Characterization of the S289A,D mutants of yeast cystathionine β-synthase

Cystathionine β-synthase (CBS) catalyzes the pyridoxal 5′-phosphate (PLP)-dependent condensation of l-serine and l-homocysteine to form l-cystathionine in the first step of the reverse transsulfuration pathway. Residue S289 of yeast CBS, predicted to form a hydrogen bond with the pyridine nitrogen of the PLP cofactor, was mutated to alanine and aspartate. The k_cat/K_m^l-Ser of the S289A mutant is reduced by a factor of ～ 800 and the β-replacement activity of the S289D mutant is undetectable. Fluorescence energy transfer between tryptophan residue(s) of the enzyme and the PLP cofactor, observed in the wild-type enzyme and diminished in the S289A mutant, is absent in S289D. These results demonstrate that residue S289 is essential in maintaining the properties and orientation of the pyridine ring of the PLP cofactor. The reduction in activity of ytCBS-S289A suggests that ytCBS catalyzes the α,β-elimination of l-Ser via an E1cB mechanism.     

“Uno,nessuno e centomila”: the different faces of the budding yeast kinetochore

“One, no one and one hundred thousand” is a masterpiece of Italian literature, written by Luigi Pirandello. The central theme is that in each individual there are multiple personalities, since one’s perception of one’s self differs from the view of others. As a consequence, a unique identity does not exist, but rather one hundred thousand. This concept can be very well applied to the kinetochore, one of the largest macromolecular complexes conserved in eukaryotes. The kinetochore is essential during cell division and fulfills different sophisticated functions, including linking chromosomes to spindle microtubules and delaying anaphase onset in case of incorrect bi-orientation. In order to perform these tasks, the kinetochore shapes its structure by recruiting different subunits, such as the components of the spindle assembly checkpoint (SAC) or the monopolin complex during meiosis. It also modifies its internal organization by rearranging intramolecular connections and acquiring a distinct identity at different time points of cell division. In this review, we describe recent insights into the changes in composition and configuration of the kinetochore in mitosis and meiosis, focusing on the kinetochore of Saccharomyces cerevisiae.     

β-glucanases of the yeast Pichia polymorpha

Fractionation of proteins secreted into the culture medium by intact cells and protoplasts of Pichia polymorpha showing enzyme activity against laminarin, pustulan or p-nitrophenyl--d-glucopyranoside has been performed, and the results compared with those obtained with cell-free extracts and lysed protoplasts. Fractionation with DEAE Sephadex A₅₀ has proved to be the best method, yielding at least three fractions which hydrolyse laminarin. One of these fractions was active on both laminarin and pustulan. Filtration on Sephadex G-100 column only yielded one active preparation. Evidence supporting the conclusion that there are three different -glucanases located in the periplasmic space is presented.     

Telomeric position effect: From the yeast paradigm to human pathologies?

Alteration of the epigenome is associated with a wide range of human diseases. Therefore, deciphering the pathways that regulate the epigenetic modulation of gene expression is a major milestone for the understanding of diverse biological mechanisms and subsequently human pathologies. Although often evoked, little is known on the implication of telomeric position effect, a silencing mechanism combining telomere architecture and classical heterochromatin features, in human cells. Nevertheless, this particular silencing mechanism has been investigated in different organisms and several ingredients are likely conserved during evolution. Subtelomeres are highly dynamic regions near the end of the chromosomes that are prone to recombination and may buffer or facilitate the spreading of silencing that emanates from the telomere. Therefore, the composition and integrity of these regions also concur to the propensity of telomeres to regulate the expression, replication and recombination of adjacent regions. Here we describe the similarities and disparities that exist among the different species at chromosome ends with regard to telomeric silencing regulation with a special accent on its implication in numerous human pathologies.     

Atg9 reservoirs,a new organelle of the yeast endomembrane system?

Despite all the advances in understanding the roles and the regulation of autophagy in health and disease realized during the past decade, the key question about the origin of the initial autophagosomal membranes remains largely unknown. Among the 16 autophagy-related (Atg) proteins composing the conserved machinery required for autophagy, Atg9 is the only integral membrane component and it is one of the first Atg proteins to be recruited to the phagophore assembly site (PAS) emphasizing its relevance in the early stages of autophagosome biogenesis. Because it is

intrinsically associated with lipid bilayers, Atg9 has all the prerequisites to be a major factor in regulating the supply of at least part of the membranes necessary for the formation and expansion of nascent autophagosomes.     

Atg9 reservoirs, a new organelle of the yeast endomembrane system?

Despite all the advances in understanding the roles and the regulation of autophagy in health and disease realized during the past decade, the key question about the origin of the initial autophagosomal membranes remains largely unknown. Among the 16 autophagy-related (Atg) proteins composing the conserved machinery required for autophagy, Atg9 is the only integral membrane component and it is one of the first Atg proteins to be recruited to the phagophore assembly site (PAS) emphasizing its relevance in the early stages of autophagosome biogenesis. Because it is: intrinsically associated with lipid bilayers, Atg9 has all the prerequisites to be a major factor in regulating the supply of at least part of the membranes necessary for the formation and expansion of nascent autophagosomes.     

