Adaptation of Candida albicans to growth on sorbose via monosomy of chromosome 5 accompanied by duplication of another chromosome carrying a gene responsible for sorbose utilization.

Candida albicans, a fungus that normally inhabits the digestive tract and other mucosal surfaces, can become a pathogen in immunocompromised individuals, causing severe or even fatal infection. Mechanisms by which C. albicans can evade commonly used antifungal agents are not fully understood.

N-terminal acetylome analyses and functional insights of the N-terminal acetyltransferase NatB.

Protein N-terminal acetylation (Nt-acetylation) is an important mediator of protein function, stability, sorting, and localization. Although the responsible enzymes are thought to be fairly well characterized, the lack of identified in vivo substrates, the occurrence of Nt-acetylation substrates displaying yet uncharacterized N-terminal acetyltransferase (NAT) specificities, and emerging evidence of posttranslational Nt-acetylation, necessitate the use of genetic models and quantitative proteomics. NatB, which targets Met-Glu-, Met-Asp-, and Met-Asn-starting protein N termini, is presumed to Nt-acetylate 15% of all yeast and 18% of all human proteins.

Functional Subunits of Eukaryotic Chaperonin CCT/TRiC in Protein Folding.

Molecular chaperones are a class of proteins responsible for proper folding of a large number of polypeptides in both prokaryotic and eukaryotic cells. Newly synthesized polypeptides are prone to nonspecific interactions, and many of them make toxic aggregates in absence of chaperones. The eukaryotic chaperonin CCT is a large, multisubunit, cylindrical structure having two identical rings stacked back to back.

N(α)-Acetylation of yeast ribosomal proteins and its effect on protein synthesis.

N(α)-Acetyltransferases (NATs) cause the N(α)-acetylation of the majority of eukaryotic proteins during their translation, although the functions of this modification have been largely unexplored. In yeast (Saccharomyces cerevisiae), four NATs have been identified: NatA, NatB, NatC, and NatD. In this study, the N(α)-acetylation status of ribosomal protein was analyzed using NAT mutants combined with two-dimensional difference gel electrophoresis (2D-DIGE) and mass spectrometry (MS).

A synopsis of eukaryotic Nalpha-terminal acetyltransferases: nomenclature, subunits and substrates.

We have introduced a consistent nomenclature for the various subunits of the NatA-NatE N-terminal acetyltransferases from yeast, humans and other eukaryotes.

Proteomics analyses reveal the evolutionary conservation and divergence of N-terminal acetyltransferases from yeast and humans.

N(alpha)-terminal acetylation is one of the most common protein modifications in eukaryotes. The COmbined FRActional DIagonal Chromatography (COFRADIC) proteomics technology that can be specifically used to isolate N-terminal peptides was used to determine the N-terminal acetylation status of 742 human and 379 yeast protein N termini, representing the largest eukaryotic dataset of N-terminal acetylation. The major N-terminal acetyltransferase (NAT), NatA, acts on subclasses of proteins with Ser-, Ala-, Thr-, Gly-, Cys- and Val- N termini.

Properties of Nat4, an N(alpha)-acetyltransferase of Saccharomyces cerevisiae that modifies N termini of histones H2A and H4.

Nat4, also designated NatD, was previously shown to acetylate the N termini of histones H2A and H4, which have SGGKG and SGRGK N termini (O. K. Song, X.

Overexpressed ribosomal proteins suppress defective chaperonins in Saccharomyces cerevisiae.

The chaperonin Cct complex of the yeast Saccharomyces cerevisiae is composed of eight different subunits encoded by eight essential genes, CCT1-CCT8. This Cct complex is responsible for the folding of a number of proteins including actin and tubulin. We have isolated and characterized 22 multicopy suppressors of the temperature-sensitive allele, cct4-1, which encodes an altered protein with a G345D replacement that diminishes ATP hydrolysis.

Methylation of proteins involved in translation.

Methylation is one of the most common protein modifications. Many different prokaryotic and eukaryotic proteins are methylated, including proteins involved in translation, including ribosomal proteins (RPs) and translation factors (TFs). Positions of the methylated residues in six Escherichia coli RPs and two Saccharomyces cerevisiae RPs have been determined.

Yeast N(alpha)-terminal acetyltransferases are associated with ribosomes.

N-terminal acetylation is one of the most common modifications, occurring on the vast majority of eukaryotic proteins. Saccharomyces cerevisiae contains three major NATs, designated NatA, NatB, and NatC, with each having catalytic subunits Ard1p, Nat3p, and Mak3p, respectively. Gautschi et al.

Mutant LYS2 mRNAs retained and degraded in the nucleus of Saccharomyces cerevisiae.

