The Vault Exterior Shell Is a Dynamic Structure that Allows Incorporation of Vault-Associated Proteins into Its Interior

Abstract

Vaults are 13 million Dalton ribonucleoprotein particles with a highly conserved structure. Expression and assembly by multimerization of an estimated 96 copies of a single protein, termed the major vault protein (MVP), is sufficient to form the minimal structure and entire exterior shell of the barrel-shaped vault particle. Multiple copies of two additional proteins, VPARP and TEP1, and a small untranslated vault RNA are also associated with vaults. We used the Sf9 insect cell expression system to form MVP-only recombinant vaults and performed a series of protein-mixing experiments to test whether this particle shell is able to exclude exogenous proteins from interacting with the vault interior. Surprisingly, we found that VPARP and TEP1 are able to incorporate into vaults even after the formation of the MVP vault particle shell is complete. Electrospray molecular mobility analysis and spectroscopic studies of vault-interacting proteins were used to confirm this result. Our results demonstrate that the protein shell of the recombinant vault particle is a dynamic structure and suggest a possible mechanism for in vivo assembly of vault-interacting proteins into preformed vaults. Finally, this study suggests that the vault interior may be functionally interactive with the cellular milieu.Vaults are structurally conserved ribonucleoprotein (RNP) 1 particles that have been implicated in multidrug resistance, nucleocytoplasmic transport, and as scaffolds for both epidermal growth factor signaling and interferon-gamma activated JAK/STAT signaling pathways; however, their precise function remains unclear (1-9). The particles have a capped-barrel morphology with dimensions of approximately 41 × 41 × 72.5 nm (10), and at 13 million Daltons, are the largest known RNP. Vaults have 8-fold symmetry around their longitudinal axis and each half-vault appears identical (8-2-2 symmetry). When plated onto poly-L-lysine coated electron microscopy grids and visualized by freeze-etch platinum shadowing, vaults † This research was supported by grants from the National Science Foundation (MCB-0210690 and CHE-0507929) and the G. Harold and Leila Y. Mathers Charitable Foundation (01124244). The UCLA Functional Proteomics Center was established and equipped by a grant from the W. M. Keck Foundation. JAL acknowledges support from the Jonsson Comprehensive Cancer Center at UCLA, the UCLA-DOE Institute for Genomics and Proteomics, and the National Institutes of Health (RR 20004).*To whom all correspondence should be addressed: Leonard H. Rome, Department of Biological Chemistry, University of California, Los Angeles, 33-131 CHS mail code #173717, 10833 Le Conte Avenue, Los Angeles, California 90095-1737, Tel. 310 825-0709; Fax. 310 206-5272; Email: lrome@mednet.ucla.edu. 1 ABBREVIATIONS: RNP, ribonucleoprotein; MVP, major vault protein; TEP1, telomerase associated protein 1; VPARP, vault poly (ADP-ribose) polymerase; VR, vault RNA; cryoEM, cryoelectron microscopy; TEM, transmission electron microscopy; M-INT, minimal MVP interactio...

Electrospray–differential mobility analysis of bionanoparticles

“…It was demonstrated that the monomers of different proteins can be differentiated from their respective oligomers (dimers, trimers) and that there was a strong correlation between the molecular weight and the mobility diameter determined using ES-DMA. Since then, the molecular weightmobility diameter empirical correlation plotted in Figure 3.1 has been confirmed by several groups of researchers for both globular, as well as non-globular proteins ranging from a few kDa [11,19,56,57] to several MDa [56]. The actual expression is later going to be used in Chapter 8.…”

“…It has also been used to gain insight into the function of a number of protein complexes such as vault proteins [56], 20S…”

Pendular proteins in gases and new avenues for characterization of macromolecules by ion mobility spectrometry

“…In particular, peptide and protein folding (2,4,(9)(10)(11)(12)(13)(14) and amyloidogenic misfolding (15) were explored. Application of IMS and IMS/MS is expanding to ever-larger macromolecules, including noncovalent assemblies with mass (m) Ͼ 1 MDa (16)(17)(18). Such studies have used both drift tube (DT) IMS (1)(2)(3)(4)(5)(7)(8)(9)(10)(11)(12)(13)(14)(15) and differential mobility analyzers (DMA) (17,18) to separate ions by absolute mobility (K).…”

“…Application of IMS and IMS/MS is expanding to ever-larger macromolecules, including noncovalent assemblies with mass (m) Ͼ 1 MDa (16)(17)(18). Such studies have used both drift tube (DT) IMS (1)(2)(3)(4)(5)(7)(8)(9)(10)(11)(12)(13)(14)(15) and differential mobility analyzers (DMA) (17,18) to separate ions by absolute mobility (K). In DT IMS, ions are separated while being pulled through still gas by an electric field.…”

“…In DT IMS, ions are separated while being pulled through still gas by an electric field. In DMA (17)(18)(19) ions traverse a space between 2 electrodes while being displaced laterally by gas flow. Only ions of specific K (determined by the voltage across the gap and flow speed) exit the gap, and mobility spectra are obtained by scanning the voltage.…”