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

ABSTRACT: 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.

SUBMITTER: Misonou Y

PROVIDER: S-EPMC4021361 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Robust assembly of the aldehyde dehydrogenase Ald4p in Saccharomyces cerevisiae.

Project description:As part of our studies of yeast aldehyde dehydrogenase (Ald4p) assembly, we identified a population of transformants (SWORD strain) that show more robust filament formation of GFP-tagged Ald4p (Ald4p-GFP) than that of a wild type ALD4::GFP strain. Sequencing of the ALD4 gene in the SWORD strain showed that the increased assembly was not due to changes to the ALD4 coding sequence, suggesting that a second mutation site was altering Ald4p assembly. Using short-read whole-genome sequencing, we identified spontaneous mutations in FLO9. Introduction of the SWORD allele of FLO9 into a wild-type ALD4::GFP yeast strain revealed that the changes to FLO9 were a contributor to the increased length of Ald4p-GFP filaments we observe in the SWORD strain and that this effect was not due to an increase in Ald4p protein levels. However, the expression of the FLO9 (SWORD) allele in wild-type yeast did not fully recapitulate the length control defect we observed in SWORD strains, arguing that there are additional genes contributing to the filament length phenotype. For our future work, this FLO9 from SWORD will be tested whether it could show global effect, promoting the assembly of some other filament-forming enzymes.

| S-EPMC10602002 | biostudies-literature

Gis4, a new component of the ion homeostasis system in the yeast Saccharomyces cerevisiae.

Project description:Gis4 is a new component of the system required for acquisition of salt tolerance in Saccharomyces cerevisiae. The gis4Delta mutant is sensitive to Na(+) and Li(+) ions but not to osmotic stress. Genetic evidence suggests that Gis4 mediates its function in salt tolerance, at least partly, together with the Snf1 protein kinase and in parallel with the calcineurin protein phosphatase. When exposed to salt stress, mutants lacking gis4Delta display a defect in maintaining low intracellular levels of Na(+) and Li(+) ions and exporting those ions from the cell. This defect is due to diminished expression of the ENA1 gene, which encodes the Na(+) and Li(+) export pump. The protein sequence of Gis4 is poorly conserved and does not reveal any hints to its molecular function. Gis4 is enriched at the cell surface, probably due to C-terminal farnesylation. The CAAX box at the C terminus is required for cell surface localization but does not seem to be strictly essential for the function of Gis4 in salt tolerance. Gis4 and Snf1 seem to share functions in the control of ion homeostasis and ENA1 expression but not in glucose derepression, the best known role of Snf1. Together with additional evidence that links Gis4 genetically and physically to Snf1, it appears that Gis4 may function in a pathway in which Snf1 plays a specific role in controlling ion homeostasis. Hence, it appears that the conserved Snf1 kinase plays roles in different pathways controlling nutrient as well as stress response.

| S-EPMC1595338 | biostudies-literature

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| S-EPMC8305157 | biostudies-literature

Heterologous Expression of Aldehyde Dehydrogenase from Saccharomyces cerevisiae in Klebsiella pneumoniae for 3-Hydroxypropionic Acid Production from Glycerol.

Project description:3-Hydroxypropionic acid (3-HP) is a commercially valuable platform compound. Klebsiella pneumoniae has been concerned as an appropriate host for 3-HP production because of its robust capacity to metabolize glycerol. Glycerol conversion to 3-HP in K. pneumoniae comprises two successive reactions: glycerol dehydratase catalyzes glycerol to 3-hydroxypropionaldehyde (3-HPA); aldehyde dehydrogenase catalyzes 3-HPA to 3-HP. Previous studies focusing on inducible expression of aldehyde dehydrogenase have shown defects of high cost of inducer and low catalytic activity due to inclusion body. Here we show a different strategy that a native promoter in the host K. pneumoniae was used to drive the heterologous expression of aldehyde dehydrogenase gene ald4 from Saccharomyces cerevisiae. The 3-HP yield of the recombinant reached a peak of 4.23 g/L at log phase, but it decreased during later period of fermentation. Except the validation of high activity of ald4, particularly, the 3-HP formation was uncovered to be closely coupled with cell division, and the lacking of NAD and ATP at latter fermentation phase became the bottleneck for cell growth and 3-HP accumulation. Furthermore, 3-HP is postulated to be converted to 3-HPA via feedback inhibition or other metabolite via unknown mechanism. Since glycerol dissimilation is a common mechanism in a variety of bacteria, the expression strategy using native promoter and implications may provide significant insight into the metabolic engineering for 3-HP production.

| S-EPMC3460132 | biostudies-literature

High-voltage electron tomography of spindle pole bodies and early mitotic spindles in the yeast Saccharomyces cerevisiae.

