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.

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.

No abstract

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.

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 a-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.R ibosomes perform their role in translation in the cytoplasm, but ribosome assembly occurs predominantly in a specialized nuclear compartment, the nucleolus (1-4). The construction of ribosomes follows an ordered assembly of~80 ribosomal proteins (r-proteins) and four ribosomal RNAs (rRNAs) into a small (40S) and large (60S) ribosomal subunit. This process is spatially and temporally coordinated, starting with cotranscriptional assembly of a first preribosomal particle (90S) in the nucleolus that is subsequently separated into pre-40S and pre-60S ribosomes, which follow independent processing and maturation steps before export into the cytoplasm (5-8). R-proteins are synthesized in the cytoplasm and are imported into the nucleus by nuclear import receptors of the importinb/karyopherin family (9, 10). These transport receptors recognize different types of nuclear localization sequences (NLSs), and hence a number of import receptors have been implicated in decoding NLSs of r-proteins in a redundant way (9, 11). After nuclear import and before incorporation into nascent ribosomes, r-proteins are released from the transport receptor by its interaction with RanGTP (12). At present, it is thought that each r-protein is individually transported into the nucleus by its import receptor. However, a number of r-proteins form functional clusters on the ribosomal surface or assemble at distinct temporal or spatial entry points during ribosome formation (13-15), thus raising the possibility of coordinated nuclear import and assembly of r-proteins.One such pair of functionally related r-proteins is Rpl5 and Rpl11, which are close to each other on the mature 60S subunit and bind to opposite sites on the 5S rRNA ( Fig.