In vivo analysis of chromosome condensation in Saccharomyces cerevisiae

Although chromosome condensation in the yeast Saccharomyces cerevisiae has been widely studied, visualization of this process in vivo has not been achieved. Using Lac operator sequences integrated at two loci on the right arm of chromosome IV and a Lac repressor-GFP fusion protein, we were able to visualize linear condensation of this chromosome arm during G2/M phase. As previously determined in fixed cells, condensation in yeast required the condensin complex. Not seen after fixation of cells, we found that topoisomerase II is required for linear condensation. Further analysis of perturbed mitoses unexpectedly revealed that condensation is a transient state that occurs before anaphase in budding yeast. Blocking anaphase progression by activation of the spindle assembly checkpoint caused a loss of condensation that was dependent on Mad2, followed by a delayed loss of cohesion between sister chromatids. Release of cells from spindle checkpoint arrest resulted in recondensation before anaphase onset. The loss of condensation in preanaphase-arrested cells was abrogated by overproduction of the aurora B kinase, Ipl1, whereas in ipl1-321 mutant cells condensation was prematurely lost in anaphase/telophase. In vivo analysis of chromosome condensation has therefore revealed unsuspected relationships between higher order chromatin structure and cell cycle control.     

Deletion of the COX7 gene in Saccharomyces cerevisiae reveals a role for cytochrome c oxidase subunit VII In assembly of remaining subunits

Cytochrome c oxidase from Saccharomyces cerevisiae is composed of nine subunits. Subunits I, II and III are products of mitochondrial genes, while subunits IV, V, VI, VII, VIIa and VIII are products of nuclear genes. To investigate the role of cytochrome c oxidase subunit VII in biogenesis or functioning of the active enzyme complex, a null mutation in the COX7 gene, which encodes subunit VII, was generated, and the resulting cox7 mutant strain was characterized. The strain lacked cytochrome c oxidase activity and haem a/a3 spectra. The strain also lacked subunit VII, which should not be synthesized owing to the nature of the cox7 mutation generated in this strain. The amounts of remaining cytochrome c oxidase subunits in the cox7 mutant were examined. Accumulation of subunit I, which is the product of the mitochondrial COX1 gene, was found to be decreased relative to other mitochondrial translation products. Results of pulse-chase analysis of mitochondrial translation products are consistent with either a decreased rate of translation of COX1 mRNA or a very rapid rate of degradation of nascent subunit I. The synthesis, stability or mitochondrial localization of the remaining nuclear-encoded cytochrome c oxidase subunits were not substantially affected by the absence of subunit VII. To investigate whether assembly of any of the remaining cytochrome c oxidase subunits is impaired in the mutant strain, the association of the mitochondrial-encoded subunits I, II and III with the nuclear-encoded subunit IV was investigated.(ABSTRACT TRUNCATED AT 250 WORDS)     

The AUG start codon of the Saccharomyces cerevisiae NFS1 gene can be substituted for by UUG without increased initiation of translation at downstream codons.

The selection of the site for initiation of translation for the Saccharomyces cerevisiae NFS1 gene was examined using mutated AUG1, AUG2 and AUG3 codons. When AUG1 of the yeast NFS1 gene was mutated to UUG and the resulting mRNA was translated in vitro using a reticulocyte system, initiation from the mutated codon was abolished and occurred instead at downstream codons at increased rates. When the same mRNA was translated using a yeast extract, translation initiated at the mutated codon, albeit at a reduced rate, and there was no increased translation at downstream AUG codons. The NFS1 gene in which AUG1 was replaced by UUG was also able to substitute for the wild-type gene in vivo in yeast. Western blots confirmed that the encoded protein was the same size as that encoded by the wild-type gene and that both the wild-type and mutated proteins localized to mitochondria. This is apparently the first example of a yeast protein where mutagenesis of AUG1 does not lead to alternate use of a downstream AUG.     

