Morphology of mitochondrial nucleoids in respiratory-deficient yeast cells varies depending on the unit length of the mitochondrial DNA sequence.

We investigated the morphology of mitochondrial nucleoids (mt-nucleoids) and mitochondria in Saccharomyces cerevisiae rho(+) and rho(-) cells with DAPI staining and mitochondria-targeted GFP. Whereas the mt-nucleoids appeared as strings of beads in wild-type rho(+) cells at log phase, the mt-nucleoids in hypersuppressive rho(-) cells (HS40 rho(-) cells) appeared as distinct punctate structures. In order to elucidate whether the punctate mt-nucleoids are common to other rho(-) cells, we observed the mt-nucleoids in rho(-) strains that retain different unit lengths of the mitochondrial DNA (mtDNA) sequence. As a result, rho(-) cells that have long mtDNA sequences, of more than 30 kb, had mt-nucleoids with a strings-of-beads appearance in tubular mitochondria. In contrast, rho(-) cells that have short mtDNA sequences, of <1 kb, had punctate mt-nucleoids in tubular mitochondria. This indicates that the morphology of mt-nucleoids in rho(-) cells significantly varies depending on the unit length of their mtDNA sequence. Analyses of mt-nucleoids suggest that the punctate mt-nucleoids in HS40 rho(-) cells consist of concatemeric mtDNAs and oligomeric circular mtDNAs associated with Abf2p and other nucleoid proteins.

Aldehyde dehydrogenase, Ald4p, is a major component of mitochondrial fluorescent inclusion bodies in the yeast Saccharomyces cerevisiae.

When Saccharomyces cerevisiae strain 3626 was cultured to the stationary phase in a medium that contained glucose, needle-like structures that emitted autofluorescence were observed in almost all cells by fluorescence microscopy under UV excitation. The needle-like structures completely overlapped with the profile of straight elongated mitochondria. Therefore, these structures were designated as mitochondrial fluorescent inclusion bodies (MFIBs). The MFIB-enriched mitochondrial fractions were successfully isolated and 2D-gel electrophoresis revealed that a protein of 54 kDa was only highly concentrated in the fractions. Determination of the N-terminal amino acid sequence of the 54-kDa protein identified it as a mitochondrial aldehyde dehydrogenase, Ald4p. Immunofluorescence microscopy showed that anti-Ald4p antibody specifically stained MFIBs. Freeze-substitution electron microscopy demonstrated that cells that retained MFIBs had electron-dense filamentous structures with a diameter of 10 nm in straight elongated mitochondria. Immunoelectron microscopy showed that Ald4p was localized to the electron-dense filamentous structures in mitochondria. These results together showed that a major component of MFIBs is Ald4p. In addition, we demonstrate that MFIBs are common features that appear in mitochondria of many species of yeast.

Rim15p-mediated regulation of sucrose utilization during molasses fermentation using Saccharomyces cerevisiae strain PE-2.

Inherited loss-of-function mutations in the Rim15p-mediated stress-response pathway contribute to the high fermentation rate of sake yeast strains. In the present study, we found that disruption of the RIM15 gene in ethanol-producing Saccharomyces cerevisiae strain PE-2 accelerated molasses fermentation through enhanced sucrose utilization following glucose starvation.

Ras/cAMP-dependent protein kinase (PKA) regulates multiple aspects of cellular events by phosphorylating the Whi3 cell cycle regulator in budding yeast.

The Start/G1 phase in the cell cycle is an important period during which cells determine their developmental fate, onset of mitotic progression, or the switch to developmental stages in response to both external and internal signals. In the budding yeast Saccharomyces cerevisiae, Whi3, a negative regulator of the G1 cyclins, has been identified as a positive regulator of cell size control and is involved in the regulation of Start. However, the regulatory pathway of Whi3 governing the response to multiple signals remains largely unknown. Here, we show that Whi3 is phosphorylated by the Ras/cAMP-dependent protein kinase (PKA) and that phosphorylation of Ser-568 in Whi3 by PKA plays an inhibitory role in Whi3 function. Phosphorylation of Whi3 by PKA led to its decreased interaction with CLN3 G1 cyclin mRNA and was required for the promotion of G1/S progression. Furthermore, we demonstrate that the phosphomimetic S568D mutation of Whi3 prevented the developmental fate switch to sporulation or invasive growth. Thus, PKA modulated the function of Whi3 by phosphorylation, thus implicating PKA-mediated modulation of Whi3 in multiple cellular events.

Overexpression of the yeast transcription activator Msn2 confers furfural resistance and increases the initial fermentation rate in ethanol production.