Project description:The spindle pole body (SPB) is the major microtubule-organizing center of budding yeast and is the functional equivalent of the centrosome in higher eukaryotic cells. We used fast-frozen, freeze-substituted cells in conjunction with high-voltage electron tomography to study the fine structure of the SPB and the events of early spindle formation. Individual structures were imaged at 5-10 nm resolution in three dimensions, significantly better than can be achieved by serial section electron microscopy. The SPB is organized in distinct but coupled layers, two of which show ordered two-dimensional packing. The SPB central plaque is anchored in the nuclear envelope with hook-like structures. The minus ends of nuclear microtubules (MTs) are capped and are tethered to the SPB inner plaque, whereas the majority of MT plus ends show a distinct flaring. Unbudded cells containing a single SPB retain 16 MTs, enough to attach to each of the expected 16 chromosomes. Their median length is approximately 150 nm. MTs growing from duplicated but not separated SPBs have a median length of approximately 130 nm and interdigitate over the bridge that connects the SPBs. As a bipolar spindle is formed, the median MT length increases to approximately 300 nm and then decreases to approximately 30 nm in late anaphase. Three-dimensional models confirm that there is no conventional metaphase and that anaphase A occurs. These studies complement and extend what is known about the three-dimensional structure of the yeast mitotic spindle and further our understanding of the organization of the SPB in intact cells.

| S-EPMC25406 | biostudies-literature

Yeast prions form infectious amyloid inclusion bodies in bacteria.

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| S-EPMC3520751 | biostudies-literature

Mitochondrial NAD dependent aldehyde dehydrogenase either from yeast or human replaces yeast cytoplasmic NADP dependent aldehyde dehydrogenase for the aerobic growth of yeast on ethanol.

Project description:BackgroundIn a previous study, we deleted three aldehyde dehydrogenase (ALDH) genes, involved in ethanol metabolism, from yeast Saccharomyces cerevisiae and found that the triple deleted yeast strain did not grow on ethanol as sole carbon source. The ALDHs were NADP dependent cytosolic ALDH1, NAD dependent mitochondrial ALDH2 and NAD/NADP dependent mitochondrial ALDH5. Double deleted strain ΔALDH2+ΔALDH5 or ΔALDH1+ΔALDH5 could grow on ethanol. However, the double deleted strain ΔALDH1+ΔALDH2 did not grow in ethanol.MethodsTriple deleted yeast strain was used. Mitochondrial NAD dependent ALDH from yeast or human was placed in yeast cytosol.ResultsIn the present study we found that a mutant form of cytoplasmic ALDH1 with very low activity barely supported the growth of the triple deleted strain (ΔALDH1+ΔALDH2+ΔALDH5) on ethanol. Finding the importance of NADP dependent ALDH1 on the growth of the strain on ethanol we examined if NAD dependent mitochondrial ALDH2 either from yeast or human would be able to support the growth of the triple deleted strain on ethanol if the mitochondrial form was placed in cytosol. We found that the NAD dependent mitochondrial ALDH2 from yeast or human was active in cytosol and supported the growth of the triple deleted strain on ethanol.ConclusionThis study showed that coenzyme preference of ALDH is not critical in cytosol of yeast for the growth on ethanol.General significanceThe present study provides a basis to understand the coenzyme preference of ALDH in ethanol metabolism in yeast.

| S-EPMC3669398 | biostudies-literature

P bodies promote stress granule assembly in Saccharomyces cerevisiae.

Project description:Recent results indicate that nontranslating mRNAs in eukaryotic cells exist in distinct biochemical states that accumulate in P bodies and stress granules, although the nature of interactions between these particles is unknown. We demonstrate in Saccharomyces cerevisiae that RNA granules with similar protein composition and assembly mechanisms as mammalian stress granules form during glucose deprivation. Stress granule assembly is dependent on P-body formation, whereas P-body assembly is independent of stress granule formation. This suggests that stress granules primarily form from mRNPs in preexisting P bodies, which is also supported by the kinetics of P-body and stress granule formation both in yeast and mammalian cells. These observations argue that P bodies are important sites for decisions of mRNA fate and that stress granules, at least in yeast, primarily represent pools of mRNAs stalled in the process of reentry into translation from P bodies.

| S-EPMC2575786 | biostudies-literature

Rapid purification and properties of potassium-activated aldehyde dehydrogenase from Saccharomyces cerevisiae.

Project description:A method for the purification of yeast K+-activated aldehyde dehydrogenase is presented which can be completed in substantially less time than other published procedures. The enzyme has a different N-terminal amino acid from preparations previously reported, and other small differences in amino acid content. These differences may be the result of differential proteolytic digestion rather than a different protein in vivo. A purification step involves the biospecific adsorption on affinity columns containing immobilized nucleotides in the absence of the substrate aldehyde. Direct binding studies with the coenzyme in the absence of aldehyde reveal 4 NAD sites per tetrameric molecule, each with a dissociation constant of 120 micron. These results conflict with properties of preparations previously reported and may conflict with kinetic models that have aldehyde as the leading substrate. Binding to Blue Dextran affinity columns suggests the presence of a dinucleotide fold in common with other dehydrogenases and kinases.

| S-EPMC1185843 | biostudies-other

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