clearly visible in the images without statistical treatment. The nature of the SPEN signal intensity changes in such experiments was unambiguously linked to olfaction, via single-nostril experiments. These experiments highlighted specific activation regions in the external plexiform region and in glomeruli in the lateral part of the bulb, when stimulated by aversive or appetitive odors, respectively. These strong signal activations were non-linear with concentration, and shed light on how chemosensory signals reaching the olfactory epithelium react in response to different cues. Second-level analyses highlighted clear differences among the appetitive, aversive and neutral odor maps; no such differences were evident upon comparing male against female olfactory activation regions.

Sex-dependent control of pheromones on social organization within groups of wild house mice.

Dominance hierarchy is a fundamental social phenomenon in a wide range of mammalian species, critically affecting fitness and health. Here, we investigate the role of pheromone signals in the control of social hierarchies and individual personalities within groups of wild mice. For this purpose, we combine high-throughput behavioral phenotyping with computational tools in freely interacting groups of wild house mice, males and females, in an automated, semi-natural system. We show that wild mice form dominance hierarchies in both sexes but use sex-specific strategies, displaying distinct male-typical and female-typical behavioral personalities that were also associated with social ranking. Genetic disabling of VNO-mediated pheromone detection generated opposite behavioral effects within groups, enhancing social interactions in males and reducing them in females. Behavioral personalities in the mutated mice displayed mixtures of male-typical and female-typical behaviors, thus blurring sex differences. In addition, rank-associated personalities were abolished despite the fact that both sexes of mutant mice formed stable hierarchies. These findings suggest that group organization is governed by pheromone-mediated sex-specific neural circuits and pave the way to investigate the mechanisms underlying sexual dimorphism in dominance hierarchies under naturalistic settings.

RNA-controlled nucleocytoplasmic shuttling of mRNA decay factors regulates mRNA synthesis and a novel mRNA decay pathway.

mRNA level is controlled by factors that mediate both mRNA synthesis and decay, including the 5' to 3' exonuclease Xrn1. Here we show that nucleocytoplasmic shuttling of several yeast mRNA decay factors plays a key role in determining both mRNA synthesis and decay. Shuttling is regulated by RNA-controlled binding of the karyopherin Kap120 to two nuclear localization sequences (NLSs) in Xrn1, location of one of which is conserved from yeast to human. The decaying RNA binds and masks NLS1, establishing a link between mRNA decay and Xrn1 shuttling. Preventing Xrn1 import, either by deleting KAP120 or mutating the two Xrn1 NLSs, compromises transcription and, unexpectedly, also cytoplasmic decay, uncovering a cytoplasmic decay pathway that initiates in the nucleus. Most mRNAs are degraded by both pathways - the ratio between them represents a full spectrum. Importantly, Xrn1 shuttling is required for proper responses to environmental changes, e.g., fluctuating temperatures, involving proper changes in mRNA abundance and in cell proliferation rate.

Mechanical unfolding of acylphosphatase studied by single-molecule force spectroscopy and MD simulations.

Single-molecule manipulation methods provide a powerful means to study protein transitions. Here we combined single-molecule force spectroscopy and steered molecular-dynamics simulations to study the mechanical properties and unfolding behavior of the small enzyme acylphosphatase (AcP). We find that mechanical unfolding of AcP occurs at relatively low forces in an all-or-none fashion and is decelerated in the presence of a ligand, as observed in solution measurements. The prominent energy barrier for the transition is separated from the native state by a distance that is unusually long for alpha/beta proteins. Unfolding is initiated at the C-terminal strand (beta(T)) that lies at one edge of the beta-sheet of AcP, followed by unraveling of the strand located at the other. The central strand of the sheet and the two helices in the protein unfold last. Ligand binding counteracts unfolding by stabilizing contacts between an arginine residue (Arg-23) and the catalytic loop, as well as with beta(T) of AcP, which renders the force-bearing units of the protein resistant to force. This stabilizing effect may also account for the decelerated unfolding of ligand-bound AcP in the absence of force.