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Objectives: Sonography is a clinical tool being incorporated in multiple medical specialties with evidence of improved patient care and cost. Some schools have begun implementing ultrasound curricula. We hope to build upon that foundation and provide another potential framework of incorporation. There are several barriers, including curricular space, equipment and physical space, adequate faculty, and performing assessment., Methods: At West Virginia University, we began a longitudinal ultrasound curriculum in 2012 with incorporation of didactic and practical sessions into gross anatomy, our systems-based second-year curriculum, physical diagnosis course, and clinical rotations. We included both written and practical assessment from the onset. After the initial 4 years, the first graduates were surveyed on their perceptions of the curriculum. Responses were correlated with specialty choice and clinical campus site., Results: Based on our survey (90% response rate), students felt sonography was useful for anatomical understanding and patient care. Overall, 93% of our respondents reviewed the curriculum favorably. Qualitative feedback was very positive, with students desiring more ultrasound education and more required components, specifically in clinical rotations., Conclusions: Based on these results, some changes have already been implemented, including decreased student-to-instructor ratios, more open scan time, and more required components. The breadth of formal assessment has increased. Multiple pilot programs for clinical rotations are being developed. There is an ongoing need for faculty development and continued assessment of ultrasound competency., (© 2018 by the American Institute of Ultrasound in Medicine.)

Class III adenylate cyclases (ACs) are widespread signaling proteins, which translate diverse intracellular and extracellular stimuli into a uniform intracellular signal. They are typically composed of an N-terminal array of input domains and transducers, followed C-terminally by a catalytic domain, which, as a dimer, generates the second messenger cAMP. The input domains, which receive stimuli, and the transducers, which propagate the signals, are often found in other signaling proteins. The nature of stimuli and the regulatory mechanisms of ACs have been studied experimentally in only a few cases, and even in these, important questions remain open, such as whether eukaryotic ACs regulated by G protein-coupled receptors can also receive stimuli through their own membrane domains. Here we survey the current knowledge on regulation and intramolecular signal propagation in ACs and draw comparisons to other signaling proteins. We highlight the pivotal role of a recently identified cyclase-specific transducer element located N-terminally of many AC catalytic domains, suggesting an intramolecular signaling capacity., (Copyright © 2018. Published by Elsevier Inc.)

Intermediate filaments (IFs) are principal components of the cytoskeleton, a dynamic integrated system of structural proteins that provides the functional architecture of metazoan cells. They are major contributors to the elasticity of cells and tissues due to their high mechanical stability and intrinsic flexibility. The basic building block for the assembly of IFs is a rod-like, 60-nm-long tetrameric complex made from two antiparallel, half-staggered coiled coils. In low ionic strength, tetramers form stable complexes that rapidly assemble into filaments upon raising the ionic strength. The first assembly products, "frozen" by instantaneous chemical fixation and viewed by electron microscopy, are 60-nm-long "unit-length" filaments (ULFs) that apparently form by lateral in-register association of tetramers. ULFs are the active elements of IF growth, undergoing longitudinal end-to-end annealing with one another and with growing filaments. Originally, we have employed quantitative time-lapse atomic force and electron microscopy to analyze the kinetics of vimentin-filament assembly starting from a few seconds to several hours. To obtain detailed quantitative insight into the productive reactions that drive ULF formation, we now introduce a "stopped-flow" approach in combination with static light-scattering measurements. Thereby, we determine the basic rate constants for lateral assembly of tetramers to ULFs. Processing of the recorded data by a global fitting procedure enables us to describe the hierarchical steps of IF formation. Specifically, we propose that tetramers are consumed within milliseconds to yield octamers that are obligatory intermediates toward ULF formation. Although the interaction of tetramers is diffusion controlled, it is strongly driven by their geometry to mediate effective subunit targeting. Importantly, our model conclusively reflects the previously described occurrence of polymorphic ULF and mature filaments in terms of their number of tetramers per cross section., (Copyright © 2018 Biophysical Society. Published by Elsevier Inc. All rights reserved.)

