No abstract

The ascomycete Candida albicans is the most common fungal pathogen in immunocompromised patients . Its ability to change morphology, from yeast to filamentous forms, in response to host environmental cues is important for virulence . Filamentation is mediated by second messengers such as cyclic adenosine 3',5'-monophosphate (cAMP) synthesized by adenylyl cyclase . The distantly related basidiomycete Cryptococcus neoformans is an encapsulated yeast that predominantly infects the central nervous system in immunocompromised patients . Similar to the morphological change in C. albicans, capsule biosynthesis in C. neoformans, a major virulence attribute, is also dependent upon adenylyl cyclase activity . Here we demonstrate that physiological concentrations of CO2/HCO3- induce filamentation in C. albicans by direct stimulation of cyclase activity. Furthermore, we show that CO2/HCO3- equilibration by carbonic anhydrase is essential for pathogenesis of C. albicans in niches where the available CO2 is limited. We also demonstrate that adenylyl cyclase from C. neoformans is sensitive to physiological concentrations of CO2/HCO3-. These data demonstrate that the link between cAMP signaling and CO2/HCO3- sensing is conserved in fungi and reveal CO2 sensing to be an important mediator of fungal pathogenesis. Novel therapeutic agents could target this pathway at several levels to control fungal infections.

The structure of yeast phosphoglycerate kinase has been determined with data obtained from amino acid sequence, nucleotide sequence, and X-ray crystallographic studies. The substrate binding sites, as deduced from electron density maps, are compatible with known substrate specificity and the stereochemical requirements for the enzymic reaction. A carboxyl-imidazole interaction appears to be involved in controlling the transition between the open and closed forms of the enzyme.

The cytoplasmic heritable determinant [PSI ؉ ] of the yeast Saccharomyces cerevisiae reflects the prion-like properties of the chromosome-encoded protein Sup35p. This protein is known to be an essential eukaryote polypeptide release factor, namely eRF3. In a [PSI ؉ ] background, the prion conformer of Sup35p forms large oligomers, which results in the intracellular depletion of functional release factor and hence inefficient translation termination. We have investigated the process by which the [ curing ͉ Sup35p ͉ cytoplasmic determinant R ecent biochemical evidence (1-5) has supported the hypothesis that the [PSI ϩ ] phenotype of Saccharomyces cerevisiae reflects the prion-like properties of the SUP35 gene product (6, 7). The essential chromosome-encoded protein Sup35p is known to be one of two eukaryote polypeptide release factors, namely eRF3 (8, 9). Sup35p associates with Sup45p (eRF1) in vivo to mediate translation termination (8). In vitro, Sup35p forms highly ordered fibers, whose appearance resembles that of fibrils formed by other amyloidogenic polypeptides (3,4 , a second prion determinant of S. cerevisiae (7). Other reagents including methanol, ethylene glycol, and hypertonic conditions, have been reported to exhibit curing properties; however, none of these cure with the neartotal efficiency of GuHCl (13,14).Two hypotheses have been proposed to account for the curing properties of GuHCl. First, the elimination of the prion might arise directly from the ability of GuHCl to denature proteins. However, the concentrations of GuHCl effective in curing [PSI ϩ ] are in the millimolar range, rather than the molar range typically required for the denaturation of proteins in vitro (13). Alternatively, GuHCl may actually promote the expression of an ancillary factor, namely the stress protein Hsp104p, which indirectly results in the reactivation of Sup35p and consequently the loss of the prion (15). Unlike other heat shock proteins, Hsp104p does not act to protect proteins against stress (i.e., heat denaturation), rather Hsp104p actively promotes the recovery of stress-denatured aggregated proteins by facilitating their refolding back into functional, native conformations (16,17). Overexpression of Hsp104p might lead to the total refolding of Sup35p from the aberrant prion conformation to its native structure, thereby mediating prion loss. To test both hypotheses, we have examined the kinetics of prion elimination upon growth in the presence of GuHCl and assessed the influence of stress on the curing process. Materials and MethodsStrain. The genotype of the strain used in this study was BSC783͞4a: , MATa. Growth Media. BSC783͞4a was grown at 30°C on 1 ⁄4YEPD solid medium [4% (wt͞vol) glucose, 1% (wt͞vol) Bacto-peptone, 0.25% (wt͞vol) yeast extract, 2% (wt͞vol) agar]. Most liquid cultures also were grown at 30°C in YEPD complete medium [2% (wt͞vol) glucose, 1% (wt͞vol) Bacto-peptone, 1% (wt͞vol) yeast extract], with or without 3 mM GuHCl. For studies using ethanol-supplemented media, strains were grown in f...

