Different point mutations in the nucleolar protein fibrillarin (Nop1p in Saccharomyces cerevisiae) can inhibit different steps in ribosome synthesis. A screen for mutations that are synthetically lethal (sl) with the nop1-5 allele, which inhibits pre-rRNA processing, identified NOP56. An independent sl mutation screen with nop1-3, which inhibits pre-rRNA methylation, identified a mutation in NOP58. Strikingly, Nop56p and Nop58p are highly homologous (45% identity). Both proteins were found to be essential and localized to the nucleolus. A temperature-sensitive lethal mutant allele, nop56-2, inhibited many steps in pre-rRNA processing, particularly on the pathway of 25S/5.8S rRNA synthesis, and led to defects in 60S subunit assembly. Epitope-tagged constructs show that both Nop56p and Nop58p are associated with Nop1p in complexes, Nop56p and Nop1p exhibiting a stoichiometric association. These physical interactions presumably underlie the observed sl phenotypes. Well-conserved homologs are present in a range of organisms, including humans (52% identity between human hNop56p and yeast Nop56p), suggesting that these complexes have been conserved in evolution.Most steps of ribosome biogenesis occur in the nucleolus, a specialized subnuclear structure (for reviews, see references 14, 34, 39, 45, and 53). In eukaryotes including Saccharomyces cerevisiae and humans, a large precursor rRNA transcript (prerRNA) is processed into the mature 18S, 5.8S, and 25S/28S rRNAs. During transcription and processing, these rRNAs associate with approximately 80 ribosomal proteins and with the 5S rRNA. In addition, the mature rRNA regions of the prerRNA undergo extensive covalent nucleotide modification, mainly base modification of uridine to pseudouridine and methylation of the ribose 2Ј-hydroxyl (2Ј-O methylation) (reviewed in reference 27). The large number of concerted reactions occurring during rRNA processing and ribosome assembly has made it difficult to analyze single steps in ribosome synthesis. Over recent years, the analysis of yeast mutants defective in ribosome biogenesis has proved to be a powerful approach (reviewed in reference 51), particularly when combined with in vitro analyses using purified components (8,26,30). Despite this progress, our understanding of the detailed mechanisms of eukaryotic rRNA processing remains poor.The small nucleolar RNAs (snoRNAs) play important roles in the covalent processing of the pre-rRNAs (reviewed in references 2, 28, and 48). With the exception of RNase MRP, which is an endonuclease structurally related to RNase P, the very large numbers of snoRNAs present in eukaryotes can be divided into two groups based on conserved sequence and structural features (3, 12; reviewed in reference 48). Most of the box CϩD snoRNAs direct the site-specific 2Ј-O methylation of the pre-rRNA (19), while most of the box HϩACA snoRNAs select the sites of pseudouridine formation (6,11,33). In addition, a few members of each group of snoRNAs do not appear to select sites of pre-rRNA modification but are re...

Stress within the endoplasmic reticulum (ER) induces a coordinated response, namely the unfolded protein response (UPR), devoted to helping the ER cope with the accumulation of misfolded proteins. Failure of the UPR plays an important role in several human diseases. Recent studies report that intracellular accumulation of saturated fatty acids (SFAs) and cholesterol, seen in diseases of high incidence, such as obesity or atherosclerosis, results in ER stress. In the present study, we evaluated the effects of perturbations to lipid homeostasis on ER stress/UPR induction in the model eukaryote Saccharomyces cerevisiae. We show that SFA originating from either endogenous (preclusion of fatty acid desaturation) or exogenous (feeding with extracellular SFA) sources trigger ER stress and that ergosterol, the major sterol in yeast, acts synergistically with SFA in this process. This latter effect is connected to ergosterol accumulation within microsomal fractions from SFA-accumulating cells, which display highly saturated phospholipid content. Moreover, treating the cells with the molecular chaperone 4-phenyl butyrate abolishes UPR induction, suggesting that lipid-induced ER stress leads to an overload of misfolded protein that acts, in turn, as the molecular signal for induction of the UPR. The present data are discussed in the context of human diseases that involve lipid deregulation.

Azole resistance has been insufficiently investigated in the yeast Candida tropicalis. Here we determined the molecular mechanisms responsible for azole resistance in a clinical isolate of this pathogenic yeast. Antifungal susceptibility testing performed by a disk diffusion method showed resistance or markedly decreased susceptibility to azoles, which was confirmed by determination of MICs. Considering the relationship between azole susceptibility and the respiration reported for other yeast species, the respiratory activity of this isolate was investigated. Flow cytometry using rhodamine 123 and oxygraphy demonstrated an increased respiratory activity, which was not linked to an overexpression or increased number of copies of the mitochondrial genome. Among previously described resistance mechanisms, an increased activity of efflux pumps was investigated by flow cytometry using rhodamine 6G. However, the efflux of rhodamine 6G was lower in the resistant isolate than in susceptible ones. Likewise, real-time reverse transcription-PCR quantification of the expression of C. tropicalis MDR1 (CtMDR1), which encodes an efflux protein belonging to the major facilitator superfamily, did not show overexpression of this gene. In contrast, the resistant isolate overexpressed the CtERG11 gene coding for lanosterol 14␣-demethylase. This was in agreement with the larger amount of ergosterol found in this isolate. Moreover, sequencing of CtERG11 showed a point mutation leading to a tyrosine substitution in the protein sequence, which might lead to decreased binding affinity for azoles. In conclusion, overexpression of CtERG11 associated with a missense mutation in this gene seemed to be responsible for the acquired azole resistance of this clinical isolate.

The nucleolar protein fibrillarin (encoded by the NOP1 gene in yeast), is required for many post‐transcriptional steps in yeast ribosome synthesis. A screen for mutations showing synthetic lethality with a temperature sensitive nop1‐5 allele led to the identification of the NOP77 gene. NOP77 is essential for viability and encodes a nucleolar protein with a predicted molecular weight of 77 kDa. Depletion of NOP77p impairs both the processing and methylation of the pre‐rRNA. The processing defect is greatest for the pathway leading to 25S rRNA synthesis, and is distinctly different from that observed for mutations in other nucleolar components. NOP77p contains three canonical RNA recognition motifs (RRMs), suggesting that it is an RNA binding protein. The NOP77 allele which complements the synthetic lethal nop1 strains has an alanine at position 308, predicted to lie in helix alpha 1 of RRM3, whereas the non‐complementing nop77‐1 allele contains a proline at the corresponding position. We propose that NOP77p mediates specific interactions between NOP1p and the pre‐rRNA.