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. 2014 Nov 25;8(11):11552-9.
doi: 10.1021/nn504778h. Epub 2014 Oct 30.

Polyribosomes are molecular 3D nanoprinters that orchestrate the assembly of vault particles

Jan Mrazek  1 , Daniel Toso, Sergey Ryazantsev, Xing Zhang, Z Hong Zhou, Beatriz Campo Fernandez, Valerie A Kickhoefer, Leonard H Rome
Affiliations
Free PMC article

Polyribosomes are molecular 3D nanoprinters that orchestrate the assembly of vault particles

Jan Mrazek et al. ACS Nano. .
Free PMC article

Abstract

Ribosomes are molecular machines that function in polyribosome complexes to translate genetic information, guide the synthesis of polypeptides, and modulate the folding of nascent proteins. Here, we report a surprising function for polyribosomes as a result of a systematic examination of the assembly of a large ribonucleoprotein complex, the vault particle. Structural and functional evidence points to a model of vault assembly whereby the polyribosome acts like a 3D nanoprinter to direct the ordered translation and assembly of the multi-subunit vault homopolymer, a process which we refer to as polyribosome templating. Structure-based mutagenesis and cell-free in vitro expression studies further demonstrated the critical importance of the polyribosome in vault assembly. Polyribosome templating prevents chaos by ensuring efficiency and order in the production of large homopolymeric protein structures in the crowded cellular environment and might explain the origin of many polyribosome-associated molecular assemblies inside the cell.

Keywords: cryo-electron tomography; nanoprinting; polyribosome function; structure-based mutagenesis; vault particle.

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Figures

Figure 1
Figure 1
Systematic mutagenesis of the vault structure. (A) Scheme of MVP configuration within the vault structure. (B) MVP N-terminal modifications. (C) Outcome of the structure-based mutagenesis: (check mark) = positive, (x) = negative.
Figure 2
Figure 2
Representative structures of the 6-His-MVP mutant. Electron micrographs of uranyl acetate stained supernatants of lysates from infected Sf9 cells. (A) Wild-type MVP vaults with a close-up view from its red inset. (B) Staggered rolls of MVP chains. Close-up view of the rolls aligned with a vault particle from the crystal structure. The vault cap (C), shoulder (S), and waist (W) regions are indicated by white dashed lines. (C) Long sheet of an unraveled MVP roll. Close-up view of the sheet superimposed with several individual MVP chains from the crystal structure.
Figure 3
Figure 3
Cryo-electron tomography of 6-His-MVP mutant rolled structures. (A,B) Two frames from a cryo-ET cut series (Supporting Information movie S1) corresponding to different sample depths through a multiple 6-His-MVP roll. (C,D) Vault particle is superimposed over the center of each roll, shown at the same magnification.
Figure 4
Figure 4
Model for vault assembly by polyribosome templating. (A) Schematic representation of a fully assembled polyribosome; as translation continues, MVP chains emerge (red); when two opposing MVP chains are long enough (red arrow), the N-termini interact to form a dimer; as translation of the MVP dimers nears completion, side-to-side interactions between neighboring MVP dimers begin to occur to give rise to an MVP tetramer (blue arrow). These side-to-side interactions of sequentially incoming MVP dimers begin to form a sheet (B,C), initiating the vault body to take its unique structure (D). Once 39 MVP dimers emerge, a pinch-off event occurs, leading to formation of an intact vault particle (E). All components of the model (MVP, vaults, and the 80S ribosome),– were drawn to scale.
Figure 5
Figure 5
Experimental evidence for polyribosome templating. (A) Electron micrograph of in vitro synthesized vaults. Samples were negatively stained with uranyl acetate. (B) Scheme of a differential centrifugation experiment. (C) Western blot analysis of S20, S100, and P100 fractions from the differential centrifugation.
Figure 6
Figure 6
Additional supportive evidence for polyribosome templating. (A–C) Coexpression of two different MVP mRNAs leads to two types of vaults. Electron micrograph of (A) VSVG-MVP full vaults expressed from a single promoter plasmid, (B) mCherry-MVP half vaults expressed from a single promoter plasmid, and (C) coexpression of mCherry/VSVG-MVP half and full vaults using a dual promoter plasmid. (D,E) Visible association of 6-His-MVP mutant structures with polyribosomes. Electron micrographs of purified polyribosomes from Sf9 cells expressing 6-His-MVP for 48 h (D) or MVP for 24 h (E). Samples were negatively stained with uranyl acetate. Scale bar 100 nm.
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