Abstract

Vaults are large macromolecular assemblies composed of the 100 kDa major vault protein (MVP), the 193 kDa vault poly(ADP)ribose polymerase (VPARP), the 240 kDa telomerase/vault asssociated protein (TEP1), and one or more related small RNAs (vRNA). About 75% of the particle mass is composed of the MVP. Their ubiquitous expression, high copy number, conserved morphology, and composition all indicate they have an important yet undefined cellular function. The vault ribonucleoprotein complex is primarily localized to the cytoplasm and about ~5% are thought to associate with the nucleus near nuclear pore complexes. VPARP is a member of the PARP family of proteins. In purified vaults VPARP retains its enzymatic activity and ADP ribosylates itself and MVP. VPARP is the only vault component identified to date with an enzymatic activity and has been shown to produce short polymers of ADP-ribose units. Our hypothesis is that the sequestration of VPARP in the vault particle is a powerful means of regulating its activity and substrate availability. We have developed a vault assembly system where expression of MVP alone is sufficient to form the basic vault particle using baculovirus expression in insect cells. In this system, co-expression of MVP with either VPARP, or TEP1, or both leads to production of uniform, stable vault-like particles. An analysis of VLP structure and VPARP catalytic activity using this vault assembly system will be presented. The major goal of our research is to determine VPARPs contribution to vault function and thus elucidate the overall function of the vault particle.

Keywords: vaults, VPARP, baculovirus