Functional mapping of the U3 small nucleolar RNA from the yeast Saccharomyces cerevisiae

The 2 f1-f2 distortion product otoacoustic emission (DP) was measured in 20 normal hearing subjects and 15 patients with moderate cochlear hearing loss and compared to the pure-tone hearing threshold, measured with the same probe system at the f2 frequencies. DPs were elicited over a wide primary tone level range between L2 = 20 and 65 dB SPL. With decreasing L2, the L1-L2 primary tone level difference was continuously increased according to L1 = 0.4L2 + 39 dB, to account for differences of the primary tone responses at the f2 place. Above 1.5 kHz, DPs were measurable with that paradigm on average within 10 dB of the average hearing threshold in both subject groups. The growth of the DP was compressive in normal hearing subjects, with strong saturation at moderate primary tone levels. In cases of cochlear impairment, reductions of the DP level were greatest at lowest, but smallest at highest stimulus levels, such that the growth of the DP became linearized. The correlation of the DP level to the hearing threshold was found to depend on the stimulus level. Maximal correlations were found in impaired ears at moderate primary tone levels around L2 = 45 dB SPL, but at lowest stimulus levels in normal hearing (L2 = 25 dB SPL). At these levels, 17/20 impaired ears and 14/15 normally hearing ears showed statistically significant correlations. It is concluded that for a clinical application and prediction of the hearing threshold, DPs should be measured not only at high, but also at lower primary tone levels.     

Mutations in the Treacher Collins syndrome gene lead to mislocalization of the nucleolar protein treacle

Treacher Collins syndrome (TCS) is an autosomal dominant disorder of craniofacial development, the features of which include conductive hearing loss and cleft palate. The TCS gene ( TCOF1 ), which is localized to chromosome 5q32-q33.1, recently has been identified by positional cloning. Analysis of TCOF1 revealed that the majority of TCS mutations result in the creation of a premature termination codon. The function of the predicted protein, treacle, is unknown, although indirect evidence from database analyses suggests that it may function as a shuttling nucleolar phosphoprotein. In the current study, we provide the first direct evidence that treacle is a nucleolar protein. An antibody generated against treacle shows that it localizes to the nucleolus. Fusion proteins tagged to a green fluorescent protein reporter were shown to localize to different compartments of the cell when putative nuclear localization signals were deleted. Parallel experiments using conserved regions of the murine homologue of TCOF1 confirmed these results. Site-directed mutagenesis has been used to recreate mutations observed in individuals with TCS. The resulting truncated proteins are mislocalized within the cell, which further supports the hypothesis that an integral part of treacle's function involves shuttling between the nucleolus and the cytoplasm. TCS is, therefore, the first Mendelian disorder resulting from mutations which lead to aberrant expression of a nucleolar protein.     

Processing of the intron-encoded U18 small nucleolar RNA in the yeast Saccharomyces cerevisiae relies on both exo- and endonucleolytic activities

Many small nucleolar RNAs (snoRNAs) are encoded within introns of protein-encoding genes and are released by processing of their host pre-mRNA. We have investigated the mechanism of processing of the yeast U18 snoRNA, which is found in the intron of the gene coding for translational elongation factor EF-1beta. We have focused our analysis on the relationship between splicing of the EF-1beta pre-mRNA and production of the mature snoRNA. Mutations inhibiting splicing of the EF-1beta pre-mRNA have been shown to produce normal U18 snoRNA levels together with the accumulation of intermediates deriving from the pre-mRNA, thus indicating that the precursor is an efficient processing substrate. Inhibition of 5'-->3' exonucleases obtained by insertion of G cassettes or by the use of a rat1-1 xrn1Delta mutant strain does not impair U18 release. In the Exo- strain, 3' cutoff products, diagnostic of an endonuclease-mediated processing pathway, were detected. Our data indicate that biosynthesis of the yeast U18 snoRNA relies on two different pathways, depending on both exonucleolytic and endonucleolytic activities: a major processing pathway based on conversion of the debranched intron and a minor one acting by endonucleolytic cleavage of the pre-mRNA.     

