scholarly journals Thermodynamic characterization of yeast triosephosphate isomerase refolding: insights into the interplay between function and stability as reasons for the oligomeric nature of the enzyme

2003 ◽  
Vol 370 (3) ◽  
pp. 785-792 ◽  
Author(s):  
Hugo NÁJERA ◽  
Miguel COSTAS ◽  
D. Alejandro FERNÁNDEZ-VELASCO
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Triosephosphate Isomerase ◽  
Guanidinium Chloride ◽  
Yeast Saccharomyces Cerevisiae ◽  
Thermodynamic Characterization ◽  
Model Simulations ◽  
State Process ◽  
The Stability ◽  
Slow Dissociation

The reasons underlying the oligomeric nature of some proteins such as triosephosphate isomerase (TIM) are unclear. It has been proposed that this enzyme is an oligomer, mainly because of its stability rather than for functional reasons. To address this issue, the reversible denaturation and renaturation of the homodimeric TIM from baker's yeast (Saccharomyces cerevisiae) induced by guanidinium chloride and urea have been characterized by spectroscopic, functional and hydrodynamic techniques. The unfolding and refolding of this enzyme are not coincident after ‘conventional’ equilibrium times. Unfolding experiments did not reach equilibrium, owing to a very slow dissociation and/or unfolding process. By contrast, equilibrium was reached in the refolding direction. The simplest equilibrium pathway compatible with the obtained data was found to be a three-state process involving an inactive and expanded monomer. The Gibbs energy changes for monomer folding (ΔGfold0 = −16.6±0.7kJ·mol-1) and monomer association (ΔGassoc0 = −70.3±1.1kJ·mol-1) were calculated from data obtained in the two denaturants. From an analysis of the present data and data from the literature on the stability of TIM from different species and for other β/α barrels, and model simulations on the effect of stability in the catalytic activity of the enzyme, it is concluded that the low stability of the monomers is neither the only, nor the main, cause for the dimeric nature of TIM. There is interplay between function and stability.

scholarly journals Cloning and characterization of four SIR genes of Saccharomyces cerevisiae.

1986 ◽  
Vol 6 (2) ◽  
pp. 688-702 ◽  
Author(s):  
J M Ivy ◽  
A J Klar ◽  
J B Hicks
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Mating Type ◽  
Blot Analysis ◽  
Transcriptional Control ◽  
Genomic Library ◽  
Yeast Saccharomyces Cerevisiae ◽  
Mating Type Genes ◽  
Rna Blots

Mating type in the yeast Saccharomyces cerevisiae is determined by the MAT (a or alpha) locus. HML and HMR, which usually contain copies of alpha and a mating type information, respectively, serve as donors in mating type interconversion and are under negative transcriptional control. Four trans-acting SIR (silent information regulator) loci are required for repression of transcription. A defect in any SIR gene results in expression of both HML and HMR. The four SIR genes were isolated from a genomic library by complementation of sir mutations in vivo. DNA blot analysis suggests that the four SIR genes share no sequence homology. RNA blots indicate that SIR2, SIR3, and SIR4 each encode one transcript and that SIR1 encodes two transcripts. Null mutations, made by replacement of the normal genomic allele with deletion-insertion mutations created in the cloned SIR genes, have a Sir- phenotype and are viable. Using the cloned genes, we showed that SIR3 at a high copy number is able to suppress mutations of SIR4. RNA blot analysis suggests that this suppression is not due to transcriptional regulation of SIR3 by SIR4; nor does any SIR4 gene transcriptionally regulate another SIR gene. Interestingly, a truncated SIR4 gene disrupts regulation of the silent mating type loci. We propose that interaction of at least the SIR3 and SIR4 gene products is involved in regulation of the silent mating type genes.

scholarly journals The Identification and Characterization of ADR6, a Gene Required for Sporulation and for Expression of the Alcohol Dehydrogenase II Isozyme From Saccharomyces cerevisiae

Genetics ◽  
1987 ◽  
Vol 116 (4) ◽  
pp. 523-530
Author(s):  
Aileen K W Taguchi ◽  
Elton T Young
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Alcohol Dehydrogenase ◽  
Isocitrate Lyase ◽  
Carbon Sources ◽  
Recessive Mutation ◽  
Yeast Saccharomyces Cerevisiae ◽  
Upstream Activating Sequence ◽  
Adh Activity ◽  
Identification And Characterization

