The essential, conserved yeast nucleolar protein Ytm1 is one of 17 proteins in ribosome assembly intermediates that contain WD40 protein-protein interaction motifs. Such proteins may play key roles in organizing other molecules necessary for ribosome biogenesis. Ytm1 is present in four consecutive 66S preribosomes containing 27SA 2 , 27SA 3 , 27SB, and 25.5S plus 7S pre-rRNAs plus ribosome assembly factors and ribosomal proteins. Ytm1 binds directly to Erb1 and is present in a heterotrimeric subcomplex together with Erb1 and Nop7, both within preribosomes and independently of preribosomes. However, Nop7 and Erb1 assemble into preribosomes prior to Ytm1. Mutations in the WD40 motifs of Ytm1 disrupt binding to Erb1, destabilize the heterotrimer, and delay pre-rRNA processing and nuclear export of preribosomes. Nevertheless, 66S preribosomes lacking Ytm1 remain otherwise intact.Biogenesis of eukaryotic ribosomes is a highly regulated and dynamic process that begins in the nucleolus with transcription of a precursor rRNA (pre-rRNA) that is rapidly packaged into the 90S ribonucleoprotein particle containing ribosomal proteins, nonribosomal proteins, and snoRNA-containing ribonucleoprotein particles (snoRNPs). The 90S pre-RNPs are converted into 43S and 66S ribosome assembly intermediates, which ultimately give rise to mature 40S and 60S ribosomal subunits (Fig. 1).Molecular genetic approaches in yeast identified more than 70 trans-acting factors required for ribosome assembly (12,14,46). Subsequent advances in proteomics enabled purification of pre-rRNPs from yeast and identification of an additional 80 assembly factors present in preribosomes, as well as most of those proteins previously discovered using genetic screens (3,7,11,17,20,21,24,26,37,38,41,(49)(50)(51). Metazoan homologues of most of the yeast ribosome assembly factors were discovered by proteomic analysis of purified nucleoli (2, 52).Among the assembly factors found in yeast preribosomes are 17 proteins containing WD40 motifs (14). These motifs function as protein-protein interaction domains (53). Therefore, such WD40-containing proteins may nucleate assembly of preribosomes by interacting sequentially or simultaneously with other assembly factors or ribosomal proteins. Previously, we identified the WD40 protein Ytm1 as a constituent of purified 66S pre-rRNPs and showed that depletion of Ytm1 results in a deficiency of 60S ribosomal subunits (21).In this study, we have further investigated the role of Ytm1 in ribosome biogenesis. Ytm1 is a constituent of multiple consecutive 66S preribosomes containing 27SA 2 , 27SA 3 , 27SB, 25.5S, and 7S pre-rRNAs plus a collection of ribosomal and nonribosomal proteins. Ytm1 is present in a heterotrimer with two other assembly factors, Nop7 and Erb1, both within 66S pre-rRNPs and as a subcomplex independent of preribosomes. Mutations in Ytm1 disrupt interactions between Ytm1 and Erb1, destabilize the heterotrimer, and significantly reduce association of these three proteins with 66S preribosomes. These 66S pre-rRNPs ...