Lignocellulosic biomass is a promising source for bioethanol production, because it is abundant worldwide and has few competing uses. However, the treatment of lignocelllulosic biomass with weak acid to release cellulose and hemicellulose generates many kinds of byproducts including furfural and 5-hydroxymethylfurfural, which inhibit fermentation by yeast, because they generate reactive oxygen species (ROS) in cells. In order to acquire high tolerance to oxidative stress in bioethanol yeast strains, we focused on the transcription activator Msn2 of Saccharomyces cerevisiae, which regulates numerous genes involved in antioxidative stress responses, and constructed bioethanol yeast strains that overexpress Msn2 constitutively. The Msn2-overexpressing bioethanol strains showed tolerance to oxidative stress, probably due to the high-level expression of various antioxidant enzyme genes. Unexpectedly, these strains showed ethanol sensitivity compared with the control strain, probably due to imbalance of the expression level between Msn2 and Msn4. In the presence of furfural, the engineered strains exhibited reduced intracellular ROS levels, and showed rapid growth compared with the control strain. The fermentation test in the presence of furfural revealed that the Msn2-overexpressing strains showed improvement of the initial rate of fermentation. Our results indicate that overexpression of the transcription activator Msn2 in bioethanol yeast strains confers furfural tolerance by reducing the intracellular ROS levels and enhances the initial rate of fermentation in the presence of furfural, suggesting that these strains are capable of adapting rapidly to various compounds that inhibit fermentation by inducing ROS accumulation. Our results not only promise to improve bioethanol production from lignocellulosic biomass, but also provide novel insights for molecular breeding of industrial yeast strains.

Identification of Tup1 and Cyc8 mutations defective in the responses to osmotic stress.

In the yeast Saccharomyces cerevisiae, Tup1, in association with Cyc8 (Ssn6), functions as a general transcriptional corepressor. This repression is mediated by recruitment of the Tup1-Cyc8 complex to target promoters through sequence-specific DNA-binding proteins such as Sko1, which mediates the HOG pathway-dependent regulation. We identified tup1 and cyc8 mutant alleles as the suppressor of osmo-sensitivity of the hog1Delta strain. In these mutants, although the expression of the genes under the control of DNA-binding proteins other than Sko1 was apparently normal, the Sko1-regulated genes GRE2 and AHP1 were derepressed under non-stress conditions, suggesting that the Tup1 and Cyc8 mutant proteins were specifically defective in the repression of the Sko1-dependent genes. Chromatin immunoprecipitation analyses of the GRE2 promoter in the mutants demonstrated that the Sko1-Tup1-Cyc8 complex was localized to the promoter, together with Gcn5/SAGA, suggesting that the erroneous recruitment of SAGA to the promoter led to the derepression.

Interplay between chromatin and trans-acting factors on the IME2 promoter upon induction of the gene at the onset of meiosis.

The IME2 gene is one of the key regulators of the initiation of meiosis in budding yeast. This gene is repressed during mitosis through the repressive chromatin structure at the promoter, which is maintained by the Rpd3-Sin3 histone deacetylase (HDAC) complex. IME2 expression in meiosis requires Gcn5/histone acetyltransferase, the transcriptional activator Ime1, and the chromatin remodeler RSC; however, the molecular basis of IME2 activation had not been previously defined. We found that, during mitotic growth, a nucleosome masked the TATA element of IME2, and this positioning depended on HDAC. This chromatin structure was remodeled at meiosis by RSC that was recruited to TATA by Ime1. Stable tethering of Ime1 to the promoter required the presence of Gcn5. Interestingly, Ime1 binding to the promoter was kept at low levels during the very early stages in meiosis, even when the levels of Ime1 and histone H3 acetylation at the promoter were at their highest, making a 4- to 6-h delay of the IME2 expression from that of IME1. HDAC was continuously present at the promoter regardless of the transcriptional condition of IME2, and deletion of RPD3 allowed the IME2 expression shortly after the expression of IME1, suggesting that HDAC plays a role in regulating the timing of IME2 expression.

RSC Nucleosome-remodeling complex plays prominent roles in transcriptional regulation throughout budding yeast gametogenesis.

RSC is a nucleosome-remodeling complex of Saccharomyces cerevisiae essential for growth that can alter histone-DNA interaction by using the energy of ATP hydrolysis. Nps1p/Sth1p is an ATPase subunit of RSC. A mutation in the conserved ATPase domain of Nps1p causes a sporulation defect with decreased expression of early meiotic genes, especially IME2. This defect is partially suppressed by the overexpression of either IME1 or IME2. A homozygous diploid of a novel temperature-sensitive nps1 mutation, nps1-13, harboring amino acid substitutions within the bromodomain, was unable to sporulate. Overexpression of IME, IME2, or both of these genes allowed the completion of meiosis I and meiosis II in nps1-13 but not the formation of mature asci. In nps1-13 carrying YEpIME1, the expression of a group of sporulation-specific genes, which express at the middle stages of sporulation and are required for spore-wall formation, notably diminished, and several late sporulation genes expressed at the early stages of sporulation. These results suggest that Nps1p/RSC plays important roles during the spore development process by controlling gene expression for initiating both meiosis and spore morphogenesis, and ensures proper expression timing of late meiotic genes.