Dictyostelium vaults: disruption of the major proteins reveals growth and morphological defects and uncovers a new associated protein.
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.
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Department of Biological Chemistry, University of California School of Medicine, Los Angeles 90024-1737, USA.
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D000595:Amino Acid Sequence; D000818:Animals; D002460:Cell Line; D018076:DNA, Complementary / Q000302:isolation & purification; D004023:Dictyostelium / Q000254:growth & development* / Q000648:ultrastructure; D005656:Fungal Proteins / Q000235:genetics / Q000502:physiology*; D008969:Molecular Sequence Data; D009154:Mutation; D015800:Protozoan Proteins; D015183:Restriction Mapping; D012261:Ribonucleoproteins / Q000235:genetics / Q000502:physiology*; D020394:Vault Ribonucleoprotein Particles; D000099187:Major Vault Protein
D018076:DNA, Complementary; D005656:Fungal Proteins; D015800:Protozoan Proteins; D012261:Ribonucleoproteins; D020394:Vault Ribonucleoprotein Particles; C082331:MvpA protein, Dictyostelium discoideum; D000099187:Major Vault Protein; C094738:MvpB protein, Dictyostelium discoideum