Ribosome biogenesis begins in the nucleolus with the formation of 90S pre-ribosomes, from which pre-40S and pre-60S particles arise that subsequently follow separate maturation pathways. Here, we show how structurally related assembly factors, the KH domain proteins Krr1 and Dim2, participate in ribosome assembly. Initially, Dim2 (Pno1) orchestrates an early step in small subunit biogenesis through its binding to a distinct region of the 90S pre-ribosome. This involves Utp1 of the UTP-B module, and Utp14, an activator of the DEAH-box helicase Dhr1 that catalyzes the removal of U3 snoRNP from the 90S. Following this dismantling reaction, the pre-40S subunit emerges, but Dim2 relocates to the pre-40S platform domain, previously occupied in the 90S by the other KH factor Krr1 through its interaction with Rps14 and the UTP-C module. Our findings show how the structurally related Krr1 and Dim2 can control stepwise ribosome assembly during the 90S-to-pre-40S subunit transition.

The biogenesis of eukaryotic ribosomes is a complicated process during which the transcription, modification, folding, and processing of the rRNA is coupled with the ordered assembly of ∼80 ribosomal proteins (r-proteins). Ribosome synthesis is catalyzed and coordinated by more than 200 biogenesis factors as the preribosomal subunits acquire maturity on their path from the nucleolus to the cytoplasm. Several biogenesis factors also interconnect the progression of ribosome assembly with quality control of important domains, ensuring that only functional subunits engage in translation. With the recent visualization of several assembly intermediates by cryoelectron microscopy (cryo-EM), a structural view of ribosome assembly begins to emerge. In this review we integrate these first structural insights into an updated overview of the consecutive ribosome assembly steps., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Adenylate cyclases (ACs) are signaling proteins that produce the second messenger cAMP. Class III ACs comprise four groups (class IIIa-d) of which class IIIa and IIIb ACs have been identified in bacteria and eukaryotes. Many class IIIa ACs are anchored to membranes via hexahelical domains. In eukaryotic ACs, membrane anchors are well conserved, suggesting that this region possesses important functional characteristics that are as yet unknown. To address this question, we replaced the hexahelical membrane anchor of the mycobacterial AC Rv1625c with the hexahelical quorum-sensing receptor from Legionella, LqsS. Using this chimera, we identified a novel 19-amino-acid cyclase transducer element (CTE) located N-terminally to the catalytic domain that links receptor stimulation to effector activation. Coupling of the receptor to the AC was possible at several positions distal to the membrane exit, resulting in stimulatory or inhibitory responses to the ligand Legionella autoinducer-1. In contrast, on the AC effector side functional coupling was only successful when starting with the CTE. Bioinformatics approaches established that distinct CTEs are widely present in class IIIa and IIIb ACs and in vertebrate guanylate cyclases. The data suggest that membrane-delimited receiver domains transduce regulatory signals to the downstream catalytic domains in an engineered AC model system. This may suggest a previously unknown mechanism for cellular cAMP regulation., (© 2017 The Authors. The FEBS Journal published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies.)

α-Helical coiled coils were described more than 60 years ago as simple, repetitive structures mediating oligomerization and mechanical stability. Over the past 20 years, however, they have emerged as one of the most diverse protein folds in nature, enabling many biological functions beyond mechanical rigidity, such as membrane fusion, signal transduction, and solute transport. Despite this great diversity, their structures can be described by parametric equations, making them uniquely suited for rational protein design. Far from having been exhausted as a source of structural insight and a basis for functional engineering, coiled coils are poised to become even more important for protein science in the coming decades., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Ribosome biogenesis in eukaryotic cells is a highly dynamic and complex process innately linked to cell proliferation. The assembly of ribosomes is driven by a myriad of biogenesis factors that shape pre-ribosomal particles by processing and folding the ribosomal RNA and incorporating ribosomal proteins. Biochemical approaches allowed the isolation and characterization of pre-ribosomal particles from Saccharomyces cerevisiae, which lead to a spatiotemporal map of biogenesis intermediates along the path from the nucleolus to the cytoplasm. Here, we cloned almost the entire set (∼180) of ribosome biogenesis factors from the thermophilic fungus Chaetomium thermophilum in order to perform an in-depth analysis of their protein-protein interaction network as well as exploring the suitability of these thermostable proteins for structural studies. First, we performed a systematic screen, testing about 80 factors for crystallization and structure determination. Next, we performed a yeast 2-hybrid analysis and tested about 32,000 binary combinations, which identified more than 1000 protein-protein contacts between the thermophilic ribosome assembly factors. To exemplary verify several of these interactions, we performed biochemical reconstitution with the focus on the interaction network between 90S pre-ribosome factors forming the ctUTP-A and ctUTP-B modules, and the Brix-domain containing assembly factors of the pre-60S subunit. Our work provides a rich resource for biochemical reconstitution and structural analyses of the conserved ribosome assembly machinery from a eukaryotic thermophile., (© 2017 The Protein Society.)

