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.
Nucleocytoplasmic transport occurs through nuclear pore complexes (NPCs) embedded in the nuclear envelope. Here, we discovered an unexpected role for yeast dynein light chain (Dyn2) in the NPC. Dyn2 is a previously undescribed nucleoporin that functions as molecular glue to dimerize and stabilize the Nup82-Nsp1-Nup159 complex, a module of the cytoplasmic pore filaments. Biochemical analyses showed that Dyn2 binds to a linear motif (termed DID(Nup159)) inserted between the Phe-Gly repeat and coiled-coil domain of Nup159. Electron microscopy revealed that the reconstituted Dyn2-DID(Nup159) complex forms a rigid rod-like structure, in which five Dyn2 homodimers align like 'pearls on a string' between two extented DID(Nup159) strands. These findings imply that the rigid 20 nm long Dyn2-DID(Nup159) filament projects the Nup159 Phe-Gly repeats from the Nup82 module. Thus, it is possible that dynein light chain plays a role in organizing natively unfolded Phe-Gly repeats within the NPC scaffold to facilitate nucleocytoplasmic transport.
Vacuolar ATPases (V-ATPases) are ATP-dependent proton pumps that maintain the acidity of cellular compartments. They are composed of a membrane-integrated proton-translocating V(0) and an extrinsic cytoplasmic catalytic domain V(1), joined by several connecting subunits. To clarify the arrangement of these peripheral connections and their interrelation with other subunits of the holocomplex, we have determined the solution structures of isolated EG and EGC connecting subcomplexes by small angle X-ray scattering and the 3D map of the yeast V-ATPase by electron microscopy. In solution, EG forms a slightly kinked rod, which assembles with subunit C into an L-shaped structure. This model is supported by the microscopy data, which show three copies of EG with two of these linked by subunit C. However, the relative arrangement of the EG and C subunits in solution is more open than that in the holoenzyme, suggesting a conformational change of EGC during regulatory assembly and disassembly.
Highlights► The CCR4-NOT complex was purified from yeast. ► The complex was stabilized by cross-linking. ► Electron microscopic studies revealed that the complex is L-shaped. ► The arms of the complex form an accessible cavity.
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