Pet111p, an inner membrane-bound translational activator that limits expression of the Saccharomyces cerevisiae mitochondrial gene COX2

The protein specified by the Saccharomyces cerevisiae nuclear gene PET111 specifically activates translation of the mitochondrially coded mRNA for cytochrome c oxidase subunit II (Cox2p). We found Pet111p specifically in mitochondria of both wild-type cells and cells expressing a chromosomal gene for a functional epitope-tagged form of Pet111p. Pet111p was associated with mitochondrial membranes and was highly resistant to extraction with alkaline carbonate. Pet111p was protected from proteolytic digestion by the mitochondrial inner membrane. Thus, it is exposed only on the matrix side, where it could participate directly in organellar translation and localize Cox2p synthesis by virtue of its functional interaction with the COX2 mRNA 5'-untranslated leader. We also found that Pet111p is present at levels limiting the synthesis of Cox2p by examining the effect of altered PET111 gene dosage in the nucleus on expression of a reporter gene, cox2::ARG8(m), that was inserted into mitochondrial DNA. The level of the reporter protein, Arg8p, was one-half that of wild type in a diploid strain heterozygous for a pet111 deletion mutation, whereas it was increased 2.8-fold in a strain bearing extra copies of PET111 on a high-copy plasmid. Thus, Pet111p could play dual roles in both membrane localization and regulation of Cox2p synthesis within mitochondria.     

In vivo N-glycosylation of the mep2 high-affinity ammonium transporter of Saccharomyces cerevisiae reveals an extracytosolic N-terminus

Saccharomyces cerevisiae possesses three related ammonium transporters, Mep1, Mep2 and Mep3, differing in their kinetic properties and in the level and regulation of their gene expression. The three Mep proteins belong to a family conserved in bacteria, plants and animals, which also includes proteins of the rhesus blood group family. In addition to its role in scavenging extracellular ammonium, the Mep2 protein has been proposed to act as an ammonium sensor, essential to pseudohyphal differentiation in response to ammonium limitation. To pursue the biochemical study of the Mep transporters, we raised polyclonal antibodies against the C-terminal tail of each Mep protein. When electrophoresed on SDS-polyacrylamide gel, the Mep1 and Mep3 proteins migrate as expected from their predicted size, whereas the Mep2 protein migrates as a high-molecular-weight smear. Protein deglycosylation with peptide-N-glycosidase F (PNGase F) indicates that, in contrast to Mep1 and Mep3, Mep2 is an asparagine-linked glycoprotein. Site-directed mutagenesis of the four potential N-glycosylation sites of Mep2 shows that Asn-4 of the protein's N-terminal tail is the only site that binds oligosaccharides. This provides evidence for the extracytosolic location of the Mep2 N-terminus. Consistently, treatment of intact protoplasts with proteinase K leads to specific proteolysis of the N-terminal tail of Mep2. The protein's C-terminus, on the other hand, is protected against protease degradation under these conditions, but digested after protoplast permeabilization, suggesting a cytoplasmic location for this part of the protein. Mep2 glycosylation is not required for pseudohyphal differentiation in response to ammonium starvation, and its absence causes only a slight reduction in the affinity of the transporter for its substrate.     

Translational regulation of nuclear gene COX4 expression by mitochondrial content of phosphatidylglycerol and cardiolipin in Saccharomyces cerevisiae

Previous results indicated that translation of four mitochondrion-encoded genes and one nucleus-encoded gene (COX4) is repressed in mutants (pgs1Delta) of Saccharomyces cerevisiae lacking phosphatidylglycerol and cardiolipin. COX4 translation was studied here using a mitochondrially targeted green fluorescence protein (mtGFP) fused to the COX4 promoter and its 5' and 3' untranslated regions (UTRs). Lack of mtGFP expression independent of carbon source and strain background was established to be at the translational level. The translational defect was not due to deficiency of mitochondrial respiratory function but was rather caused directly by the lack of phosphatidylglycerol and cardiolipin in mitochondrial membranes. Reintroduction of a functional PGS1 gene under control of the ADH1 promoter restored phosphatidylglycerol synthesis and expression of mtGFP. Deletion analysis of the 5' UTR(COX4) revealed the presence of a 50-nucleotide fragment with two stem-loops as a cis-element inhibiting COX4 translation. Binding of a protein factor(s) specifically to this sequence was observed with cytoplasm from pgs1Delta but not PGS1 cells. Using HIS3 and lacZ as reporters, extragenic spontaneous recessive mutations that allowed expression of His3p and beta-galactosidase were isolated, which appeared to be loss-of-function mutations, suggesting that the genes mutated may encode the trans factors that bind to the cis element in pgs1Delta cells.     

