The 90S preribosomal particle is required for the production of the 18S rRNA from a pre-rRNA precursor. Despite the identification of the protein components of this particle, its mechanism of assembly and structural design remain unknown. In this work, we have combined biochemical studies, proteomic techniques, and bioinformatic analyses to shed light into the rules of assembly of the yeast 90S preribosome. Our results indicate that several protein subcomplexes work as discrete assembly subunits that bind in defined steps to the 35S pre-rRNA. The assembly of the t-UTP subunit is an essential step for the engagement of at least five additional subunits in two separate, and mutually independent, assembling routes. One of these routes leads to the formation of an assembly intermediate composed of the U3 snoRNP, the Pwp2p/UTP-B, subunit and the Mpp10p complex. The other assembly route involves the stepwise binding of Rrp5p and the UTP-C subunit. We also report the use of a bioinformatic approach that provides a model for the topological arrangement of protein components within the fully assembled particle. Together, our data identify the mechanism of assembly of the 90S preribosome and offer novel information about its internal architecture.The formation of eukaryotic ribosomes involves the production and correct assembly of four rRNAs and Ϸ80 ribosomal proteins. Due to its amenability for genetic and proteomic analyses, Saccharomyces cerevisiae is the organism where the different steps of this pathway have been best characterized (4,11,13,18,32,34). Thus, it is known that three of the four mature rRNAs that form the ribosome structure are generated from a common 35S pre-rRNA polycystronic precursor. After being transcribed in the nucleolus, this precursor is chemically modified and cleaved at three positions (known as the A 0 , A 1 , and A 2 sites) of its 5Ј-terminal end to generate the intermediate 33S, 32S, 27SA 2 , and 20S pre-rRNA precursors (see Fig. S1 in the supplemental material). The 20S and 27SA 2 pre-rRNAs then follow two independent maturation routes that lead to the generation of either the 18S rRNA (a component of the 40S ribosomal subunit) or the 5.8S and 25S rRNAs (two components of the 60S ribosomal subunit), respectively (see Fig. S1 in the supplemental material). These pre-rRNA maturation steps require the involvement of Ϸ170 nonribosomal proteins and 70 small nucleolar ribonucleoproteins (snoRNPs) (4,11,13,32). Different subsets of these molecules form large ribonucleoprotein complexes with specific pre-rRNA precursors that, according to their specific Svedberg coefficients in gradient ultracentrifugation experiments, were initially referred to as 90S, 66S, and 43S preribosomal particles (31, 33). The 90S particle, also known as the "small-subunit processome," contains the 35S pre-rRNA and assembly/processing factors needed for the early cleavage of the 35S pre-rRNA precursor at A 0 , A 1 , and A 2 sites, which is strictly required for the production of 40S ribosomal subunits. The 66S and 43S pa...

We have used an extensive mutagenesis approach to study the specific role of the eight structural domains of Vav during both the activation and signaling steps of this Rac1 exchange factor. Our results indicate that several Vav domains (Dbl homology, pleckstrin homology, and zinc finger) are essential for all the biological activities tested, whereas others are required for discrete, cell type-specific biological effects. Interestingly, we have found that Vav domains have no unique functions. Thus, the calponin homology domain mediates the inhibition of Vav both in vitro and in vivo but, at the same time, exerts effector functions in lymphocytes upon receptor activation. The Vav SH2 and SH3 regions play regulatory roles in the activation of Vav in fibroblasts, mediating both its phosphorylation and translocation to the plasma membrane. In contrast, the Vav SH2 and SH3 regions act as scaffolding platforms in T-cells, ensuring the proper phosphorylation of Vav and the subsequent engagement of downstream effectors. We also provide evidence indicating that the zinc finger region exerts at least three different functional roles in Vav, aiding in the down-regulation of its basal activity, the engagement of substrates, and the induction of ancillary pathways required for cell transformation. Finally, the results obtained are consistent with a new regulatory model for Vav, in which the calponin homology region inhibits the basal activity of Vav through interactions with the zinc finger region.Vav proteins are