Kai-Uwe Fröhlich

Aged mother cells of Saccharomyces cerevisiae show markers of oxidative stress and apoptosis

Recently, we and others have shown that genetic and environmental changes that increase the load of yeast cells with reactive oxygen species (ROS) lead to a shortening of the life span of yeast mother cells. Deletions of yeast genes coding for the superoxide dismutases or the catalases, as well as changes in atmospheric oxygen concentration, considerably shortened the life span. The presence of the physiological antioxidant glutathione, on the other hand, increased the life span of yeast cells. Taken together, these results pointed to a role for oxygen in the yeast ageing process. Here, we show by staining with dihydrorhodamine that old yeast mother cells isolated by elutriation, but not young cells, contain ROS that are localized in the mitochondria. A relatively large proportion of the old mother cells shows phenotypic markers of yeast apoptosis, i.e. TUNEL (TdT‐mediated dUTP nick end labelling) and annexin V staining. Although it has been shown previously that apoptosis in yeast can be induced by a cdc48 allele, by expressing pro‐apoptotic human cDNAs or by stressing the cells with hydrogen peroxide, we are now showing a physiological role for apoptosis in unstressed but aged wild‐type yeast mother cells.

Apoptosis in yeast – a monocellular organism exhibits altruistic behaviour

Apoptosis is a highly regulated form of programmed cell death crucial for life and health in metazoan animals. Apoptosis is defined by a set of cytological alterations. The recent discovery of these markers in yeast indicates the presence of the basic mechanisms of apoptosis already in unicellular eukaryotes. Oxygen radicals regulate both mammalian and yeast apoptosis. We suggest that apoptosis originated in unicellular organisms as an altruistic response to severe oxidative damage. Later, cells developed mechanisms to purposely produce reactive oxygen species as a regulator of apoptosis. Yeast may become an important model to investigate the conserved steps of apoptosis.

Mammalian Bax triggers apoptotic changes in yeast

Apoptosis is co‐regulated by the conserved family of Bcl‐2‐related proteins, which includes both its agonists (Bax) and antagonists (Bcl‐XL). A mutant strain of the yeast Saccharomyces cerevisiae has been shown to express all morphological signs of apoptosis. Overexpression of Bax is lethal in S. cerevisiae, whereas simultaneous overexpression of Bcl‐XL rescues the cells. We report that overexpression of mammalian Bax in a S. cerevisiae wild type strain triggers morphological changes similar to those of apoptotic metazoan cells: the loss of asymmetric distribution of plasma membrane phosphatidylserine, plasma membrane blebbing, chromatin condensation and margination, and DNA fragmentation. Simultaneous overexpression of Bcl‐XL prevents these changes. We demonstrate that Bax triggers phenotypic alterations in yeast strongly resembling those it causes in metazoan apoptotic cells.