Deletion analysis of the SUP2 gene in Saccharomyces cerevisiae

The sup2 mutations of the yeast Saccharomyces cerevisiae or plasmid-mediated amplification of the wild type SUP2 gene lead to suppression of different types of nonsense mutations. The Sup2 protein includes a C-terminal region homologous to elongation factor EF-1 alpha and an unique N-terminal region. The SUP2 is an essential gene. The functional role of different regions of the SUP2 gene was investigated, by deleting them without disruption of the reading frame. Such constructs were maintained in yeast on episomal or centromeric plasmids. It was shown that the region, homologous to EF-1 alpha is necessary for viability, while the remaining N-terminal part is nonessential. The region of the first 154 amino acids is necessary and sufficient for the suppressor effect, caused by plasmid-mediated amplification of the SUP2 gene.     

Interactions among COX1, COX2, and COX3 mRNA-specific translational activator proteins on the inner surface of the mitochondrial inner membrane of Saccharomyces cerevisiae

The core of the cytochrome c oxidase complex is composed of its three largest subunits, Cox1p, Cox2p, and Cox3p, which are encoded in mitochondrial DNA of Saccharomyces cerevisiae and inserted into the inner membrane from the inside. Mitochondrial translation of the COX1, COX2, and COX3 mRNAs is activated mRNA specifically by the nuclearly coded proteins Pet309p, Pet111p, and the concerted action of Pet54p, Pet122p, and Pet494p, respectively. Because the translational activators recognize sites in the 5'-untranslated leaders of these mRNAs and because untranslated mRNA sequences contain information for targeting their protein products, the activators are likely to play a role in localizing translation. Herein, we report physical associations among the mRNA-specific translational activator proteins, located on the matrix side of the inner membrane. These interactions, detected by coimmune precipitation and by two-hybrid experiments, suggest that the translational activator proteins could be organized on the surface of the inner membrane such that synthesis of Cox1p, Cox2p, and Cox3p would be colocalized in a way that facilitates assembly of the core of the cytochrome c oxidase complex. In addition, we found interactions between Nam1p/Mtf2p and the translational activators, suggesting an organized delivery of mitochondrial mRNAs to the translation system.     

In vivo robustness analysis of cell division cycle genes in Saccharomyces cerevisiae

Intracellular biochemical parameters, such as the expression level of gene products, are considered to be optimized so that a biological system, including the parameters, works effectively. Those parameters should have some permissible range so that the systems have robustness against perturbations, such as noise in gene expression. However, little is known about the permissible range in real cells because there has been no experimental technique to test it. In this study, we developed a genetic screening method, named “genetic tug-of-war” (gTOW) that evaluates upper limit copy numbers of genes in a model eukaryote Saccharomyces cerevisiae, and we applied it for 30 cell-cycle related genes (CDC genes). The experiment provided unique quantitative data that could be used to argue the system-level properties of the cell cycle such as robustness and fragility. The data were used to evaluate the current computational model, and refinements to the model were suggested.     

In vivo involvement of mutated initiation factor IF1 in gene expression control at the translational level

The influence in vivo of mutated forms of translation initiation factor (IF1) on the expression of the lacZ or 3A' reporter genes, with different initiation and/or +2 codons, has been investigated. Reporter gene expression in these infA(IF1) mutants is similar to the wild-type strain. The results do not support the longstanding hypothesis that IF1 could perform discriminatory functions while blocking the aminoacyl-tRNA acceptor site (A-site) of the ribosome. One cold-sensitive IF1 mutant shows a general overexpression, in particular at low temperatures, of both reporter genes at the protein but not mRNA level.     

