Direct visualization of vaults within intact cells by electron cryo-tomography.

The vault complex is the largest cellular ribonucleoprotein complex ever characterized and is present across diverse Eukarya. Despite significant information regarding the structure, composition and evolutionary conservation of the vault, little is know about the complex's actual biological function. To determine if intracellular vaults are morphologically similar to previously studied purified and recombinant vaults, we have used electron cryo-tomography to characterize the vault complexes found in the thin edges of primary human cells growing in tissue culture. Our studies confirm that intracellular vaults are similar in overall size and shape to purified and recombinant vaults previously analyzed. Results from subtomogram averaging indicate that densities within the vault lumen are not ordered, but randomly distributed. We also observe that vaults located in the extreme periphery of the cytoplasm predominately associate with granule-like structures and actin. Our ultrastructure studies augment existing biochemical, structural and genetic information on the vault, and provide important intracellular context for the ongoing efforts to understand the biological function of the native cytoplasmic vault.

Molecular cloning and characterization of major vault protein of Echinococcus multilocularis.

The cDNA clone coding a major vault protein (MVP)-like protein was derived from Echinococcus multilocularis cysts. MVP is a main component of vault particles, which are the largest cytoplasmic ribonucleoprotein particles in eukaryotic cells. We sequenced and characterized E. multilocularis MVP (EmMVP). The nucleotide sequence of the emmvp cDNA clone was 2607 bp in the full length open reading frame and its deduced amino acid sequence had several signature motifs which were specific to MVP families. Immunoblot analysis with mouse anti-EmMVP antiserum revealed that crude antigens of E. multilocularis included EmMVP protein. Furthermore, our results showed that the expression of EmMVP protein in an Sf9 insect cell line using a baculovirus vector directed the formation of particles that shared similar biochemical characteristics with other vault proteins and the distinct vault-like morphology when negatively stained and examined by electron microscopy.

Assembly of nuclear pore complexes mediated by major vault protein.

During interphase growth of eukaryotic cells, nuclear pore complexes (NPCs) are continuously incorporated into the intact nuclear envelope (NE) by mechanisms that are largely unknown. De novo formation of NPCs involves local fusion events between the inner and outer nuclear membrane, formation of a transcisternal membranous channel of defined diameter and the coordinated assembly of hundreds of nucleoporins into the characteristic NPC structure. Here we have used a cell-free system based on Xenopus egg extract, which allows the experimental separation of nuclear-membrane assembly and NPC formation. Nuclei surrounded by a closed double nuclear membrane, but devoid of NPCs, were first reconstituted from chromatin and a specific membrane fraction. Insertion of NPCs into the preformed pore-free nuclei required cytosol containing soluble nucleoporins or nucleoporin subcomplexes and, quite unexpectedly, major vault protein (MVP). MVP is the main component of vaults, which are ubiquitous barrel-shaped particles of enigmatic function. Our results implicate MVP, and thus also vaults, in NPC biogenesis and provide a functional explanation for the association of a fraction of vaults with the NE and specifically with NPCs in intact cells.

A vault ribonucleoprotein particle exhibiting 39-fold dihedral symmetry.

Vault is a 12.9 MDa ribonucleoprotein particle with a barrel-like shape, two protruding caps and an invaginated waist structure that is highly conserved in a wide variety of eukaryotes. Multimerization of the major vault protein (MVP) is sufficient to assemble the entire exterior shell of the barrel-shaped vault particle. Multiple copies of two additional proteins, vault poly(ADP-ribose) polymerase (VPARP) and telomerase-associated protein 1 (TEP1), as well as a small vault RNA (vRNA), are also associated with vault. Here, the crystallization of vault particles is reported. The crystals belong to space group C2, with unit-cell parameters a = 708.0, b = 385.0, c = 602.9 angstroms, beta = 124.8 degrees . Rotational symmetry searches based on the R factor and correlation coefficient from noncrystallographic symmetry (NCS) averaging indicated that the particle has 39-fold dihedral symmetry.

Preliminary analysis of two and three dimensional crystals of vault ribonucleoprotein particles.