α-Helical coiled coils constitute one of the most diverse folds yet described. They range in length over two orders of magnitude; they form rods, segmented ropes, barrels, funnels, sheets, spirals, and rings, which encompass anywhere from two to more than 20 helices in parallel or antiparallel orientation; they assume different helix crossing angles, degrees of supercoiling, and packing geometries. This structural diversity supports a wide range of biological functions, allowing them to form mechanically rigid structures, provide levers for molecular motors, project domains across large distances, mediate oligomerization, transduce conformational changes and facilitate the transport of other molecules. Unlike almost any other protein fold known to us, their structure can be computed from parametric equations, making them an ideal model system for rational protein design. Here we outline the principles by which coiled coils are structured, review the determinants of their folding and stability, and present an overview of their diverse architectures.

Adenylate cyclases convert intra- and extracellular stimuli into a second messenger cAMP signal. Many bacterial and most eukaryotic ACs possess membrane anchors with six transmembrane spans. We replaced the anchor of the AC Rv1625c by the quorum-sensing receptor from Vibrio harveyi which has an identical 6TM design and obtained an active, membrane-anchored AC. We show that a canonical class III AC is ligand-regulated in vitro and in vivo. At 10 µM, the cholera-autoinducer CAI-1 stimulates activity 4.8-fold. A sequence based clustering of membrane domains of class III ACs and quorum-sensing receptors established six groups of potential structural and functional similarities. The data support the notion that 6TM AC membrane domains may operate as receptors which directly regulate AC activity as opposed and in addition to the indirect regulation by GPCRs in eukaryotic congeners. This adds a completely novel dimension of potential AC regulation in bacteria and vertebrates., Competing Interests: The author declares that no competing interests exist.

Coiled coils are the best-understood protein fold, as their backbone structure can uniquely be described by parametric equations. This level of understanding has allowed their manipulation in unprecedented detail. They do not seem a likely source of surprises, yet we describe here the unexpected formation of a new type of fiber by the simple insertion of two or six residues into the underlying heptad repeat of a parallel, trimeric coiled coil. These insertions strain the supercoil to the breaking point, causing the local formation of short β-strands, which move the path of the chain by 120° around the trimer axis. The result is an α/β coiled coil, which retains only one backbone hydrogen bond per repeat unit from the parent coiled coil. Our results show that a substantially novel backbone structure is possible within the allowed regions of the Ramachandran space with only minor mutations to a known fold.

Ribosome synthesis is catalyzed by ∼200 assembly factors, which facilitate efficient production of mature ribosomes. Here, we determined the cryo-EM structure of a Saccharomyces cerevisiae nucleoplasmic pre-60S particle containing the dynein-related 550-kDa Rea1 AAA(+) ATPase and the Rix1 subcomplex. This particle differs from its preceding state, the early Arx1 particle, by two massive structural rearrangements: an ∼180° rotation of the 5S ribonucleoprotein complex and the central protuberance (CP) rRNA helices, and the removal of the 'foot' structure from the 3' end of the 5.8S rRNA. Progression from the Arx1 to the Rix1 particle was blocked by mutational perturbation of the Rix1-Rea1 interaction but not by a dominant-lethal Rea1 AAA(+) ATPase-ring mutant. After remodeling, the Rix1 subcomplex and Rea1 become suitably positioned to sense correct structural maturation of the CP, which allows unidirectional progression toward mature ribosomes.

The exosome regulates the processing, degradation, and surveillance of a plethora of RNA species. However, little is known about how the exosome recognizes and is recruited to its diverse substrates. We report the identification of adaptor proteins that recruit the exosome-associated helicase, Mtr4, to unique RNA substrates. Nop53, the yeast homolog of the tumor suppressor PICT1, targets Mtr4 to pre-ribosomal particles for exosome-mediated processing, while a second adaptor Utp18 recruits Mtr4 to cleaved rRNA fragments destined for degradation by the exosome. Both Nop53 and Utp18 contain the same consensus motif, through which they dock to the "arch" domain of Mtr4 and target it to specific substrates. These findings show that the exosome employs a general mechanism of recruitment to defined substrates and that this process is regulated through adaptor proteins., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Despite the therapeutic potential of sphingolipids, the ability to develop this class of compounds as active pharmaceutical ingredients has been hampered by issues of solubility and delivery. Beyond these technical hurdles, significant challenges in completing the necessary preclinical studies to support regulatory review are necessary for commercialization. This review seeks to identify the obstacles and potential solutions in the translation of a novel liposomal technology from the academic bench to investigational new drug (IND) stage by discussing the preclinical development of the Ceramide NanoLiposome (CNL), which is currently being developed as an anticancer drug for the initial indication of hepatocellular carcinoma (HCC).