Regulation of cellular Mg2+ by Saccharomyces cerevisiae

Regulation of cellular Mg2+ by S. cerevisiae was investigated. The minimal concentration of Mg2+ that results in optimal growth of S. cerevisiae is about 30 microM and a half-maximum growth rate is attained at about 5 microM Mg2+. Since the plasma membrane has an electrical potential greater than 100 mV, passive equilibration of Mg2+ across the plasma membrane would provide sufficient cytosolic Mg2+ (0.1-1 mM). The total cellular Mg2+ of cells grown in synthetic medium containing 1 mM Mg2+ is about 400 nmol/mg protein, most of which is bound to polyphosphate, nucleic acids, and ATP. Total cellular Mg2+ decreases to about 80 nmol/mg protein as the Mg2+ in synthetic growth medium is reduced to 0.02 mM, but remains relatively constant in growth medium containing 1 to 100 mM Mg2+. Cells shifted into Mg(2+)-free medium continue to grow by utilizing the vacuolar Mg2+ stores. Mg(2+)-starved cells replenish vacuolar Mg2+ stores with a halftime of 30 min. following the addition of 1 mM Mg2+ to the growth medium. The data indicate that cytosolic Mg2+ is maintained by the regulation of Mg2+ fluxes across both the vacuolar and plasma membranes.     

Modifications to the ADH1 promoter of Saccharomyces cerevisiae for efficient production of heterologous proteins

The promoter of alcohol dehydrogenase I of the yeast Saccharomyces cerevisiae was studied using Bacillus amyloliquefaciens alpha-amylase as a marker protein. On glucose, activity of the original ADH1 promoter decreases during late exponential, ethanol production growth phase. When 1100 bp (from -414 bp to -1500 bp) of the upstream sequence are deleted, activity increases into the late ethanol consumption phase but the promoter becomes active only after ethanol production growth phase (Ruohonen et al. (1991) Yeast 7, 337-346). We have now restored 300 bp (from -414 bp to -700 bp) upstream of the deletion site and obtained expression from the ADH1 promoter throughout the yeast growth cycle. The restored sequence allowed alpha-amylase expression to start during early exponential growth phase indicating that it is required for activation of the ADH1 promoter during ethanol production growth phase, possibly through glucose induction. On ethanol, all the promoters were active, but the short promoter was temporally activated first, suggesting that the restored sequence is not required for promoter activity during early oxidative growth.     

Construction of a sorbitol-based vector for expression of heterologous proteins in Saccharomyces cerevisiae

A new inducible yeast expression vector, pXS7, was constructed by using the promoter and terminator sequences from the Saccharomyces cerevisiae SOR1 gene, which codes for the sorbitol dehydrogenase protein. We cloned the coding sequence of the Saccharomyces YEF3 gene in this vector and demonstrated an increase in YEF3 protein levels when cells were grown in the presence of the sugar sorbitol.     

Screening for glycosylphosphatidylinositol (GPI)-dependent cell wall proteins in Saccharomyces cerevisiae

Open reading frames in the genome of Saccharomyces cerevisiae were screened for potential glycosylphosphatidylinositol (GPI)-attached proteins. The identification of putative GPI-attached proteins was based on three criteria: the presence of a GPI-attachment signal sequence, a signal sequence for secretion and a serine- or threonine-rich sequence. In all, 53 ORFs met these three criteria and 38 were further analyzed as follows. The sequence encoding the 40 C-terminal amino acids of each was fused with the structural gene for a reporter protein consisting of a secretion signal, alpha-galactosidase and a hemagglutinin (HA) epitope, and examined for the ability to become incorporated into the cell wall. On this basis, 14 of fusion proteins were classified as GPI-dependent cell wall proteins because cells expressing these fusion proteins: (i) had high levels of alpha-galactosidase activity on their surface; (ii) released significant amounts of the fusion proteins from the membrane on treatment with phosphatidylinositol-specific phospholipase C (PI-PLC); and (iii) released fusion proteins from the cell wall following treatment with laminarinase. Of the 14 identified putative GPI-dependent cell wall proteins, 12 had novel ORFs adjacent to their GPI-attachment signal sequence. Amino acid sequence alignment of the C-terminal sequences of the 12 ORFs, together with those of known cell wall proteins, reveals some sequence similarities among them.     

