Cristina Mazzoni

THE SLT2(MPK1) MAP KINASE IS ACTIVATED DURING PERIODS OF POLARIZED CELL GROWTH IN YEAST

The SLT2(MPK1) mitogen‐activated protein kinase signal transduction pa thway has been implicated in several biological processes in Saccharomyces cerevisiae, including the regulation of cytoskeletal and cell wall structure, polarized cell growth, and response to nutrient availability, hypo‐osmotic shock and heat shock. We examined the conditions under which the SLT2 pathway is activated. We found that the SLT2 kinase is tyrosine phosphorylated and activated during periods in which yeast cells are undergoing polarized cell growth, namely during bud formation of vegetative cell division and during projection formation upon treatment with mating pheromone. BCK1(SLK1), a MEK kinase, is required for SLT2 activation in both of these situations. Upstream of BCK1(SLK1), we found that the STE20 kinase was required for SLT2 activation by mating pheromone, but was unnecessary for its activation during the vegetative cell cycle. Finally, SLT2 activation during vegetative growth was partially dependent on CDC28 in that the stimulation of SLT2 tyrosine phosphorylation was significantly reduced directly after a temperature shift in cdc28 ts mutants. Our data are consistent with a role for SLT2 in promoting polarized cell growth.

Caspase-dependent apoptosis in yeast

Damaging environment, certain intracellular defects or heterologous expression of pro-apoptotic genes induce death in yeast cells exhibiting typical markers of apoptosis. In mammals, apoptosis can be directed by the activation of groups of proteases, called caspases, that cleave specific substrates and trigger cell death. In addition, in plants, fungi, Dictyostelium and metazoa, paracaspases and metacaspases have been identified that share some homologies with caspases but showing different substrate specificity. In the yeast Saccharomyces cerevisiae, a gene (MCA1/YCA1) has been identified coding for a metacaspase involved in the induction of cell death. Metacaspases are not biochemical, but sequence and functional homologes of caspases, as deletion of them rescues entirely different death scenarios. In this review we will summarize the current knowledge in S. cerevisiae on apoptotic processes, induced by internal and external triggers, which are dependent on the metacaspase gene YCA1.

Yeast caspase 1 links messenger RNA stability to apoptosis in yeast.

During the past years, yeasts have been successfully established as models to study the mechanisms of apoptotic regulation. We recently showed that mutations in the LSM4 gene, which is involved in messenger RNA decapping, lead to increased mRNA stability and apoptosis in yeast. Here, we show that mitochondrial function and YCA1, which encodes a budding yeast metacaspase, are necessary for apoptosis triggered by stabilization of mRNAs. Deletion of YCA1 in yeast cells mutated in the LSM4 gene prevents mitochondrial fragmentation and rapid cell death during chronological ageing of the culture, diminishes reactive oxygen species accumulation and DNA breakage, and increases resistance to H2O2 and acetic acid. mRNA levels in lsm4 mutants deleted for YCA1 are still increased, positioning the Yca1 budding yeast caspase as a downstream executor of cell death induced by mRNA perturbations. In addition, we show that mitochondrial function is necessary for fast death during chronological ageing, as well as in LSM4 mutated and wild‐type cells.

Characterization of human VDAC isoforms: A peculiar function for VDAC3?

VDACs are a family of pore-forming proteins mainly located in the mitochondrial outer membrane. In mammals three isoforms exist. In this work we review the information available about them with the addition of new results. We have compared the human VDACs transformed in a yeast strain lacking the endogenous porin. VDAC1 and 2 are able to complement the lack of porin in mitochondrial respiration and modulation of ROS. VDAC3 has a limited ability to support the mitochondrial respiration and has no influence in the control of ROS production. The over-expression of VDAC isoforms in wild type yeast strain led to a dramatic sensitivity to oxidative stress, especially for VDAC3, and a shorter lifespan in respiratory conditions. Real-time PCR comparison of the isoforms indicated that in HeLa cells VDAC1 is 10 times more abundant than VDAC2 and 100 times than VDAC3. The over-expression of any single isoform caused a 10 times increase of the transcripts of VDAC2 and VDAC3, while VDAC1 is not changed by the over-expression of the other isoforms. Models of VDAC2 and VDAC3 isoform structure showed that they could be made of a 19-strand beta-barrel and an N-terminal sequence with variable features. In this work we show for the first time a functional characterization of VDAC3 in a cellular context.

Recombinant expression of Pleurotus ostreatus laccases in Kluyveromyces lactis and Saccharomyces cerevisiae

Heterologous expression of Pleurotus ostreatus POXC and POXA1b laccases in two yeasts, Kluyveromyces lactis and Saccharomyces cerevisiae, was performed. Both transformed hosts secreted recombinant active laccases, although K. lactis was much more effective than S. cerevisiae. rPOXA1b transformants always had higher secreted activity than rPOXC transformants did. The lower tendency of K. lactis with respect to S. cerevisiae to hyperglycosylate recombinant proteins was confirmed. Recombinant laccases from K. lactis were purified and characterised. Specific activities of native and recombinant POXA1b are similar. On the other hand, rPOXC specific activity is much lower than that of the native protein, perhaps due to incomplete or incorrect folding. Both recombinant laccase signal peptides were correctly cleaved, with rPOXA1b protein having two C-terminal amino acids removed. The availability of the established recombinant expression system provides better understanding of laccase structure–function relationships and allows the development of new oxidative catalysts through molecular evolution techniques.