Nuclear pore complexes (NPCs) are huge assemblies formed from ∼30 different nucleoporins, typically organized in subcomplexes. One module, the conserved Nup82 complex at the cytoplasmic face of NPCs, is crucial to terminate mRNA export. To gain insight into the structure, assembly, and function of the cytoplasmic pore filaments, we reconstituted in yeast the Nup82-Nup159-Nsp1-Dyn2 complex, which was suitable for biochemical, biophysical, and electron microscopy analyses. Our integrative approach revealed that the yeast Nup82 complex forms an unusual asymmetric structure with a dimeric array of subunits. Based on all these data, we developed a three-dimensional structural model of the Nup82 complex that depicts how this module might be anchored to the NPC scaffold and concomitantly can interact with the soluble nucleocytoplasmic transport machinery., (© 2015 Gaik et al.)

Trimeric autotransporter adhesins (TAAs) are modular, highly repetitive outer membrane proteins that mediate adhesion to external surfaces in many Gram-negative bacteria. In recent years, several TAAs have been investigated in considerable detail, also at the structural level. However, in their vast majority, putative TAAs in prokaryotic genomes remain poorly annotated, due to their sequence diversity and changeable domain architecture. In order to achieve an automated annotation of these proteins that is both detailed and accurate we have taken a domain dictionary approach, in which we identify recurrent domains by sequence comparisons, produce bioinformatic descriptors for each domain type, and connect these to structural information where available. We implemented this approach in a web-based platform, daTAA, in 2008 and demonstrated its applicability by reconstructing the complete fiber structure of a TAA conserved in enterobacteria. Here we review current knowledge on the domain structure of TAAs., (Copyright © 2014 The Authors. Published by Elsevier GmbH.. All rights reserved.)

The thermophilic fungus Chaetomium thermophilum holds great promise for structural biology. To increase the efficiency of its biochemical and structural characterization and to explore its thermophilic properties beyond those of individual proteins, we obtained transcriptomics and proteomics data, and integrated them with computational annotation methods and a multitude of biochemical experiments conducted by the structural biology community. We considerably improved the genome annotation of Chaetomium thermophilum and characterized the transcripts and expression of thousands of genes. We furthermore show that the composition and structure of the expressed proteome of Chaetomium thermophilum is similar to its mesophilic relatives. Data were deposited in a publicly available repository and provide a rich source to the structural biology community., (© The Author(s) 2014. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Eukaryotic ribosome biogenesis involves ∼200 assembly factors, but how these contribute to ribosome maturation is poorly understood. Here, we identify a network of factors on the nascent 60S subunit that actively remodels preribosome structure. At its hub is Rsa4, a direct substrate of the force-generating ATPase Rea1. We show that Rsa4 is connected to the central protuberance by binding to Rpl5 and to ribosomal RNA (rRNA) helix 89 of the nascent peptidyl transferase center (PTC) through Nsa2. Importantly, Nsa2 binds to helix 89 before relocation of helix 89 to the PTC. Structure-based mutations of these factors reveal the functional importance of their interactions for ribosome assembly. Thus, Rsa4 is held tightly in the preribosome and can serve as a "distribution box," transmitting remodeling energy from Rea1 into the developing ribosome. We suggest that a relay-like factor network coupled to a mechano-enzyme is strategically positioned to relocate rRNA elements during ribosome maturation., (© 2014 Baßler et al.)

