Dictyostelium Vaults: Disruption of the Major Proteins Reveals Growth and Morphological Defects and Uncovers a New Associated Protein

Abstract

Vaults are large cytoplasmic ribonucleoprotein particles that are highly conserved in both morphology and protein composition. Protein components of vaults isolated from Dictyostelium discoideum migrate on SDS-polyacrylamide gels as two bands, one at 94 kDa (MvpA) and the other at 92 kDa (MvpB). An MvpB cDNA clone was isolated from a Dictyostelium expression library. MvpB shares 60% identity with MvpA at the amino acid level. This cDNA has been used to disrupt the single mvpB gene in both wild-type and mvpA- genetic backgrounds. Although the mvp- mutant lines are viable, they show that loss of MvpA and/or MvpB interferes with vault function sufficiently to impede growth under conditions of nutritional stress. The resulting mutant cell lines reach stationary phase in suspension culture at one-third of the density of wild-type cells. Ovoid structures isolated from mvp- single mutant lines represent what remains of vaults in these cells. Similar ovoid structures isolated from the mvpA- mvpB- line copurify with a newly identified protein of 92 kDa (MvpC), which lacks cross-reactivity with currently available anti-vault antibodies. Our results indicate that the major vault proteins are necessary for optimal cell growth in Dictyostelium and reveal an unanticipated complexity in vault composition.