New arylthioindoles and related bioisosteres at the sulfur bridging group. 4. Synthesis, tubulin polymerization, cell growth inhibition, and molecular modeling studies.

New arylthioindoles along with the corresponding ketone and methylene compounds were potent tubulin assembly inhibitors. As growth inhibitors of MCF-7 cells, sulfur derivatives were superior or sometimes equivalent to the ketones, while methylene derivatives were substantially less effective. Esters 24, 27-29, 36, 39, and 41 showed approximately 50% of inhibition on human HeLa and HCT116/chr3 cells at 0.5 microM, and these compounds inhibited the growth of HEK, M14, and U937 cells with IC(50)s in the 78-220 nM range. While murine macrophage J744.1 cell growth was significantly less affected (20% at higher concentrations), four other nontransformed cell lines remained sensitive to these esters. The effect of drug treatment on cell morphology was examined by time-lapse microscopy. In a protocol set up to evaluate toxicity on the Saccharomyces cerevisiae BY4741 wild type strain, compounds 24 and 54 strongly reduced cell growth, and 29, 36, and 39 also showed significant inhibition.

Ethanol-induced and glucose-insensitive alcohol dehydrogenase activity in the yeast Kluyveromyces lactis

The alcohol dehydrogenase (ADH) system in the yeast Kluyveromyces lactis is encoded by four ADH genes. In this paper we report evidence that at least three of these genes are transcribed and transcribed into protein. KIADH1 and KIADH2, which encode cytoplasmic activities, are preferentially expressed in glucose‐grown cells with respect to ethanol‐grown cells. KIADH4, which encodes one of the two activities localized within mitochondria, is induced at the transcriptional level in the presence of ethanol as is the ADH2 gene in Saccharomyces cerevlslae. However the regulation of the expression of the K. lactis gene is completely different from that of ADH2 and of other known ADH genes in that KIADH4 is insensitive to glucose repression and is not expressed on non‐fermentabie carbon sources other than ethanol. This km6 of regulation can be clearly observed in non‐fermenting strains, where the induction of KIADH4 is dependent on the addition of ethanol to the medium. On the contrary, in fermenting strains KIADH4 is always induced by ethanol or acetaldehyde produced endocellularly and this results in constitutive expression of the gene aiso in the presence of glucose. The mitochondrial localization of the activity encoded by KIADH4 and the peculiar regulation of this gene could be related to the fact that K. lactis is a petite negative yeast in which some mitochondrial functions seem to be essential for cell viability.

Swapping of the N-terminus of VDAC1 with VDAC3 restores full activity of the channel and confers anti-aging features to the cell

Voltage‐dependent anion‐selective channels (VDACs) are pore‐forming proteins allowing the permeability of the mitochondrial outer membrane. The VDAC3 isoform is the least abundant and least active in a complementation assay performed in a yeast strain devoid of porin‐1. We swapped the VDAC3 N‐terminal 20 amino acids with homologous sequences from the other isoforms. The substitution of the VDAC3 N‐terminus with the VDAC1 N‐terminus caused the chimaera to become more active than VDAC1. The VDAC2 N‐terminus improved VDAC3 activity, though to a lesser extent. The VDAC3 carrying the VDAC1 N‐terminus was able to complement the lack of the yeast porin in mitochondrial respiration and in modulation of reactive oxygen species (ROS). This chimaera increased life span, indicating a more efficient bioenergetic metabolism and/or a better protection from ROS.

Apoptosis and aging in mitochondrial morphology mutants ofS. cerevisiae

Cell viability during chronological aging and after apoptotic stimuli in some yeast mutants with altered mitochondrial morphology was followed; a function for the corresponding genes in the apoptotic process was assessed.MDM30 andDNM1, the genes encoding an F-box protein and the dynamin-related GTPase, respectively, are involved in triggering aging and apoptosis. In contrast,YME1, encoding a subunit of the mitochondrial inner membrane i-AAA proteinase complex, has a protective role in these processes.FIS1, the mitochondrial fission gene, might play a protective role after an apoptotic insult while it seems to promote cell death in aging cells.

HOW TO BRING ORPHAN GENES INTO FUNCTIONAL FAMILIES

In the framework of the B1 Consortium of the EUROFAN‐1 project, we set up a series of simple phenotypic tests that can be performed on a large number of strains at a time. This methodological approach was intended to help assign functions of putative genes coding for unknown proteins to several specific aspects of cell biology. The tests were chosen to study phenotypes which should be affected by numerous genes. In this report, we examined the sensitivity/resistance or the adaptation of the cell to physical or chemical stresses (thermotolerance, osmotolerance and ethanol sensitivity), the effects of the alteration of the level of protein phosphorylation (sensitivity or resistance to compounds affecting the activity of protein kinases or phosphatases) and the effects of compounds interfering with synthesis of nucleic acids or proteins. Deletions in 66 genes of unknown function have been tested in 21 different conditions. In many deletant strains, phenotypes were observed and, for the most promising candidates, tetrad analysis was performed in order to verify co‐segregation of the deletion marker with the phenotype. Copyright