Eukaryotic ribosome biogenesis requires more than 150 auxiliary proteins, which transiently interact with pre-ribosomal particles. Previous studies suggest that several of these biogenesis factors function together as modules. Using a heterologous expression system, we show that the large ribosomal subunit (LSU) biogenesis factor Noc1p of Saccharomyces cerevisiae can simultaneously interact with the LSU biogenesis factor Noc2p and Rrp5p, a factor required for biogenesis of the large and the small ribosomal subunit. Proteome analysis of RNA polymerase-I-associated chromatin and chromatin immunopurification experiments indicated that all members of this protein module and a specific set of LSU biogenesis factors are co-transcriptionally recruited to nascent ribosomal RNA (rRNA) precursors in yeast cells. Further ex vivo analyses showed that all module members predominantly interact with early pre-LSU particles after the initial pre-rRNA processing events have occurred. In yeast strains depleted of Noc1p, Noc2p or Rrp5p, levels of the major LSU pre-rRNAs decreased and the respective other module members were associated with accumulating aberrant rRNA fragments. Therefore, we conclude that the module exhibits several binding interfaces with pre-ribosomes. Taken together, our results suggest a co- and post-transcriptional role of the yeast Rrp5p-Noc1p-Noc2p module in the structural organization of early LSU precursors protecting them from non-productive RNase activity.

The nuclear export of the preribosomal 60S (pre-60S) subunit is coordinated with late steps in ribosome assembly. Here, we show that Bud20, a conserved C(2)H(2)-type zinc finger protein, is an unrecognized shuttling factor required for the efficient export of pre-60S subunits. Bud20 associates with late pre-60S particles in the nucleoplasm and accompanies them into the cytoplasm, where it is released through the action of the Drg1 AAA-ATPase. Cytoplasmic Bud20 is then reimported via a Kap123-dependent pathway. The deletion of Bud20 induces a strong pre-60S export defect and causes synthetic lethality when combined with mutant alleles of known pre-60S subunit export factors. The function of Bud20 in ribosome export depends on a short conserved N-terminal sequence, as we observed that mutations or the deletion of this motif impaired 60S subunit export and generated the genetic link to other pre-60S export factors. We suggest that the shuttling Bud20 is recruited to the nascent 60S subunit via its central zinc finger rRNA binding domain to facilitate the subsequent nuclear export of the preribosome employing its N-terminal extension.

The biogenesis of ribosomes is a fundamental cellular process, which provides the molecular machines that synthesize all cellular proteins. The assembly of eukaryotic ribosomes is a highly complex multi-step process that requires more than 200 ribosome biogenesis factors, which mediate a broad spectrum of maturation reactions. The participation of many energy-consuming enzymes (e.g. AAA-type ATPases, RNA helicases, and GTPases) in this process indicates that the expenditure of energy is required to drive ribosome assembly. While the precise function of many of these enzymes remains elusive, recent progress has revealed that the three AAA-type ATPases involved in 60S subunit biogenesis are specifically dedicated to the release and recycling of distinct biogenesis factors. In this review, we will highlight how the molecular power of yeast Drg1, Rix7, and Rea1 is harnessed to promote the release of their substrate proteins from evolving pre-60S particles and, where appropriate, discuss possible catalytic mechanisms., (Copyright © 2011 Elsevier B.V. All rights reserved.)

The AAA(+)-ATPase Rea1 removes the ribosome biogenesis factor Rsa4 from pre-60S ribosomal subunits in the nucleoplasm to drive nuclear export of the subunit. To do this, Rea1 utilizes a MIDAS domain to bind a conserved motif in Rsa4. Here, we show that the Rea1 MIDAS domain binds another pre-60S factor, Ytm1, via a related motif. In vivo Rea1 contacts Ytm1 before it contacts Rsa4, and its interaction with Ytm1 coincides with the exit of early pre-60S particles from the nucleolus to the nucleoplasm. In vitro, Rea1's ATPase activity triggers removal of the conserved nucleolar Ytm1-Erb1-Nop7 subcomplex from isolated early pre-60S particle. We suggest that the Rea1 AAA(+)-ATPase functions at successive maturation steps to remove ribosomal factors at critical transition points, first driving the exit of early pre-60S particles from the nucleolus and then driving late pre-60S particles from the nucleus., (Copyright (c) 2010 Elsevier Inc. All rights reserved.)

