CDNA CLONING AND DISRUPTION OF THE MAJOR VAULT PROTEIN-ALPHA GENE (MVPA) IN DICTYOSTELIUM-DISCOIDEUM

Vaults are large cytoplasmic ribonucleoprotein particles found in near ly all eukaryotic cells. Dictyostelium vaults contain two major protei ns, MVPalpha (94.2 kDa) and MVPbeta (approximately 92 kDa). Using an a nti-rat vault antibody, we screened a Dictyostelium cDNA expression li brary and isolated a 2.8-kilobase pair clone that contained a single f ull-length reading frame. The identity of the clone was established by the presence of a predicted 20-amino acid sequence identical to that found in a peptide sequenced from purified MVPalpha. We have disrupted the single copy gene using homologous recombination and have demonstr ated a loss of MVPalpha. Although the cells still produce MVPbeta, the y do not contain characteristic vault particles, suggesting that MVPal pha is required for normal vault structure. These cells should be a va luable tool for elucidating the function of vaults.