phosphorylation-dependent exchange factors that catalyze the release of GDP from Rho/Rac family members, thereby facilitating their transition from the inactive (GDP-bound) to the active (GTP-bound) state (1). This activity is crucial for the coordination of developmental and mitogenic processes. Thus, the elimination of the vav gene results in impaired lymphoid development, lymphopenia, and defective immune responses in mice (2-5). Similarly, deletion of either vav2 or vav3 genes results in impaired signaling responses in activated B-cells (6 -8). It has also been demonstrated that the subversion on the normal activation/deactivation cycle of some members of the Vav family results in severe alterations of cell behavior, including tumorigenesis, changes in F-actin organization, and the acquisition of metastatic properties by transformed cells (1). Finally, the activation of Vav or Vav2 proteins by the Nef protein of the human immunodeficiency virus plays an essential role in the pathogenic cycle of this virus (9, 10).One important feature of this GEF family is the structural complexity of its members (1, 10) (see Fig. 1A). Mammalian and avian Vav proteins contain eight structural domains, including a calponin homology (CH) 1 region, an acidic (Ac) domain, the catalytic Dbl homology (DH) region, a pleckstrin homology (PH) domain, a zinc finger (ZF) region similar to those present in c-Raf and protein kinase C family members, and a SH2 domain flanked by two SH3 domains. Caenorhabditis elegans and Drosophila melanogas...

G-protein-coupled receptors (GPCRs) transduce the signals for a wide range of hormonal and sensory stimuli by activating a heterotrimeric guanine nucleotide-binding protein (G protein). The analysis of loss-offunction and constitutively active receptor mutants has helped to reveal the functional properties of GPCRs and their role in human diseases. Here we describe the identification of a new class of mutants, dominantnegative mutants, for the yeast G-protein-coupled ␣-factor receptor (Ste2p). Sixteen dominant-negative receptor mutants were isolated based on their ability to inhibit the response to mating pheromone in cells that also express wild-type receptors. Detailed analysis of two of the strongest mutant receptors showed that, unlike other GPCR interfering mutants, they were properly localized at the plasma membrane and did not alter the stability or localization of wild-type receptors. Furthermore, their dominant-negative effect was inversely proportional to the relative amount of wild-type receptors and was reversed by overexpressing the G-protein subunits, suggesting that these mutants compete with the wild-type receptors for the G protein. Interestingly, the dominant-negative mutations are all located at the extracellular ends of the transmembrane segments, defining a novel region of the receptor that is important for receptor signaling. Altogether, our results identify residues of the ␣-factor receptor specifically involved in ligand binding and receptor activation and define a new mechanism by which GPCRs can be inactivated that has important implications for the evaluation of receptor mutations in other G-protein-coupled receptors.G-protein-coupled receptors (GPCRs) comprise a large family of receptors that are found in a wide range of eukaryotic organisms from yeasts to humans (4, 10). These receptors respond to diverse stimuli including hormones, neurotransmitters, and other chemical messengers (48). GPCRs transduce their signal by stimulating the ␣ subunit of a heterotrimeric guanine nucleotide binding protein (G protein) to bind GTP (4, 16). This releases the ␣ subunit from the ␤␥ subunits, and then either the ␣ subunit or the ␤␥ subunits go on to promote signaling depending on the specific pathway (28).GPCRs are structurally similar in that they contain seven transmembrane domains (TMDs) connected by intracellular and extracellular loops. Although many techniques have been applied to study receptor function, much of our knowledge on the mechanisms of GPCR activation comes from the characterization of mutant receptors. Loss-of-function and supersensitive mutants have helped to identify receptor regions needed for ligand binding, G-protein activation, and down-regulation of signaling (4, 49). Furthermore, the study of constitutively active receptor mutations has played a key role in the development of current models for receptor activation (26). Naturally occurring GPCR mutations have also been implicated in a number of human diseases (8,25,42). Interestingly, the analysis of different mutant rec...