Ribosome biogenesis is a fundamental process that provides cells with the molecular factories for cellular protein production. Accordingly, its misregulation lies at the heart of several hereditary diseases (e.g., Diamond-Blackfan anemia). The process of ribosome assembly comprises the processing and folding of the pre-rRNA and its concomitant assembly with the ribosomal proteins. Eukaryotic ribosome biogenesis relies on a large number (>200) of non-ribosomal factors, which confer directionality and accuracy to this process. Many of these non-ribosomal factors fall into different families of energy-consuming enzymes, notably including ATP-dependent RNA helicases, AAA-ATPases, GTPases, and kinases. Ribosome biogenesis is highly conserved within eukaryotic organisms; however, due to the combination of powerful genetic and biochemical methods, it is best studied in the yeast Saccharomyces cerevisiae. This review summarizes our current knowledge on eukaryotic ribosome assembly, with particular focus on the molecular role of the involved energy-consuming enzymes.

The dynein-related AAA ATPase Rea1 is a preribosomal factor that triggers an unknown maturation step in 60S subunit biogenesis. Using electron microscopy, we show that Rea1's motor domain is docked to the pre-60S particle and its tail-like structure, harboring a metal ion-dependent adhesion site (MIDAS), protrudes from the preribosome. Typically, integrins utilize a MIDAS to bind extracellular ligands, an interaction that is strengthened under applied tensile force. Likewise, the Rea1 MIDAS binds the preribosomal factor Rsa4, which is located on the pre-60S subunit at a site that is contacted by the flexible Rea1 tail. The MIDAS-Rsa4 interaction is essential for ATP-dependent dissociation of a group of non-ribosomal factors from the pre-60S particle. Thus, Rea1 aligns with its interacting partners on the preribosome to effect a necessary step on the path to the export-competent 60S subunit.

The GTPase Arf1 is considered as a molecular switch that regulates binding and release of coat proteins that polymerize on membranes to form transport vesicles. Here, we show that Arf1-GTP induces positive membrane curvature and find that the small GTPase can dimerize dependent on GTP. Investigating a possible link between Arf dimerization and curvature formation, we isolated an Arf1 mutant that cannot dimerize. Although it was capable of exerting the classical role of Arf1 as a coat receptor, it could not mediate the formation of COPI vesicles from Golgi-membranes and was lethal when expressed in yeast. Strikingly, this mutant was not able to deform membranes, suggesting that GTP-induced dimerization of Arf1 is a critical step inducing membrane curvature during the formation of coated vesicles.

The success of the World Health Organization smallpox eradication program three decades ago resulted in termination of routine vaccination and consequent decline in population immunity. Despite concerns regarding the reintroduction of smallpox, there is little enthusiasm for large-scale redeployment of licensed live vaccinia virus vaccines because of medical contraindications and anticipated serious side effects. Therefore, highly attenuated strains such as modified vaccinia virus Ankara (MVA) are under evaluation in humans and animal models. Previous studies showed that priming and boosting with MVA provided protection for >2 years in a monkeypox virus challenge model. If variola virus were used as a biological weapon, however, the ability of a vaccine to quickly induce immunity would be essential. Here, we demonstrate more rapid immune responses after a single vaccination with MVA compared to the licensed Dryvax vaccine. To determine the kinetics of protection of the two vaccines, macaques were challenged intravenously with monkeypox virus at 4, 6, 10, and 30 days after immunization. At 6 or more days after vaccination with MVA or Dryvax, the monkeys were clinically protected (except for 1 of 16 animals vaccinated with MVA), although viral loads and number of skin lesions were generally higher in the MVA vaccinated group. With only 4 days between immunization and intravenous challenge, however, MVA still protected whereas Dryvax failed. Protection correlated with the more rapid immune response to MVA compared to Dryvax, which may be related to the higher dose of MVA that can be tolerated safely.

Shuttling transport receptors carry cargo through nuclear pore complexes (NPCs) via transient interactions with Phe-Gly (FG)-rich nucleoporins. Here, we identify Arx1, a factor associated with a late 60S preribosomal particle in the nucleus, as an unconventional export receptor. Arx1 binds directly to FG nucleoporins and exhibits facilitated translocation through NPCs. Moreover, Arx1 functionally overlaps with the other 60S export receptors, Xpo1 and Mex67-Mtr2, and is genetically linked to nucleoporins. Unexpectedly, Arx1 is structurally unrelated to known shuttling transport receptors but homologous to methionine aminopeptidases (MetAPs), however, without enzymatic activity. Typically, the MetAP fold creates a central cavity that binds the methionine. In contrast, the predicted central cavity of Arx1 is involved in the interaction with FG repeat nucleoporins and 60S subunit export. Thus, an ancient enzyme fold has been adopted by Arx1 to function as a nuclear export receptor.

