vaults: novel cell particles

Scientists: Concluding Remarks


The Rome laboratory at UCLA is examining vaults with two major aims. First to determine the function of vaults and second to engineer the vault particle as a nanocapsule with a wide range of potential functions.

Since function is frequently dictated by structure, we have numerous ongoing projects detailing the structure of the vault particle by conventional electron microscopy, cryo-EM and x-ray crystallography. Studies have led to the characterization at the molecular level of all of the core vault components. Examination of the sequenced vRNAs has revealed a striking structural similarity which has lent support to the hypothesis that the vRNA serves a functional role in the vault particle. The details of this contribution to vault function need to be resolved, possibly using antisense oligonucleotide technology or by creating a mouse knockout. The vRNA genes have turned out to be interesting RNA polymerase III substrates containing a mixture of type-2 and type-3 promoter elements whose arrangement tightly controls expression and is likely to explain the wide tissue variability of this RNA. Analysis of major vault proteins from a number of species indicates that the MVPs constitute a new class of proteins, highly related to one another but unrelated to any previously characterized cell proteins.

In dictyostelium, where there are at least 3 MVPs, disruption of two MVPs leads to an altered growth phenotype under nutritional stress. The identification of the high molecular weight vault proteins, p240, as a component of telomerase and p193, as a new PARP, reveals an intriguing complexity in this particle. Although the MVP appears to play a structural role, its upregulation in multi-drug resistant cancer cells is intriguing and supports previous predictions that vaults have a transport function. Are vaults actively transporting drugs and/or RNA and are they interacting with cellular structures, such as the nuclear pore complex? Answers to these questions will be the basis of future studies of vault function.

Studies of recombinant vaults demonstrate that nanoparticles can be produced with chemically active or fluorescent proteins sequestered within the particle cavity. The baculovirus system is robust, allowing for production and purification of 4-20 mg of vaults per liter culture of cells. The INT domain appears to be a general targeting sequence that should be able to direct a wide variety of recombinant proteins (and other molecules) into the vault. As the internal volume of the vault is 50 million cubic angstroms there is sufficient space for 100's of proteins in the vault lumen. The largest non-vault protein thus far targeted to the vault interior using the INT domain is the ~61 kDa firefly luciferase protein. Once targeted inside the vault, INT-fusion proteins appear to be both functional and stably associated. This strategy could be used to confer unique properties onto vaults by targeting other molecules (e.g. metals, nucleic acids, polynucleotides, polymers, etc.) by virtue of fusing their protein binding domains onto the INT domain. Although the thin protein shell of the vault does not prevent the entry of small ligands, there appears to be a diffusion barrier, particularly for charged molecules. The entry of charged molecules into the vault cavity might require relatively slow conformational changes to take place within the vault protein shell.

The engineering of vault particles with designed properties and functionalities represents an important direction for the emerging field of bionanotechnology. This research establishes that intrinsic optical properties of proteins can be retained when they are confined within the vault nanocapsule. It is likely that a wide range of proteins with other chemical properties may be sequestered within recombinant vaults using this approach. A number of applications for these engineered particles can be envisioned including biologically-based chemical sensors, bioreactors, and protein stabilizers, all of which can be targeted at the cellular level because of the biocompatibility of the capsule.

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