Nuclear accumulation of Saccharomyces cerevisiae Mcm3 is dependent on its nuclear localization sequence

BACKGROUND: The proteins of the Mcm2-7 family are required for the initiation of DNA replication. In Saccharomyces cerevisiae the nuclear envelope does not break down during the mitotic phase of the cell cycle. Large nuclear proteins, such as the Mcm proteins, which accumulate in the nucleus during specific portions of the cell cycle, must have regulated mechanisms to direct their entry into the nucleus. RESULTS: We have identified a nuclear localization sequence (NLS) in Mcm3, and demonstrated that it is necessary for the translocation of Mcm3 into the nucleus and sufficient for directing Escherichia coli beta-galactosidase to the nucleus. Immediately adjacent to the nuclear localization sequence are four potential sites for phosphorylation by Cdc28. Mutagenesis of all four sites has no immediate phenotypic effect on cell growth or viability, nor does it affect nuclear accumulation of Mcm3, although two-dimensional protein gel analysis has shown that at least some of these sites are normally phosphorylated in vivo. Substitution of the Mcm3 NLS by the SV40 large T-antigen NLS also directs the nuclear accumulation of the Mcm3-T-antigen protein, although cell growth is compromised. Replication activity in cells bearing either the Mcm3-Cdc28 phosphorylation site mutations or the Mcm3 T-antigen NLS substitution, as measured by plasmid stability assays, is comparable to activity in wild-type cells. CONCLUSIONS: The Mcm3 protein is imported into the nucleus by a specific NLS. The cell cycle specific nuclear accumulation of Mcm3 appears to be a result of nuclear retention or nuclear targeting, rather than nuclear import regulated through the NLS.     

Analysis of genes encoding highly conserved lysine-rich proteins in Aplysia californica and Saccharomyces cerevisiae

To isolate a gene that can be used as an internal control in studies on gene expression in Aplysia californica neurons, we have characterized a cDNA clone (pKRP-A) isolated on the basis of its high expression in A. californica neurons. This cDNA is of 850 nucleotides and codes for a putative 29-kDa lysine-rich protein. Blotting experiments revealed that the gene is expressed in all tested A. californica tissues, and in individually identified neurons of the abdominal ganglion, suggesting that this gene can be efficiently used as internal control in studies of gene expression. We have also isolated one cDNA and two different genomic clones from yeast libraries that show 59% identity with pKRP-A. Sequence comparison of genomic clones, as well as PCR and Southern blotting experiments, revealed that at least two homologous genes are present in yeast. Northern blotting experiments revealed that the expression of the gene is strongly repressed at 39 degrees C.     

Expression and biochemical characterization of iron regulatory proteins 1 and 2 in Saccharomyces cerevisiae

Iron-regulatory proteins (IRPs) 1 and 2 are cytosolic RNA-binding proteins that bind to specific stem-loop structures, termed iron-responsive elements (IREs) that are located in the untranslated regions of specific mRNAs encoding proteins involved in iron metabolism. The binding of IRPs to IREs regulates either translation or stabilization of mRNA. Although IRP1 and IRP2 are similar proteins in that they are ubiquitously expressed and are negatively regulated by iron, they are regulated by iron by different mechanisms. IRP1, the well-characterized IRP in cells, is a dual-function protein exhibiting either aconitase activity when cellular iron is abundant or RNA-binding activity when cellular iron is scarce. In contrast, IRP2 lacks detectable aconitase activity and functions exclusively as an RNA-binding protein. To study and compare the biochemical characteristics of IRP1 and IRP2, we expressed wild-type and mutant rat IRP1 and IRP2 in the yeast Saccharomyces cerevisiae. IRP1 and IRP2 expressed in yeast bind the IRE RNA with high affinity, resulting in the inhibition of translation of an IRE-reporter mRNA. Mutant IRP2s lacking a 73 amino acid domain unique to IRP2 and a mutant IRP1 containing an insertion of this domain bound RNA, but lacked detectable aconitase activity, suggesting that the presence of this domain prevents aconitase activity. Like IRP1, the RNA-binding activity of IRP2 was sensitive to inactivation by N-ethylmaleimide (NEM) or 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB), indicating IRP2 contains a cysteine(s) that is (are) necessary for RNA binding. However, unlike IRP1, where reconstitution of the 4Fe-4S cluster resulted in a loss in RNA-binding activity, the RNA-binding activity of IRP2 was unaffected using the same iron treatment. These data suggested that IRP2 does not contain a 4Fe-4S cluster similar to the cluster in IRP1, indicating that they sense iron by different mechanisms.     

