Linker Nups connect the nuclear pore complex inner ring with the outer ring and transport channel.

Nuclear pore complexes (NPCs) mediate transport between the nucleus and cytoplasm. NPCs are composed of ∼30 nucleoporins (Nups), most of which are organized in stable subcomplexes. How these modules are interconnected within the large NPC framework has been unknown. Here we show a mechanism of how supercomplexes can form between NPC modules. The Nup192 inner-pore-ring complex serves as a seed to which the Nup82 outer-ring complex and Nsp1 channel complex are tethered. The linkage between these subcomplexes is generated by short sequences within linker Nups. The conserved Nup145N is the most versatile connector of NPC modules because it has three discrete binding sites for Nup192, Nup170 and Nup82. We assembled a large part of a Chaetomium thermophilum NPC protomer in vitro, providing a step forward toward the reconstitution of the entire NPC.

The Nuclear Pore-Associated TREX-2 Complex Employs Mediator to Regulate Gene Expression.

Nuclear pore complexes (NPCs) influence gene expression besides their established function in nuclear transport. The TREX-2 complex localizes to the NPC basket and affects gene-NPC interactions, transcription, and mRNA export. How TREX-2 regulates the gene expression machinery is unknown. Here, we show that TREX-2 interacts with the Mediator complex, an essential regulator of RNA Polymerase (Pol) II. Structural and biochemical studies identify a conserved region on TREX-2, which directly binds the Mediator Med31/Med7N submodule. TREX-2 regulates assembly of Mediator with the Cdk8 kinase and is required for recruitment and site-specific phosphorylation of Pol II. Transcriptome and phenotypic profiling confirm that TREX-2 and Med31 are functionally interdependent at specific genes. TREX-2 additionally uses its Mediator-interacting surface to regulate mRNA export suggesting a mechanism for coupling transcription initiation and early steps of mRNA processing. Our data provide mechanistic insight into how an NPC-associated adaptor complex accesses the core transcription machinery.

Functional reconstitution of mitochondrial Fe/S cluster synthesis on Isu1 reveals the involvement of ferredoxin.

Maturation of iron-sulphur (Fe/S) proteins involves complex biosynthetic machinery. In vivo synthesis of [2Fe-2S] clusters on the mitochondrial scaffold protein Isu1 requires the cysteine desulphurase complex Nfs1-Isd11, frataxin, ferredoxin Yah1 and its reductase Arh1. The roles of Yah1-Arh1 have remained enigmatic, because they are not required for in vitro Fe/S cluster assembly. Here, we reconstitute [2Fe-2S] cluster synthesis on Isu1 in a reaction depending on Nfs1-Isd11, frataxin, Yah1, Arh1 and NADPH. Unlike in the bacterial system, frataxin is an essential part of Fe/S cluster biosynthesis and is required simultaneously and stoichiometrically to Yah1. Reduced but not oxidized Yah1 tightly interacts with apo-Isu1 indicating a dynamic interaction between Yah1-apo-Isu1. Nuclear magnetic resonance structural studies identify the Yah1-apo-Isu1 interaction surface and suggest a pathway for electron flow from reduced ferredoxin to Isu1. Together, our study defines the molecular function of the ferredoxin Yah1 and its human orthologue FDX2 in mitochondrial Fe/S cluster synthesis.

Structural basis of histone H2A-H2B recognition by the essential chaperone FACT.

Facilitates chromatin transcription (FACT) is a conserved histone chaperone that reorganizes nucleosomes and ensures chromatin integrity during DNA transcription, replication and repair. Key to the broad functions of FACT is its recognition of histones H2A-H2B (ref. 2). However, the structural basis for how histones H2A-H2B are recognized and how this integrates with the other functions of FACT, including the recognition of histones H3-H4 and other nuclear factors, is unknown. Here we reveal the crystal structure of the evolutionarily conserved FACT chaperone domain Spt16M from Chaetomium thermophilum, in complex with the H2A-H2B heterodimer. A novel 'U-turn' motif scaffolded onto a Rtt106-like module embraces the α1 helix of H2B. Biochemical and in vivo assays validate the structure and dissect the contribution of histone tails and H3-H4 towards Spt16M binding. Furthermore, we report the structure of the FACT heterodimerization domain that connects FACT to replicative polymerases. Our results show that Spt16M makes several interactions with histones, which we suggest allow the module to invade the nucleosome gradually and block the strongest interaction of H2B with DNA. FACT would thus enhance 'nucleosome breathing' by re-organizing the first 30 base pairs of nucleosomal histone-DNA contacts. Our snapshot of the engagement of the chaperone with H2A-H2B and the structures of all globular FACT domains enable the high-resolution analysis of the vital chaperoning functions of FACT, shedding light on how the complex promotes the activity of enzymes that require nucleosome reorganization.