Vaults are large ribonucleoprotein particles found in a wide variety of eukaryotes. When imaged by electron-microscopy vaults present a strikingly conserved barrel-shaped structure with an invaginated waist and two protruding caps. In this work, we present two dimensional (2D) and three dimensional (3D) crystals of naturally produced vaults in murine and monkey cells, respectively. The 2D-crystals presented a hexagonal packing with the lattice parameter defined by the diameter of the vault barrel. Fourier transforms from images of the negatively stained 2D-crystals showed spots till about 45 A resolution. The 3D-crystals reached about 0.15 x 0.15 x 0.02 mm3 in size and presented a flat triangular morphology with well-developed faces. The preliminary characterization of these 3D-crystals, which diffract very weakly to approximately 10 A resolution, suggests a trigonal packing with the R32 space group symmetry. The 3D-crystals appear to be formed by adding layers of vaults, which retain the hexagonal organization seen in the 2D-crystals, with relative shifts that maximize the interdigitation of particles in adjacent layers. Accurate crystal symmetry in the 2D- and 3D-crystals requires neighbor particles interacting according to a 6-fold and a 3-fold dihedral symmetry, respectively. Compatibility with the reported 8-fold symmetry would imply multiples of 24-fold rotational symmetry, in agreement with the recently proposed 48-fold dihedral symmetry for reconstituted recombinant vaults.

Nuclear localization of the major vault protein in U373 cells.

The major vault protein (MVP) is the predominant member of a large ribonucleoprotein particle, named vault. Vaults are abundant in the cytosol of mammalian cells. Mammalian MVP has previously been reported to be associated with the nucleus, particularly its cytosolic surface on which vaults are thought to dock at or near the nuclear pore complex. To date the presence of vault particles inside the nucleus has been convincingly reported only for sea urchin cells. We have addressed the potential nuclear localization of MVP in mammalian cells by employing confocal laser microscopy and cryo-immunoelectron microscopy. As revealed by immunostaining and by analysis of cells transfected with a construct encoding MVP and green fluorescent protein, MVP is present in both the cytosol and in the nucleus. Cryo-electron microscopy of human astroglioma U373 cells reveals clusters of immunogold particles at nuclear pores and in the nucleoplasm suggesting that nuclear MVP is associated with particulate structures. Quantification of the fluorescence observed in the cytosol and in the nuclei reveals that about 5% of the MVP in U373 cells is localized inside the nucleus. Our results further support the notion that part of the cellular MVP can enter the nucleus.

Engineering of vault nanocapsules with enzymatic and fluorescent properties.

One of the central issues facing the emerging field of nanotechnology is cellular compatibility. Nanoparticles have been proposed for diagnostic and therapeutic applications, including drug delivery, gene therapy, biological sensors, and controlled catalysis. Viruses, liposomes, peptides, and synthetic and natural polymers have been engineered for these applications, yet significant limitations continue to prevent their use. Avoidance of the body's natural immune system, lack of targeting specificity, and the inability to control packaging and release are remaining obstacles. We have explored the use of a naturally occurring cellular nanoparticle known as the vault, which is named for its morphology with multiple arches reminiscent of cathedral ceilings. Vaults are 13-MDa ribonucleoprotein particles with an internal cavity large enough to sequester hundreds of proteins. Here, we report a strategy to target and sequester biologically active materials within the vault cavity. Attachment of a vault-targeting peptide to two proteins, luciferase and a variant of GFP, resulted in their sequestration within the vault cavity. The targeted proteins confer enzymatic and fluorescent properties on the recombinant vaults, both of which can be detected by their emission of light. The modified vaults are compatible with living cells. The ability to engineer vault particles with designed properties and functionalities represents an important step toward development of a biocompatible nanocapsule.

Sea urchin vault structure, composition, and differential localization during development.

