Vaults are large cytoplasmic ribonucleoprotein particles that possess a distinct barrel-shaped morphology. Vaults have a mass of 13 million daltons and are composed of four proteins and a small RNA. Vaults are found in most if not all eukaryotic organisms including the lower eukaryote Dictyostelium discoideum. The ubiquitous distribution of these structures and the strong conservation of their morphology and composition suggest that vault function is essential. Although vaults are largely cytoplasmic, a subpopulation of vaults has been localized to the nuclear membrane at nuclear pore complexes (NPCs). This finding, coupled with vault shape, size and symmetry suggests that vaults may interact with the NPC. The discovery of vaults in Dictyostelium has lead to a molecular approach toward elucidation of vault function in this organism. cDNAs for the two major Dictyostelium vault proteins (MVPalpha and MVPbeta) will be cloned and used to construct gene replacement plasmids. Vault proteins will be ablated or truncated in Dictyostelium by gene targeting and double homologous recombination in order to produce cell lines devoid of vault function. We will examine important features of cellular metabolism and physiology in these mutant lines to determine the cellular functions in which vaults play a role. The recent availability of a cDNA for the rat major vault protein has allowed the design of a PCR strategy for identifying a similar protein in yeast. Once identified, the yeast AM gene will be cloned, sequenced and used to determine if yeast contain vaults and if these structures are essential to yeast growth and survival.
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