Metacaspases are caspases. Doubt no more

Caspases are cysteine proteases that cleave their substrates after an aspartate residue. Besides their multiple vital roles ranging from immune regulation to spermatogenesis, they are crucial in most cell death pathways, representing a sort of executing sword in the hands of apoptosis. In 2000, a psi-Blast in-silico approach led Uren et al. to identify two novel caspase-related families: metacaspases and paracaspases. Paracaspases are involved in the development of MALT lymphoma, but not in cell death execution, and are found both in eukaryotes owning caspases (animals), as well as in organisms lacking caspases. Metacaspases, on the other hand, are found only in eukaryotes that are devoid of caspases, for example, plants, protists and fungi. Similar to caspases, they contain a caspase-specific catalytic diad of histidine and cysteine, as well as a caspase-like secondary structure. Our lab was among the first to perform experiments on metacaspases showing that the sole metacaspase encoded by the genome of Saccharomyces cerevisiae (which we termed YCA1) is involved in oxidative stress-induced cell death. Overexpression of YCA1 caused a type of cell death that was accompanied by apoptotic markers, while deletion of YCA1 protected against apoptosis caused by reactive oxygen species or chronological aging. We thus had revealed that one metacaspase, YCA1, was involved in the same process as caspases, namely programmed cell death (PCD). Indeed, many groups subsequently unfolded the crucial contribution of metacaspases to cell death execution upon various stresses in yeast and other fungi, as well as in plants. Bozhkov, Zhivotovsky and colleagues could even demonstrate that the plant metacaspase mcII-Pa shapes the embryo of Norway spruce (Picea abies) during development, presumably through its implication in developmental cell death. Together with the unequivocal fact of a common evolutionary origin, these results made us conclude that, in functional terms, metacaspases behave like caspases, thus unchaining a stormy scientific debate. Driven by the fact that caspases occur in animals but metacaspases are present in all kingdoms except animals, many authors deduced that, although cell death is a universal and fundamental process, the implication of caspases in lethal processes is not necessarily conserved. The discovery that metacaspases have a different cleavage specificity than caspases – they hydrolyze proteins after arginine or lysine (basic residues), not after aspartate (an acidic residue) 7,9 – added further fuel to the discussion. In fact, the exclusion of metacaspases from the caspase family and their regrouping into a separate family in the CD clan of cysteine peptidases was demanded. In other words, it was implicated that metacaspases are not caspases. Nevertheless, we strongly felt that Yca1p, a protein that possesses sequence homology to human caspases, and the knockout of which rescues roughly 40% of all cell death scenarios tested in yeast, should retain the name ‘metacaspase’. Nomenclature reaches beyond particular points of divergence (such as localization, structural features or, as it is for metacaspases, the amino acid specificity) when it refers to functional groups. For instance, we use the expression ‘nucleic acids’ for DNA or RNA from prokaryotes that by definition lack nuclei. Similarly, we talk about ‘mitochondrial DNA’ even though this pool of DNA is located in the mitochondria, not in the nucleus. Admittedly, the question whether metacaspases are caspases (or not) can only be answered by enzymology rather than by the vague comparison of their implication in various lethal signaling pathways. Do caspases and metacaspases cleave similar death-related substrates? We reasoned that, although a major divergence of the amino acid specificity of caspases and metacaspases may have occurred during evolution, their target proteins should fall into similar functional groups if the role of caspases and metacaspases was conserved. Hence, do the degradomes of caspases and metacaspases overlap? In a recent paper published in Nature Cell Biology, Peter Bozhkov and colleagues identified the first metacaspase substrate, which they showed to be a functional substrate of mammalian caspase-3 as well. In their seminal study, they demonstrated that both caspases and metacaspases cleave the phylogenetically conserved protein TSN (Tudor staphylococcal nuclease). The authors revealed that TSN from P. abies (PaTSN) is cleaved by its metacaspase (mcII-Pa) at four different sites. This process was blocked either by adding a metacaspase inhibitor or by mutation of a catalytic cysteine. Importantly, PaTSN was shown to be a component of the degradome during both stress-induced and developmental PCD. During embryogenesis, metacaspase activity correlated with proteolysis of endogenous PaTSN in the different embryo stages (high in early and low in mature embryos). Consistently, knockdown of mcII-Pa via

Identification of the regions of porcine VCP preventing its function in Saccharomyces cerevisiae

Cdc48p is essential for homotypic endoplasmic reticular fusion in Saccharomyces cerevisiae. It is localized at the endoplasmic reticulum during most of the cell division cycle but concentrates in the nucleus at the G1/S-transition. Its mammalian homologue VCP alternates between the endoplasmic reticulum and the centrosome in dependence of the cell cycle. Though Cdc48p and porcine VCP show a high sequence conservation--almost 70% of their amino acid residues are identical the VCP gene fails to complement a disruption of CDC48. Complementation studies with CDC48 and VCP gene hybrids show that an exchange of the central Cdc48p domain for the central VCP domain prevents a complementation of a CDC48 disruption, although this is the best conserved region between the two proteins. Protein chimeras containing the N-terminal part of VCP only complement a disruption of CDC48 when expressed at high levels. The respective yeast strain shows a nucleus devoid of Cdc48p. In contrast to VCP, Cdc48p contains an almost perfect nuclear targeting sequence in this region. Exchange of the C-terminal Cdc48p domain for the C-terminus of VCP leads to normal viability of the cell, even at low expression levels.

Pentalenolactone-insensitive glyceraldehyde-3-phosphate dehydrogenase from Streptomyces arenae is closely related to GAPDH from thermostable eubacteria and plant chloroplasts

Streptomyces arenae produces the antibiotic pentalenolactone, a highly specific inhibitor of glyceraldehyde-3-phosphate dehydrogenase (GAPDH). During the phase of pentalenolactone production,S. arenae expresses a pentalenolactone-insensitive GAPDH isoform; otherwise, a pentalenolactone-sensitive form is expressed. The gene of the pentalenolactone-insensitive GAPDH was cloned and sequenced. Regulatory elements typical for genes encoding antibiotic resistance and production are localized upstream and downstream of the open reading frame. No expression of pentalenolactone-insensitive GAPDH was detected inStreptomyces lividans transformed with the gene. InEscherichia coli, the gene was expressed from an inducedlac promoter. Amino-terminal sequencing of the heterologously expressed GAPDH proved its identity with pentalenolactone-insensitive GAPDH fromS. arenae. Sequence comparisons with GAPDH from other organisms showed a close relationship to GAPDH of plant chloroplasts, of other gram-positive bacteria, and of thermophilic gram-negative bacteria. Pentalenolactone-insensitive GAPDH differs from all closely related GAPDHs only in a few residues, none of which are directly involved in catalysis or substrate binding. The total amino acid composition is more similar to GAPDH of thermophilic species than to that of mesophilic species. The purified enzyme was moderately thermotolerant, which could be a side effect of the structural changes causing pentalenolactone-resistance.