Many proteins can misfold into β-sheet-rich, self-seeding polymers (amyloids). Prions are exceptional among such aggregates in that they are also infectious. In fungi, prions are not pathogenic but rather act as epigenetic regulators of cell physiology, providing a powerful model for studying the mechanism of prion replication. We used prion-forming domains from two budding yeast proteins (Sup35p and New1p) to examine the requirements for prion formation and inheritance. In both proteins, a glutamine/asparagine-rich (Q/N-rich) tract mediates sequence-specific aggregation, while an adjacent motif, the oligopeptide repeat, is required for the replication and stable inheritance of these aggregates. Our findings help to explain why although Q/N-rich proteins are relatively common, few form heritable aggregates: prion inheritance requires both an aggregation sequence responsible for self-seeded growth and an element that permits chaperone-dependent replication of the aggregate. Using this knowledge, we have designed novel artificial prions by fusing the replication element of Sup35p to aggregation-prone sequences from other proteins, including pathogenically expanded polyglutamine.

] by coexpression of a dominant, ATPase-negative allele of HSP104 were similar to those observed for GdnHCl-induced elimination. Based on these and other data, we propose a two-cycle model for "prionization" of Sup35p in [PSI ؉ ] cells: cycle A is the GdnHCl-sensitive (Hsp104-dependent) replication of the prion seeds, while cycle B is a GdnHCl-insensitive (Hsp104-independent) process that converts these seeds to pelletable aggregates.[PSI ϩ ] is a non-Mendelian epigenetic element originally identified in a genetic screen by its ability to increase the efficiency of tRNA-mediated nonsense suppression in Saccharomyces cerevisiae (6, 7). Based on genetic arguments, Wickner (47) was the first to suggest that the [PSI ϩ ] determinant was a self-replicating prion form of translation termination factor Sup35p (also called eRF3 [43,50]). In accordance with Prusiner's original prion hypothesis (33), Sup35p is able to exist in one of two different states: the normal soluble form, required for translation termination, and an "infectious" (i.e., transmissible) aggregated form, i.e., as a prion. ] cells, Sup35p is largely inactivated by its sequestration into high-molecular-weight complexes, thereby precluding it from efficiently performing its role as a translation termination factor.Studies on the conversion of mammalian prion protein PrP to transmissible prion PrP Sc form have generated a number of different models to explain how a prion protein determinant can be propagated and transmitted (see reference 2 for a review). A detailed in vitro analysis of yeast Sup35p self-directed aggregation led Serio et al. (39) to suggest that a process of nucleated conformational conversion of Sup35p most probably occurs in the establishment of the [PSI ϩ ] determinant. This mechanism involves both the nucleated-polymerization and the template assembly processes proposed for PrP (2), and Serio et al. (39) proposed that conformational conversion of the native Sup35p occurs by the seeding activity of a nucleated complex. These nuclei (or intermediate oligomers) would be formed by the conformational modification of the partially unfolded Sup35p protein. Once the intermediate polymers reach a critical concentration, assembly of large polymers can occur through a templating mechanism.Physicochemical analyses of Sup35p have shown that the N-terminal portion of the protein (the so-called N domain or prion-forming domain) contains the minimal region responsible for [PSI ϩ ] propagation in vivo and directs amyloid fiber formation in vitro (16,23,32). The Sup35p amyloid fibers have features in common with the amyloid proteins implicated in several human diseases (for reviews, see references 1, 11, 34, and 48). However, it remains to be established whether the * Corresponding author. Mailing address:

Synonymous codons encode the same amino acid, but differ in other biophysical properties. The evolutionary selection of codons whose properties are optimal for a cell generates the phenomenon of codon bias. Although recent studies have shown strong effects of codon usage changes on protein expression levels and cellular physiology, no translational control mechanism is known that links codon usage to protein expression levels. Here, we demonstrate a novel translational control mechanism that responds to the speed of ribosome movement immediately after the start codon. High initiation rates are only possible if start codons are liberated sufficiently fast, thus accounting for the observation that fast codons are overrepresented in highly expressed proteins. In contrast, slow codons lead to slow liberation of the start codon by initiating ribosomes, thereby interfering with efficient translation initiation. Codon usage thus evolved as a means to optimise translation on individual mRNAs, as well as global optimisation of ribosome availability.

SummaryIn the yeast Saccharomyces cerevisiae, Sup35p (eRF3), a subunit of the translation termination complex, can take up a prion-like, self-propagating conformation giving rise to the non-