title: The vault exterior shell is a dynamic structure that allows incorporation of vault-associated proteins into its interior.
abstract: Vaults are 13 million Da 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 functionally interact with the cellular milieu.
authors.affiliation: Department of Biological Chemistry, David Geffen School of Medicine, University of California, Los Angeles, California 90095, USA.
authors.forename: Michael J
authors.initials: MJ
authors.lastname: Poderycki
authors.forename: Valerie A
authors.initials: VA
authors.lastname: Kickhoefer
authors.forename: Catherine S
authors.initials: CS
authors.lastname: Kaddis
authors.forename: Sujna
authors.initials: S
authors.lastname: Raval-Fernandes
authors.forename: Erik
authors.initials: E
authors.lastname: Johansson
authors.forename: Jeffrey I
authors.initials: JI
authors.lastname: Zink
authors.forename: Joseph A
authors.initials: JA
authors.lastname: Loo
authors.forename: Leonard H
authors.initials: LH
authors.lastname: Rome
mesh_terms: D000818:Animals; D002352:Carrier Proteins / Q000737:chemistry*; D006801:Humans; D011065:Poly(ADP-ribose) Polymerases / Q000737:chemistry*; D020836:Protein Structure, Quaternary; D016601:RNA-Binding Proteins / Q000737:chemistry*; D051381:Rats; D011994:Recombinant Proteins / Q000737:chemistry; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods; D020394:Vault Ribonucleoprotein Particles / Q000737:chemistry*; D000099187:Major Vault Protein
chemical_list: D002352:Carrier Proteins; D011065:Poly(ADP-ribose) Polymerases; D016601:RNA-Binding Proteins; D011994:Recombinant Proteins; D020394:Vault Ribonucleoprotein Particles; D000099187:Major Vault Protein; C104342:TEP1 protein, human; C449359:vault poly(ADP-ribose) polymerase