ABSTRACT The alcohol dehydrogenase II isozyme (enzyme, ADHII; structural gene, ADH2) of the yeast, Saccharomyces cerevisiae, is under stringent carbon catabolite control. This cytoplasmic isozyme exhibits negligible activity during growth in media containing fermentable carbon sources such as glucose and is maximal during growth on nonfermentable carbon sources. A recessive mutation, adr6-1, and possibly two other alleles at this locus, were selected for their ability to decrease Ty-activated ADH2-6 c expression. The adr6-1 mutation led to decreased ADHII activity in both ADH2-6c and ADH2+ strains, and to decreased levels of ADH2 mRNA. Ty transcription and the expression of two other carbon catabolite regulated enzymes, isocitrate lyase and malate dehydrogenase, were unaffected by the adr6-1 mutation. adr6-1/adr6-1strains were defective for sporulation, indicating that adr6 mutations may have pleiotropic effects. The sporulation defect was not a consequence of decreased ADH activity. Since the ADH2-6c mutation is due to insertion of a 5.6-kb Ty element at the TATAA box, it appears that the ADR6+-dependent ADHII activity required ADH2 sequences 3′ to or including the TATAA box. The ADH2 upstream activating sequence (UAS) was probably not required. The ADR6 locus was unlinked to the ADR1 gene which encodes another trans-acting element required for ADH2 expression.

scholarly journals Conservation of function and regulation within the Cdc28/cdc2 protein kinase family: characterization of the human Cdc2Hs protein kinase in Saccharomyces cerevisiae.

1989 ◽  
Vol 9 (9) ◽  
pp. 4064-4068 ◽  
Author(s):  
C Wittenberg ◽  
S I Reed
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Protein Kinase ◽  
Yeast Strain ◽  
Cell Cycle Progression ◽  
G1 Arrest ◽  
Yeast Saccharomyces Cerevisiae ◽  
Environmental Signals ◽  
Transgenic Yeast ◽  
M Phase

Whereas the Cdc28 protein kinase of the budding yeast Saccharomyces cerevisiae plays an essential role in cell cycle progression during the G1 interval, a function in the progression from the G2 interval into M phase has been inferred for its homologs, including the Cdc2Hs protein kinase of humans. To better understand these apparently disparate roles, we constructed a yeast strain in which the resident CDC28 gene was replaced by its human homolog, CDC2Hs. This transgenic yeast strain was able to perform the G1 functions attributed to the Cdc28 protein kinase, including the ability to grow and divide normally, to respond to environmental signals that induce G1 arrest, and to regulate the Cdc2Hs protein kinase appropriately in response to these signals.

Purification and characterization of wheat α-gliadin synthesized in the yeast, Saccharomyces cerevisiae

Gene ◽  
1992 ◽  
Vol 116 (2) ◽  
pp. 119-127 ◽  
Author(s):  
Ann E. Blechl ◽  
Kristin S. Thrasher ◽  
William H. Vensel ◽  
Frank C. Greene
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Purification And Characterization ◽  
Yeast Saccharomyces Cerevisiae

scholarly journals A brief history of TOR

2011 ◽  
Vol 39 (2) ◽  
pp. 437-442 ◽  
Author(s):  
Robbie Loewith
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Antifungal Agent ◽  
Budding Yeast ◽  
Intermediary Metabolism ◽  
Molecular Pathways ◽  
Central Importance ◽  
Yeast Saccharomyces Cerevisiae ◽  
Initial Characterization ◽  
History Of

The TOR (target of rapamycin) serine/threonine kinases are fascinating in that they influence many different aspects of eukaryote physiology including processes often dysregulated in disease. Beginning with the initial characterization of rapamycin as an antifungal agent, studies with yeast have contributed greatly to our understanding of the molecular pathways in which TORs operate. Recently, building on advances in quantitative MS, the rapamycin-dependent phosphoproteome in the budding yeast Saccharomyces cerevisiae was elucidated. These studies emphasize the central importance of TOR and highlight its many previously unrecognized functions. One of these, the regulation of intermediary metabolism, is discussed.

scholarly journals Cloning and characterization of DST2, the gene for DNA strand transfer protein beta from Saccharomyces cerevisiae.

1991 ◽  
Vol 11 (5) ◽  
pp. 2583-2592 ◽  
Author(s):  
C C Dykstra ◽  
K Kitada ◽  
A B Clark ◽  
R K Hamatake ◽  
A Sugino
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Intragenic Recombination ◽  
Temperature Sensitive ◽  
Strand Transfer ◽  
Gene Encoding ◽  
Yeast Saccharomyces Cerevisiae ◽  
Prolonged Incubation ◽  
Transfer Protein ◽  
Dna Strand

The gene encoding the 180-kDa DNA strand transfer protein beta from the yeast Saccharomyces cerevisiae was identified and sequenced. This gene, DST2 (DNA strand transferase 2), was located on chromosome VII. dst2 gene disruption mutants exhibited temperature-sensitive sporulation and a 50% longer generation time during vegetative growth than did the wild type. Spontaneous mitotic recombination in the mutants was reduced severalfold for both intrachromosomal recombination and intragenic gene conversion. The mutants also had reduced levels of the intragenic recombination that is induced during meiosis. Meiotic recombinants were, however, somewhat unstable in the mutants, with a decrease in recombinants and survival upon prolonged incubation in sporulation media. spo13 or spo13 rad50 mutations did not relieve the sporulation defect of dst2 mutations. A dst1 dst2 double mutant has the same phenotype as a dst2 single mutant. All phenotypes associated with the dst2 mutations could be complemented by a plasmid containing DST2.

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