BACKGROUND: Vaults are intriguing ribonucleoprotein assemblies with an unknown function that are conserved among higher eukaryotes. The Pacific coast sea urchin, Strongylocentrotus purpuratus, is an invertebrate model organism that is evolutionarily closer to humans than Drosophila and C. elegans, neither of which possesses vaults. Here we compare the structures of sea urchin and mammalian vaults and analyze the subcellular distribution of vaults during sea urchin embryogenesis. RESULTS: The sequence of the sea urchin major vault protein (MVP) was assembled from expressed sequence tags and genome traces, and the predicted protein was found to have 64% identity and 81% similarity to rat MVP. Sea urchin MVP includes seven approximately 50 residue repeats in the N-terminal half of the protein and a predicted coiled coil domain in the C-terminus, as does rat MVP. A cryoelectron microscopy (cryoEM) reconstruction of isolated sea urchin vaults reveals the assembly to have a barrel-shaped external structure that is nearly identical to the rat vault structure. Analysis of the molecular composition of the sea urchin vault indicates that it contains components that may be homologs of the mammalian vault RNA component (vRNA) and protein components (VPARP and TEP1). The sea urchin vault appears to have additional protein components in the molecular weight range of 14-55 kDa that might correspond to molecular contents. Confocal experiments indicate a dramatic relocalization of MVP from the cytoplasm to the nucleus during sea urchin embryogenesis. CONCLUSIONS: These results are suggestive of a role for the vault in delivering macromolecules to the nucleus during development.

Cryoelectron microscopy imaging of recombinant and tissue derived vaults: localization of the MVP N termini and VPARP.

The vault is a highly conserved ribonucleoprotein particle found in all higher eukaryotes. It has a barrel-shaped structure and is composed of the major vault protein (MVP); vault poly(ADP-ribose) polymerase (VPARP); telomerase-associated protein 1 (TEP1); and small untranslated RNA (vRNA). Although its strong conservation and high abundance indicate an important cellular role, the function of the vault is unknown. In humans, vaults have been implicated in multidrug resistance during chemotherapy. Recently, assembly of recombinant vaults has been established in insect cells expressing only MVP. Here, we demonstrate that co-expression of MVP with one or both of the other two vault proteins results in their co-assembly into regularly shaped vaults. Particles assembled from MVP with N-terminal peptide tags of various length are compared. Cryoelectron microscopy (cryoEM) and single-particle image reconstruction methods were used to determine the structure of nine recombinant vaults of various composition, as well as wild-type and TEP1-deficient mouse vaults. Recombinant vaults with MVP N-terminal peptide tags showed internal density that varied in size with the length of the tag. Reconstruction of a recombinant vault with a cysteine-rich tag revealed 48-fold rotational symmetry for the vault. A model is proposed for the organization of MVP within the vault with all of the MVP N termini interacting non-covalently at the vault midsection and 48 copies of MVP forming each half vault. CryoEM difference mapping localized VPARP to three density bands lining the inner surface of the vault. Difference maps designed to localize TEP1 showed only weak density inside of the caps, suggesting that TEP1 may interact with MVP via a small interaction region. In the absence of atomic-resolution structures for either VPARP or TEP1, fold recognition methods were applied. A total of 21 repeats were predicted for the TEP1 WD-repeat domain, suggesting an unusually large beta-propeller fold.

The vault complex.

Vaults are large ribonucleoprotein particles found in eukaryotic cells. They are composed of multiple copies of a Mr 100,000 major vault protein and two minor vault proteins of Mr 193,000 and 240,000, as well as small untranslated RNAs of 86-141 bases. The vault components are arranged into a highly characteristic hollow barrel-like structure of 35 x 65 nm in size. Vaults are predominantly localized in the cytoplasm where they may associate with cytoskeletal elements. A small fraction of vaults are found to be associated with the nucleus. As of yet, the precise cellular function of the vault complex is unknown. However, their distinct morphology and intracellular distribution suggest a role in intracellular transport processes. Here we review the current knowledge on the vault complex, its structure, components and possible functions.

Vaults bind directly to microtubules via their caps and not their barrels.