Consistent mutational paths predict eukaryotic thermostability.

BACKGROUND: Proteomes of thermophilic prokaryotes have been instrumental in structural biology and successfully exploited in biotechnology, however many proteins required for eukaryotic cell function are absent from bacteria or archaea. With Chaetomium thermophilum, Thielavia terrestris and Thielavia heterothallica three genome sequences of thermophilic eukaryotes have been published. RESULTS: Studying the genomes and proteomes of these thermophilic fungi, we found common strategies of thermal adaptation across the different kingdoms of Life, including amino acid biases and a reduced genome size. A phylogenetics-guided comparison of thermophilic proteomes with those of other, mesophilic Sordariomycetes revealed consistent amino acid substitutions associated to thermophily that were also present in an independent lineage of thermophilic fungi. The most consistent pattern is the substitution of lysine by arginine, which we could find in almost all lineages but has not been extensively used in protein stability engineering. By exploiting mutational paths towards the thermophiles, we could predict particular amino acid residues in individual proteins that contribute to thermostability and validated some of them experimentally. By determining the three-dimensional structure of an exemplar protein from C. thermophilum (Arx1), we could also characterise the molecular consequences of some of these mutations. CONCLUSIONS: The comparative analysis of these three genomes not only enhances our understanding of the evolution of thermophily, but also provides new ways to engineer protein stability.

Structural characterization of a eukaryotic chaperone--the ribosome-associated complex.

Ribosome-associated chaperones act in early folding events during protein synthesis. Structural information is available for prokaryotic chaperones (such as trigger factor), but structural understanding of these processes in eukaryotes lags far behind. Here we present structural analyses of the eukaryotic ribosome-associated complex (RAC) from Saccharomyces cerevisiae and Chaetomium thermophilum, consisting of heat-shock protein 70 (Hsp70) Ssz1 and the Hsp40 Zuo1. RAC is an elongated complex that crouches over the ribosomal tunnel exit and seems to be stabilized in a distinct conformation by expansion segment ES27. A unique α-helical domain in Zuo1 mediates ribosome interaction of RAC near the ribosomal proteins L22e and L31e and ribosomal RNA helix H59. The crystal structure of the Ssz1 ATPase domain bound to ATP-Mg²âº explains its catalytic inactivity and suggests that Ssz1 may act before the RAC-associated chaperone Ssb. Our study offers insights into the interplay between RAC, the ER membrane-integrated Hsp40-type protein ERj1 and the signal-recognition particle.

Synchronizing nuclear import of ribosomal proteins with ribosome assembly.

Ribosomal proteins are synthesized in the cytoplasm, before nuclear import and assembly with ribosomal RNA (rRNA). Little is known about coordination of nucleocytoplasmic transport with ribosome assembly. Here, we identify a transport adaptor, symportin 1 (Syo1), that facilitates synchronized coimport of the two 5S-rRNA binding proteins Rpl5 and Rpl11. In vitro studies revealed that Syo1 concomitantly binds Rpl5-Rpl11 and furthermore recruits the import receptor Kap104. The Syo1-Rpl5-Rpl11 import complex is released from Kap104 by RanGTP and can be directly transferred onto the 5S rRNA. Syo1 can shuttle back to the cytoplasm by interaction with phenylalanine-glycine nucleoporins. X-ray crystallography uncovered how the α-solenoid symportin accommodates the Rpl5 amino terminus, normally bound to 5S rRNA, in an extended groove. Symportin-mediated coimport of Rpl5-Rpl11 could ensure coordinated and stoichiometric incorporation of these proteins into pre-60S ribosomes.

Analysis of the yeast nucleoporin Nup188 reveals a conserved S-like structure with similarity to karyopherins.