We previously demonstrated that mRNAs retained in the nucleus of Saccharomyces cerevisiae are subjected to a degradation system-designated DRN (degradation of mRNA in the nucleus), that is diminished in cbc1-Delta or cbc2-Delta mutants lacking components of the cap-binding complex and in rrp6-Delta mutants lacking Rrp6p, a 3' to 5' nuclear exonuclease. Two mutants, lys2-187 and lys2-121, were uncovered by screening numerous lys2 mutants for suppression by cbc1-Delta and rrp6-Delta. Both mutants were identical and contained the two base changes, one of which formed a TGA nonsense codon.

Phenotypes of yeast mutants lacking the mitochondrial protein Pet20p.

The pet20-delta deletion in Saccharomyces cerevisiae causes diminished growth on media containing non-fermentable carbon sources when incubated at both above and below the 30 degrees C optimal growth temperature. Furthermore, the pet20-delta strain has a greatly reduced level of cytochrome c, especially at 37 degrees C. The pet20-delta strain was sensitive to high NaCl and CaCl2 concentrations, hydrogen peroxide, oligomycin, polymixin B, amphotericin B and fluconazole.

The yeast translation release factors Mrf1p and Sup45p (eRF1) are methylated, respectively, by the methyltransferases Mtq1p and Mtq2p.

The translation release factors (RFs) RF1 and RF2 of Escherichia coli are methylated at the N5-glutamine of the GGQ motif by PrmC methyltransferase. This motif is conserved in organisms from bacteria to higher eukaryotes. The Saccharomyces cerevisiae RFs, mitochondrial Mrf1p and cytoplasmic Sup45p (eRF1), have sequence similarities to the bacterial RFs, including the potential site of glutamine methylation in the GGQ motif.

A nuclear degradation pathway controls the abundance of normal mRNAs in Saccharomyces cerevisiae.

We previously demonstrated an increased degradation of mRNAs in mutants of Saccharomyces cerevisiae having blocks in nuclear export. The degradation activity, designated DRN (degradation of mRNA in the nucleus), requires Cbc1p, a nuclear cap-binding protein, and Rrp6p, a nuclear exosome component. Microarray procedures were used to determine the half-lives of mRNAs from normal and mutant strains, leading to the tentative identification of hundreds of normal mRNAs that were notably stabilized when either CBC1 or RRP6 were deleted.

Candida albicans SOU1 encodes a sorbose reductase required for L-sorbose utilization.

Previous work in our laboratory showed that L-sorbose utilization in Candida albicans is subject to a novel form of regulation which involves a reversible increase or decrease in the copy number of chromosome 5. Furthermore, the structural gene SOU1 is required for L-sorbose utilization and encodes a member of the short chain dehydrogenase family. However, the precise function of SOU1 was not known and neither was the pathway for L-sorbose utilization.

The concomitant expression and availability of conventional and alternative, cyanide-insensitive, respiratory pathways in Candida albicans.

The opportunistic oral pathogen Candida albicans expresses a cyanide-insensitive alternative oxidase (AOX) upon exposure to respiratory inhibitors that act downstream from coenzyme Q, and upon ageing of cells. To investigate whether the conventional pathway is retained when the alternative pathway is induced, cells were grown in the presence of sodium cyanide, a reversible inhibitor of cytochrome oxidase. AOX expression was monitored by Western blotting and the presence of cytochromes associated with complexes III and IV of the conventional pathway was monitored by recording spectra between 500 and 650 nm at 77K.

Cap-binding protein 1-mediated and eukaryotic translation initiation factor 4E-mediated pioneer rounds of translation in yeast.

Nonsense-mediated mRNA decay (NMD) in mammalian cells is restricted to newly synthesized mRNA that is bound at the 5' cap by the major nuclear cap-binding complex and at splicing-generated exon-exon junctions by exon junction complexes. This messenger ribonucleoprotein has been called the pioneer translation initiation complex and, accordingly, NMD occurs as a consequence of nonsense codon recognition during a pioneer round of translation. Here, we characterize the nature of messenger ribonucleoprotein that is targeted for NMD in Saccharomyces cerevisiae.

Physiological effects of unassembled chaperonin Cct subunits in the yeast Saccharomyces cerevisiae.

Eukaryotic chaperonins, the Cct complexes, are assembled into two rings, each of which is composed of a stoichiometric array of eight different subunits, which are denoted Cct1p-Cct8p. Overexpression of a single CCT gene in Saccharomyces cerevisiae causes an increase of the corresponding Cct subunit, but not of the Cct complex. Nevertheless, overexpression of certain Cct subunits, especially CCT6, suppresses a wide range of abnormal phenotypes, including those caused by the diverse types of conditional mutations tor2-21, lst8-2 and rsp5-9 and those caused by the concomitant overexpression of Sit4p and Sap155p.

