The yeast protein Ebp2 is required for early steps in production of 60S ribosomal subunits. To search for cofactors with which Ebp2 functions, or substrates on which it acts, we screened for mutants that were synthetically lethal (sl) with the ebp2-14 mutation. Four different mutant alleles of the 60S ribosomal subunit assembly factor Brx1 were found. To investigate defects of the double mutant, we constructed strains conditional for the ebp2-14 brx1- synthetic lethal phenotype. These ebp2-14 brx1 mutants were defective in processing of 27S pre-rRNA and production of 60S subunits, under conditions where each single mutant was not. Ebp2 and Brx1 exhibit a strong two-hybrid interaction, which is eliminated by some combinations of brx1 and ebp2 mutations. In one such mutant, Ebp2 and Brx1 can still associate with pre-ribosomes, but subunit maturation is perturbed. Depletion of either Ebp2 or Brx1 revealed that Brx1 requires Ebp2 for its stable association with pre-ribosomes, but Ebp2 does not depend on the presence of Brx1 to enter pre-ribosomes. These results suggest that assembly of 60S ribosomal subunits requires cooperation of Ebp2 with Brx1, together with other molecules present in pre-ribosomes, potentially including several found in assembly subcomplexes with Brx1 and Ebp2.

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