Vaults are large (13 Mda) ribonucleoprotein particles that are especially abundant in multidrug resistant cancer cells and have been implicated in nucleocytoplasmic drug transport. To understand how these large barrel-shaped complexes are transported through the cytosol, we examined the association of vaults with microtubules both in vitro and in vivo. Within cells, a subpopulation of vaults clearly associates with microtubules, and these vaults remain associated with tubulin dimers/oligomers when microtubules are disassembled by nocodazole treatment. In vitro, a microtubule-pull down assay using highly purified rat vaults and reassembled microtubules reveals that vaults exhibit concentration-dependent binding to microtubules that does not require the carboxyl terminal end of tubulin. Remarkably, negative staining for electron microscopy reveals that vault binding to microtubules is mediated by the vault caps; more than 82% of bound vaults attach to the microtubule lattice with their long axes perpendicular to the long axis of the microtubule. Five to six vault particles were bound per micron of microtubule, with no crosslinking of microtubules observed, suggesting that only one end of the vault can bind microtubules. Taken together, the data support the model of vaults as barrel-shaped containers that transiently interact with microtubules.

Assembly of vault-like particles in insect cells expressing only the major vault protein.

Vaults are the largest (13 megadalton) cytoplasmic ribonucleoprotein particles known to exist in eukaryotic cells. They have a unique barrel-shaped structure with 8-fold symmetry. Although the precise function of vaults is unknown, their wide distribution and highly conserved morphology in eukaryotes suggests that their function is essential and that their structure must be important for their function. The 100-kDa major vault protein (MVP) constitutes approximately 75% of the particle mass and is predicted to form the central barrel portion of the vault. To gain insight into the mechanisms for vault assembly, we have expressed rat MVP in the Sf9 insect cell line using a baculovirus vector. Our results show that the expression of the rat MVP alone can direct the formation of particles that have biochemical characteristics similar to endogenous rat vaults and display the distinct vault-like morphology when negatively stained and examined by electron microscopy. These particles are the first example of a single protein polymerizing into a non-spherically, non-cylindrically symmetrical structure. Understanding vault assembly will enable us to design agents that disrupt vault formation and hence aid in elucidating vault function in vivo.

RNA location and modeling of a WD40 repeat domain within the vault.

The vault complex is a ubiquitous 13-MDa ribonucleoprotein assembly, composed of three proteins (TEP1, 240 kDa; VPARP, 193 kDa; and MVP, 100 kDa) that are highly conserved in eukaryotes and an untranslated RNA (vRNA). The vault has been shown to affect multidrug resistance in cancer cells, and one particular component, MVP, is thought to play a role in the transport of drug from the nucleus. To locate the position of the vRNA, vaults were treated with RNases, and cryo-electron microscopy (cryo-EM) was performed on the resulting complexes. Using single-particle reconstruction techniques, 3,476 particle images were combined to generate a 22-A-resolution structure. Difference mapping between the RNase-treated vault and the previously calculated intact vault reconstructions reveals the vRNA to be at the ends of the vault caps. In this position, the vRNA may interact with both the interior and exterior environments of the vault. The finding of a 16-fold density ring at the top of the cap has allowed modeling of the WD40 repeat domain of the vault TEP1 protein within the cryo-EM vault density. Both stoichiometric considerations and the finding of higher resolution for the computationally selected and refined "barrel only" images indicate a possible symmetry mismatch between the barrel and the caps. The molecular architecture of the complex is emerging, with 96 copies of MVP composing the eightfold symmetric barrel, and the vRNA together with one copy of TEP1 and four predicted copies of VPARP comprising each cap.

Structure of the vault, a ubiquitous celular component.

BACKGROUND: The vault is a ubiquitous and highly conserved ribonucleoprotein particle of approximately 13 MDa. This particle has been shown to be upregulated in certain multidrug-resistant cancer cell lines and to share a protein component with the telomerase complex. Determination of the structure of the vault was undertaken to provide a first step towards understanding the role of this cellular component in normal metabolism and perhaps to shed some light on its role in mediating drug resistance. RESULTS: Over 1300 particle images were combined to calculate an approximately 31 A resolution structure of the vault. Rotational power spectra did not yield a clear symmetry peak, either because of the thin, smooth walls or inherent flexibility of the vault. Although cyclic eightfold (C8) symmetry was imposed, the resulting reconstruction may be partially cylindrically averaged about the eightfold axis. Our results reveal the vault to be a hollow, barrel-like structure with two protruding caps and an invaginated waist. CONCLUSIONS: Although the normal cellular function of the vault is as yet undetermined, the structure of the vault is consistent with either a role in subcellular transport, as previously suggested, or in sequestering macromolecular assemblies.