The importance of mutation, then and now: studies with yeast cytochrome c.

The development of a genetic system based on the CYC1 gene was initiated over 40 years ago, primarily because of the anticipated ease of sequencing of the corresponding encoded protein, iso-1-cytochrome c from Saccharomyces cerevisiae. The success of the iso-cytochrome c system was dependent on the early development of methods for detecting and selecting cyc1 defective mutants and CYC1 functional revertants, and of methods for fine-structure genetic mapping using deletions and single-site mutations. The nonsense codons TAA and TAG, and the initiation codon ATG, were determined from the amino acid alterations of iso-1-cytochromes c from intragenic revertants; this represented the first assignments of such codons in a eukaryotic organism.

Enhanced mitochondrial degradation of yeast cytochrome c with amphipathic structures.

The dispensable N-terminus of iso-1-cytochrome c (iso-1) in the yeast Saccharomyces cerevisiae was replaced by 11 different amphipathic structures. Rapid degradation of the corresponding iso-1 occurred, with the degree of degradation increasing with the amphipathic moments; and this amphipathic-dependent degradation was designated ADD. ADD occurred with the holo-forms in the mitochondria but not as the apo-forms in the cytosol.

Role of the 14-3-3 protein in carbon metabolism of the pathogenic yeast Candida albicans.

We previously demonstrated that the pathogenic yeast Candida albicans effectively adapts to utilize L-sorbose (Sou+) by a novel mechanism based on the loss of one copy of chromosome 5, probably due to the reduction of copy number of a negative regulator located on this chromosome. We report here another negative regulator of L-sorbose utilization, an orthologue of the Saccharomyces cerevisiae BMH1 gene, which encodes the evolutionarily conserved protein 14-3-3. This essential gene is located on chromosome 1, does not have paralogues, and is supposedly a component of the regulatory network.

Polyadenylation of rRNA in Saccharomyces cerevisiae.

In contrast to mRNAs, rRNAs are transcribed by RNA polymerase I or III and are not believed to be polyadenylated. Here we show that in Saccharomyces cerevisiae, at least a small fraction of rRNAs do have a poly(A) tail. The levels of polyadenylated rRNAs are dramatically increased in strains lacking the degradation function of Rrp6p, a component of the nuclear exosome.

Sue1p is required for degradation of labile forms of altered cytochromes C in yeast mitochondria.

Previous studies on certain altered holo-isocytochromes c revealed a rho(-)-dependent degradation (RDD) phenotype, in which certain altered holo-iso-1-cytochromes c are at normal or nearly normal levels in rho+ strains, but are at low levels or absent in rho- strains, although wild-type holo-iso-1-cytochrome c is present at normal levels in both rho+ and related rho- strains. The diminished levels of altered holo-iso-1-cytochrome c are due to the rapid degradation that is carried out by a novel proteolytic pathway in the IMS of mitochondria. SUE1, a nuclear gene that encodes a mitochondrial protein, was identified with a genetic screen for mutants that diminish RDD.

Degradation of normal mRNA in the nucleus of Saccharomyces cerevisiae.

A nuclear mRNA degradation (DRN) system was identified from analysis of mRNA turnover rates in nup116-Delta strains of Saccharomyces cerevisiae lacking the ability to export all RNAs, including poly(A) mRNAs, at the restrictive temperature. Northern blotting, in situ hybridization, and blocking transcription with thiolutin in nup116-delta strains revealed a rapid degradation of mRNAs in the nucleus that was suppressed by the rrp6-delta, rai1-delta, and cbc1-delta deletions, but not by the upf1-delta deletion, suggesting that DRN requires Rrp6p, a 3'-to-5' nuclear exonuclease, the Rat1p, a 5'-to-3' nuclear exonuclease, and Cbc1p, a component of CBC, the nuclear cap binding complex, which may direct the mRNAs to the site of degradation. We propose that certain normal mRNAs retained in the nucleus are degraded by the DRN system, similar to degradation of transcripts with 3' end formation defects in certain mutants.

Composition and function of the eukaryotic N-terminal acetyltransferase subunits.

Saccharomyces cerevisiae contains three N-terminal acetyltransferases (NATs), NatA, NatB, and NatC, composed of the following catalytic and auxiliary subunits: Ard1p and Nat1p (NatA); Nat3p and Mdm20p (NatB); and Mak3p, Mak10, and Mak31p (NatC). The overall patterns of N-terminally acetylated proteins and NAT orthologous genes suggest that yeast and higher eukaryotes have similar systems for N-terminal acetylation. The differential expression of certain NAT subunits during development or in carcinomas of higher eukaryotes suggests that the NATs are more highly expressed in cells undergoing rapid protein synthesis.