Members of the Hsp70 family of proteins in the cell wall of Saccharomyces cerevisiae

Western blot (immunoblot) analysis of cell wall and cytosolic extracts obtained from parental and ssa1 and ssa2 single- and double-mutant strains of Saccharomyces cerevisiae showed that the heat shock protein 70 (Hsp70) products of these genes, previously thought to be restricted to the cell interior, are also present in the cell wall. A cell wall location was further confirmed by indirect immunofluorescence with intact cells and biotinylation of extracellular Hsp70. Hsp70s have been implicated in translocation across the membrane and as molecular chaperones, and changes in the profile of cell wall proteins suggested that these proteins may have a similar role in the cell wall.     

Mitochondrial transport of mitoribosomal proteins, YmL8 and YmL20, in Saccharomyces cerevisiae

Two mitochondrial ribosomal (mitoribosomal) proteins, YmL8 and YmL20, of the yeast Saccharomyces cerevisiae and their derivatives were synthesized in vitro and their transport into isolated yeast mitochondria was examined. Of the two proteins, YmL20 possesses an N-terminal presequence of 18 amino acid residues, while YmL8 has no such presequence. Both proteins were found to be transported into isolated mitochondria in an energy-dependent manner. Furthermore, YmL20 protein without its N-terminal presequence was also transported, despite the fact that the presequence alone was capable of transporting a fused passenger protein, Chinese hamster dihydrofolate reductase (DHFR). Therefore, YmL20 protein appears to possess redundant transport signals in its structure. Similarly, YmL8 derivatives lacking either 40 or 86 amino acid residues from the N-terminus and/or 52 amino acid residues from the C-terminus were transported. In addition, the N-terminal segment of this protein was capable of transporting Chinese hamster DHFR into mitochondria, while its C-terminal segment was not. Thus, YmL8 protein also appears to possess two or more transport signals in its structure. Perhaps the presence of many basic amino acid residues in these proteins might, at least partly, contribute to their mitochondrial transport.     

Processing glycosidases of Saccharomyces cerevisiae

The properties of the N-glycan processing glycosidases located in the endoplasmic reticulum of Saccharomyces cerevisiae are described. alpha-Glucosidase I encoded by CWH41 cleaves the terminal alpha1, 2-linked glucose and alpha-glucosidase II encoded by ROT2 removes the two alpha1,3-linked glucose residues from the Glc3Man9GlcNAc2 oligosaccharide precursor while the alpha1,2-mannosidase encoded by MNS1 removes one specific mannose to form a single isomer of Man8GlcNAc2. Although trimming by these glycosidases is not essential for the formation of N-glycan outer chains, recent studies on mutants lacking these enzymes indicate that alpha-glucosidases I and II play an indirect role in cell wall beta1,6-glucan formation and that the alpha1,2-mannosidase is involved in endoplasmic reticulum quality control. Detailed structure-function studies of recombinant yeast alpha1,2-mannosidase are described that serve as a model for other members of this enzyme family that has been conserved through eukaryotic evolution.     

In vitro trans-splicing in Saccharomyces cerevisiae

The interactions established at the 5'-splice site during spliceosome assembly are likely to be important for both precise recognition of the upstream intron boundary and for positioning this site in the active center of the spliceosome. Definition of the RNA-RNA and the RNA-protein interactions at the 5' splice site would be facilitated by the use of a small substrate amenable to modification during chemical synthesis. We describe a trans-splicing reaction performed in Saccharomyces cerevisiae extracts in which the 5' splice site and the 3' splice site are on separate molecules. The RNA contributing the 5' splice site is only 20 nucleotides long and was synthesized chemically. The trans-splicing reaction is accurate and has the same sequence, ATP, and Mg2+ requirements as cis-splicing. We also report how deoxy substitutions around the 5'-splice site affect trans-splicing efficiency.