Nuclear pore complexes (NPCs) embedded in the double nuclear membrane mediate the entire nucleocytoplasmic transport between the nucleus and cytoplasm. Each NPC is composed of about 30 different proteins (nucleoporins or Nups), which exist in multiple (8, 16 or 32) copies within the NPC scaffold. Recently, we have identified and characterized the large structural Nups, Nup188 and Nup192, from the thermophilic eukaryote Chaetomium thermophilum, which exhibited superior properties for biochemical and structural studies, when compared to their mesophilic orthologs. Here, we study the large structural Nups from the model organism yeast Saccharomyces cerevisiae. Our data show that yeast Nup188 like its thermophilic orthologue ctNup188 exhibits a twisted S-like structure, which flexibly binds the linker nucleoporin Nic96 via a short conserved α-helix motif. Using bioinformatic methods, we have generated a pseudo-atomic structural model of Nup188 and its related Nup192, which further strengthens the view that the large α-solenoid structural Nups are related to karyopherins.

Insight into structure and assembly of the nuclear pore complex by utilizing the genome of a eukaryotic thermophile.

Despite decades of research, the structure and assembly of the nuclear pore complex (NPC), which is composed of ∼30 nucleoporins (Nups), remain elusive. Here, we report the genome of the thermophilic fungus Chaetomium thermophilum (ct) and identify the complete repertoire of Nups therein. The thermophilic proteins show improved properties for structural and biochemical studies compared to their mesophilic counterparts, and purified ctNups enabled the reconstitution of the inner pore ring module that spans the width of the NPC from the anchoring membrane to the central transport channel. This module is composed of two large Nups, Nup192 and Nup170, which are flexibly bridged by short linear motifs made up of linker Nups, Nic96 and Nup53. This assembly illustrates how Nup interactions can generate structural plasticity within the NPC scaffold. Our findings therefore demonstrate the utility of the genome of a thermophilic eukaryote for studying complex molecular machines.

The structure of Get4 reveals an alpha-solenoid fold adapted for multiple interactions in tail-anchored protein biogenesis.

Tail-anchored proteins play important roles in protein translocation, membrane fusion and apoptosis. They are targeted to the endoplasmic reticulum membrane via the guided-entry of tail-anchored proteins (Get) pathway. We present the 2A crystal structure of Get4 which participates in early steps of the Get pathway. The structure shows an alpha-solenoid fold with particular deviations from the regular pairwise arrangement of alpha-helices. A conserved hydrophobic groove accommodates the flexible C-terminal region in trans. The structural organization of the Get4 helical hairpin motifs provides a scaffold for protein-protein interactions in the Get pathway.

Structural insights into tail-anchored protein binding and membrane insertion by Get3.

Tail-anchored (TA) membrane proteins are involved in a variety of important cellular functions, including membrane fusion, protein translocation, and apoptosis. The ATPase Get3 (Asna1, TRC40) was identified recently as the endoplasmic reticulum targeting factor of TA proteins. Get3 consists of an ATPase and alpha-helical subdomain enriched in methionine and glycine residues. We present structural and biochemical analyses of Get3 alone as well as in complex with a TA protein, ribosome-associated membrane protein 4 (Ramp4). The ATPase domains form an extensive dimer interface that encloses 2 nucleotides in a head-to-head orientation and a zinc ion. Amide proton exchange mass spectrometry shows that the alpha-helical subdomain of Get3 displays considerable flexibility in solution and maps the TA protein-binding site to the alpha-helical subdomain. The non-hydrolyzable ATP analogue AMPPNP-Mg(2+)- and ADP-Mg(2+)-bound crystal structures representing the pre- and posthydrolysis states are both in a closed form. In the absence of a TA protein cargo, ATP hydrolysis does not seem to be possible. Comparison with the ADP.AlF(4)(-)-bound structure representing the transition state (Mateja A, et al. (2009) Nature 461:361-366) indicates how the presence of a TA protein is communicated to the ATP-binding site. In vitro membrane insertion studies show that recombinant Get3 inserts Ramp4 in a nucleotide- and receptor-dependent manner. Although ATP hydrolysis is not required for Ramp4 insertion per se, it seems to be required for efficient insertion. We postulate that ATP hydrolysis is needed to release Get3 from its receptor. Taken together, our results provide mechanistic insights into posttranslational targeting of TA membrane proteins by Get3.