The yeast Mex67-Mtr2 complex and its homologous metazoan counterpart TAP-p15 operate as nuclear export receptors by binding and translocating mRNA through the nuclear pore complexes. Here, we show how Mex67-Mtr2 can also function in the nuclear export of the ribosomal 60S subunit. Biochemical and genetic studies reveal a previously unrecognized interaction surface on the NTF2-like scaffold of the Mex67-Mtr2 heterodimer, which in vivo binds to pre-60S particles and in vitro can interact with 5S rRNA. Crucial structural requirements for this binding platform are loop insertions in the middle domain of Mex67 and Mtr2, which are absent from human TAP-p15. Notably, when the positively charged amino acids in the Mex67 loop are mutated, interaction of Mex67-Mtr2 with pre-60S particles and 5S rRNA is inhibited, and 60S subunits, but not mRNA, accumulate in the nucleus. Thus, the general mRNA exporter Mex67-Mtr2 contains a distinct electrostatic interaction surface for transporting 60S preribosomal cargo.

The putative yeast GTPase Nug1, which is associated with several pre-60 S particles in the nucleolus and nucleoplasm, consists of an N-terminal domain, which is found only in eukaryotic orthologues, and middle and C-terminal domains that are conserved throughout eukaryotes, bacteria, and archaea. Here, we analyzed the role of the eukaryote-specific Nug1 N-domain (Nug1-N). We show that the essential Nug1-N is sufficient and necessary for nucle(ol)ar targeting and association with pre-60 S particles. Nug1-N exhibits RNA binding activity and is genetically linked in an allele-specific way to the pre-60 S factors Noc2, Noc3, and Dbp10. In contrast, the middle domain, which exhibits a circularly permuted GTPase fold and an intrinsic GTP hydrolysis activity in vitro, is not essential for cell growth. The conserved Nug1 C-domain, which has a yet uncharacterized fold, is also essential for ribosome biogenesis. Our findings suggest that Nug1 associates with pre-60 S subunits via its essential N-terminal RNA-binding domain and exerts a non-essential regulative role in pre-60 S subunit biogenesis via its central GTPase domain.

Mitochondria perform a central function in the biogenesis of cellular iron-sulphur (Fe/S) proteins. It is unknown to date why this biosynthetic pathway is indispensable for life, the more so as no essential mitochondrial Fe/S proteins are known. Here, we show that the soluble ATP-binding cassette (ABC) protein Rli1p carries N-terminal Fe/S clusters that require the mitochondrial and cytosolic Fe/S protein biogenesis machineries for assembly. Mutations in critical cysteine residues of Rli1p abolish association with Fe/S clusters and lead to loss of cell viability. Hence, the essential character of Fe/S clusters in Rli1p explains the indispensable character of mitochondria in eukaryotes. We further report that Rli1p is associated with ribosomes and with Hcr1p, a protein involved in rRNA processing and translation initiation. Depletion of Rli1p causes a nuclear export defect of the small and large ribosomal subunits and subsequently a translational arrest. Thus, ribosome biogenesis and function are intimately linked to the crucial role of mitochondria in the maturation of the essential Fe/S protein Rli1p.

Sda1 is an essential protein required for cell cycle progression in Saccharomyces cerevisiae. Here, we show that the sda1-1 mutation causes a defect in the formation and nuclear export of 60S ribosomal subunits. Moreover, the sda1-1, but also other mutants defective in ribosome biogenesis (e.g., rix1-1 and tif6Delta), exhibit a G1 arrest, which could be the consequence of impaired ribosome biogenesis. Interestingly, additional deletion of the non-essential Swe1 kinase, the homolog of S. pombe Wee1, causes a pronounced delay in entering a new cell cycle in sda1-1, rix1-1 and tif6Delta cells, when shifted back from restrictive to permissive conditions. However, such a prolonged delay is independent of the Tyr19 phosphorylation in Cdc28. Moreover, the lack of Swe1 causes delay in budding and DNA replication in cells released from the G1 arrest due to the block of protein synthesis. Our data suggest that Swe1 is required for timely entry into cell cycle after a G1 arrest caused by impairment in pre-60S biogenesis and in protein synthesis. Therefore we propose that Swe1, which is required for coordination of cell growth and cell division in G2/M, also has a role in the beginning of the cell cycle.