The RHC21 gene of budding yeast, a homologue of the fission yeast rad21 +gene, is essential for chromosome segregation

The Saccharomyces cerevisiae gene RHC21 is a homologue of the fission yeast rad21 ⁺gene, which affects the sensitivity of cells to γ-irradiation and is essential for cell growth in S. pombe. Disruption of the RHC21 gene showed that it is also essential in S. cerevisiae. To examine its function in cell growth further, we have isolated temperature-sensitive mutants for the RHC21 gene and characterized one of them, termed rhc21-sk16. When this mutant was incubated at 36°?C, the percentage of large-budded cells was increased. Most of the large-budded cells had aberrant nuclear structures, such as unequally extended nuclear DNA with incompletely elongated spindles across the mother-daughter neck or only in a mother cell. Furthermore, a circular minichromosome is more unstable in the mutant than in the wild-type, even at 25°?C. Flow cytometry showed that the bulk of DNA replication takes place normally at the restrictive temperature in the mutant. These results indicated that the RHC21 gene is required for proper segregation of the chromosomes. In addition, we found that the mutant is sensitive not only to UV radiation and γ-rays but also to the antimicrotubule agent nocodazole at 25°?C. This suggests that the RHC21 gene is involved in the microtubule function. We discuss how the RHC21 gene product may be involved in chromosome segregation and microtubule function.     

Replication and recombination functions associated with the yeast plasmid, 2μ circle

By examining both the transformation efficiency of yeast of various plasmids containing defined regions of the 2μ circle genome and the characteristics of the resultant transformants, we have identified several regions of the 2μ circle genome which are involved in 2μ circle replication or recombination. First, by identifying those DNA fragments from the molecule which promote high frequency transformation of yeast, we have localized the origin of replication to a sequence partially within the large unique region, which, as determined by subsequent deletion analysis, extends from the middle of the inverted repeat region into the contiguous unique region. Second, by examining the relative efficiency of replication in yeast of hybrid plasmids containing either the entire 2μ circle genome or a fragment of 2μ circle encompassing the origin of replication, we have determined that efficient use of the 2μ circle origin requires some function or functions encoded in the molecule at a site away from the origin. Third, by examining the ability of a mutant 2μ circle molecule to undergo intramolecular recombination in yeast, we have identified a 2μ circle gene which codes for a product required for this process.     

Aegeline,a natural product from the plant Aegle marmelos, mimics the yeast SNARE protein Sec22p in suppressing α-synuclein and Bax toxicity in yeast

Herein, we have identified yeast Sec22p (ySec22p), a SNARE protein essential for endoplasmic reticulum to Golgi trafficking, as a suppressor of Bax-induced yeast apoptosis and corroborated published observations that ySec22p suppresses α-synuclein’s toxicity in yeast. It has been suggested that compounds which enhance expression, in neurons, of human homologues of ySec22p (Sec22Bp/Sec22p/Sec22A) would prevent synucleinopathies, such as Parkinson’s disease. With the aim of finding a small molecule that would mimic ySec22p, a library of natural products consisting of 394-compounds was screened using yeast cells that express either human α-synuclein or human Bax. The antioxidant aegeline, an alkaloid-amide occurring in the leaves of the plant Aegle marmelos Correa, was the only molecule that overcame apoptosis induced by both α-synuclein and Bax in yeast. Besides, aegeline also prevented growth block in cells expressing the more toxic A53T α-synuclein mutant. Restoration of cell growth occurred through inhibition of increased ROS levels, mitochondrial membrane potential loss and nuclear DNA fragmentation, characteristics of apoptosis manifested in α-synuclein or Bax-expressing cells. These results highlight the importance of yeast systems to identify rapidly molecules that may prevent the onset of apoptosis that occurs in Parkinson’s disease.     

Molecular diversity and intragenomic variability in the yeast genus Xanthophyllomyces: the origin of Phaffia rhodozyma?

The teleomorphic basidiomycetous yeast Xanthophyllomyces dendrorhous is important as a commercial source of astaxanthin, which is a component of feeds for mariculture. Phaffia rhodozyma is the anamorphic state of Xanthophyllomyces; however, there are conflicting reports in the literature concerning the presence of a sexual cycle in P. rhodozyma. The current study attempted to explain this enigma. Strains were obtained from the Phaff Yeast Culture Collection (University of California, Davis) and other sources in the northern hemisphere. Molecular sequences of three nuclear rDNA regions were examined: the internal transcribed spacers (ITS), intergenic spacer (IGS1) and the D1D2 region at the 5' end of the 26S gene. Different levels of genetic variability were observed in the three regions. The D1D2 differentiated major groups of strains, while an increased variability in the ITS suggested that the ITS region could be employed as an ecological marker. The greatest variability was in the IGS1 region, where strains can be defined by the presence and location of indels. Intragenomic sequence heterogeneity in the ITS and IGS1 regions led to the hypothesis that the type strain of P. rhodozyma (CBS 5905(T), UCD 67-210(T)) was derived as a mating-deficient basidiospore from the parent teleomorphic strain CBS 9090.