Sequential gene function in the initiation of Saccharomyces cerevisiae DNA synthesis

Four steps are known to be required for the initiation of DNA synthesis in Saccharomyces cerevisiae. Three of these are mediated by the products of genes cdc 4, 7, and 28 and the fourth is identified by the inhibition exerted on haploid α cells by the mating pheromone, α factor. These four steps have been ordered by a combination of two methods and found to be:

initiation of DNA synthesis The two sequencing methods are described in detail. Experiments involving the shift of mutant cells from the restrictive to the permissive temperature in the presence of cycloheximide demonstrated that the protein synthesis requirement for yeast DNA replication can be completed before the cdc 7-mediated step.     

Genetic evidence that different functional domains of the PET54 gene product facilitate expression of the mitochondrial genes COX1 and COX3 in Saccharomyces cerevisiae.

Expression of the yeast mitochondrial genes COX1 and COX3, which encode subunits I and III of cytochrome oxidase, respectively, is controlled by a common nuclear-encoded trans-acting factor. This protein, encoded by the PET54 gene, controls expression of COX1 at the level of RNA splicing and COX3 at the level of mRNA translation. While the steps of COX1 and COX3 gene expression affected by the PET54 gene product are different, it is possible that the PET54 protein is monofunctional and affects expression of each gene by a single mechanism, such as modulation of RNA secondary structure. The goal of this study was to address whether the PET54 protein is monofunctional or multifunctional with respect to its role in COX1 and COX3 gene expression. Ten insertion mutations, which each resulted in the in-frame addition of four amino acids within the PET54 polypeptide, were generated, and the resulting mutants were characterized for respiration phenotype and mitochondrial gene expression. Five of the ten mutants were respiration deficient. Two of these five mutants were defective in expression of COX3 but not in expression of COX1, while two other mutants had the opposite phenotype (primarily defective in expression of COX1). The fifth mutant was equally defective in expression of both genes. These results demonstrate that the two functions of PET54 are genetically separable and support the idea that the PET54 protein is multifunctional.     

Cloning of the LEU2 gene of Saccharomyces cerevisiae by in vivo recombination

We describe a convenient method for the in vivo construction of large plasmids that possess a multitude of restriction sites. A large (23 kbases) circular self-replicating plasmid carrying a partial LEU2-d gene was cotransformed with a circular non-replicating plasmid carrying the entire LEU2 gene. In vivo recombination results preferentially in a plasmid that carries both the LEU2-d and the entire LEU2 gene. In addition we also found one plasmid with a tandem LEU2 insertion and one plasmid where the LEU2-d gene was replaced by the entire LEU2 gene.     

In vivo analysis of cohesin architecture using FRET in the budding yeast Saccharomyces cerevisiae

Cohesion between sister chromatids in eukaryotes is mediated by the evolutionarily conserved cohesin complex. Cohesin forms a proteinaceous ring, large enough to trap pairs of replicated sister chromatids. The circumference consists of the Smc1 and Smc3 subunits, while Scc1 is thought to close the ring by bridging the Smc (structural maintenance of chromosomes) ATPase head domains. Little is known about two additional subunits, Scc3 and Pds5, and about possible conformational changes of the complex during the cell cycle. We have employed fluorescence resonance energy transfer (FRET) to analyse interactions within the cohesin complex in live budding yeast. These experiments reveal an unexpected geometry of Scc1 at the Smc heads, and suggest that Pds5 plays a role at the Smc hinge on the opposite side of the ring. Key subunit interactions, including close proximity of the two ATPase heads, are constitutive throughout the cell cycle. This depicts cohesin as a stable molecular machine undergoing only transient conformational changes during binding and dissociation from chromosomes. Using FRET, we did not observe interactions between more than one cohesin complex in vivo.