Formation and nuclear export of 60 S pre-ribosomes requires many factors including the heterodimeric Noc1-Noc2 and Noc2-Noc3 complexes. Here, we report another Noc complex with a specific role in 40 S subunit biogenesis. This complex consists of Noc4p, which exhibits the conserved Noc domain and is homologous to Noc1p, and Nop14p, a nucleolar protein with a role in 40 S subunit formation. Moreover, noc4 thermosensitive mutants are defective in 40 S biogenesis, and rRNA processing is inhibited at early cleavage sites A(0), A(1), and A(2). Using a fluorescence-based visual assay for 40 S subunit export, we observe a strong nucleolar accumulation of the Rps2p-green fluorescent protein reporter in noc4 ts mutants, but 60 S subunit export was normal. Thus, Noc4p and Nop14p form a novel Noc complex with a specific role in nucleolar 40 S subunit formation and subsequent export to the cytoplasm.

60S ribosomes undergo initial assembly in the nucleolus before export to the cytoplasm and recent analyses have identified several nucleolar pre-60S particles. To unravel the steps in the pathway of ribosome formation, we have purified the pre-60S ribosomes associated with proteins predicted to act at different stages as the pre-ribosomes transit from the nucleolus through the nucleoplasm and are then exported to the cytoplasm for final maturation. About 50 non-ribosomal proteins are associated with the early nucleolar pre-60S ribosomes. During subsequent maturation and transport to the nucleoplasm, many of these factors are removed, while others remain attached and additional factors transiently associate. When the 60S precursor particles are close to exit from the nucleus they associate with at least two export factors, Nmd3 and Mtr2. As the 60S pre-ribosome reaches the cytoplasm, almost all of the factors are dissociated. These data provide an initial biochemical map of 60S ribosomal subunit formation on its path from the nucleolus to the cytoplasm.

We report the characterization of early pre-ribosomal particles. Twelve TAP-tagged components each showed nucleolar localization, sedimented at approximately 90S on sucrose gradients, and coprecipitated both the 35S pre-rRNA and the U3 snoRNA. Thirty-five non-ribosomal proteins were coprecipitated, including proteins associated with U3 (Nop56p, Nop58p, Sof1p, Rrp9, Dhr1p, Imp3p, Imp4p, and Mpp10p) and other factors required for 18S rRNA synthesis (Nop14p, Bms1p, and Krr1p). Mutations in components of the 90S pre-ribosomes impaired 40S subunit assembly and export. Strikingly, few components of recently characterized pre-60S ribosomes were identified in the 90S pre-ribosomes. We conclude that the 40S synthesis machinery predominately associates with the 35S pre-rRNA factors, whereas factors required for 60S subunit synthesis largely bind later, showing an unexpected dichotomy in binding.

A nuclear GTPase, Nug1p, was identified in a genetic screen for components linked to 60S ribosomal subunit export. Nug1p cosedimented with nuclear 60S preribosomes and was required for subunit export to the cytoplasm. Tagged Nug1p coprecipitated with proteins of the 60S subunit, late precursors to the 25S and 5.8S rRNAs, and at least 21 nonribosomal proteins. These included a homologous nuclear GTPase, Nug2p, the Noc2p/Noc3p heterodimer, Rix1p, and Rlp7p, each of which was implicated in 60S subunit export. Other known ribosome synthesis factors and proteins of previously unknown function, including the 559 kDa protein Ylr106p, also copurified. Eight of these proteins were copurified with nuclear pore complexes, suggesting that this complex represents the transport intermediate for 60S subunit export.

It is not known how Mex67p and Mtr2p, which form a heterodimer essential for mRNA export, transport mRNPs through the nuclear pore. Here, we show that the Mex67p/Mtr2p complex binds to all of the repeat types (GLFG, FXFG, and FG) found in nucleoporins. For this interaction, complex formation between Mex67p and Mtr2p has to occur. MEX67 and MTR2 also genetically interact with different types of repeat nucleoporins, such as Nup116p, Nup159p, Nsp1p, and Rip1p/Nup40p. These data suggest a model in which nuclear mRNA export requires the Mex67p/Mtr2p heterodimeric complex to directly contact several repeat nucleoporins, organized in different nuclear pore complex subcomplexes, as it carries the mRNP cargo through the nuclear pore.