We have cloned a new gene, SCP160, from Saccharomyces cerevisiae, the deduced amino acid sequence of which does not exhibit overall similarity to any known yeast protein. A weak resemblance between the C-terminal part of the Scp160 protein and regulatory subunits of cAMP-dependent protein kinases from eukaryotes as well as the pstB protein of Escherichia coli was observed. The SCP160 gene resides on the left arm of chromosome X and codes for a polypeptide of molecular weight around 160 kDa. By immunofluorescence microscopy the Scp160 protein appears to be localized to the nuclear envelope and to the endoplasmic reticulum (ER). However, no signal sequence or membrane-spanning region exists, suggesting that the Scp160 protein is attached to the cytoplasmic surface of the ER-nuclear envelope membranes. Disruption of the SCP160 gene is not lethal but results in cells of decreased viability, abnormal morphology and increased DNA content. This phenotype is not reversible by transformation with a plasmid carrying the wild-type gene. Crosses of SCP160 deletion mutant strains among each other or with unrelated strains lead to irregular segregation of genetic markers. Taken together the data suggest that the Scp160 protein is required during cell division for faithful partitioning of the ER-nuclear envelope membranes which in S. cerevisiae enclose the duplicated chromosomes.

Cells may use multiple pathways to commit suicide. In certain contexts, dying cells generate large amounts of autophagic vacuoles and clear large proportions of their cytoplasm, before they finally die, as exemplified by the treatment of human mammary carcinoma cells with the anti-estrogen tamoxifen (TAM, <= 1 mu M). Protein analysis during autophagic cell death revealed distinct proteins of the nuclear fraction including CST-pi and some proteasomal subunit constituents to be affected during autophagic cell death. Depending on the functional status of caspase-3, MCF-7 cells may switch between autophagic and apoptotic features of cell death [Fazi, B., Bursch, W., Fimia, G.M., Nardacci R., Piacentini, M., Di Sano, F., Piredda. L., 2008. Fenretinide induces autophagic cell death in caspase-defective breast cancer cells. Autophagy 4(4), 435-441]. Furthermore, the self-destruction of MCF-7 cells was found to be completed by phagocytosis of cell residues [Petrovski, G., Zahuczky, G., Katona, K., Vereb, G., Martinet. W., Nemes, Z., Bursch, W., Fesus, L., 2007. Clearance of dying autophagic cells of different origin by professional and non-professional phagocytes. Cell Death Diff. 14 (6),1117-1128]. Autophagy also constitutes a cell's strategy of defense upon cell damage by eliminating damaged bulk proteins/organelles. This biological condition may be exemplified by the treatment of MCF-7 cells with a necrogenic TAM-dose (10 mu M), resulting in the lysis of almost all cells within 24 h. However, a transient (1 h) challenge of MCF-7 cells with the same dose allowed the recovery of cells involving autophagy. Enrichment of chaperones in the insoluble cytoplasmic protein fraction indicated the formation of aggresomes, a potential trigger for autophagy. In a further experimental model HL60 cells were treated with TAM, causing dose-dependent distinct responses: 1-5 mu M TAM, autophagy predominant; 7-9 mu M, apoptosis predominant; 15 mu M, necrosis. These phenomena might be attributed to the degree of cell damage caused by tamoxifen, either by generating ROS, increasing membrane fluidity or forming DNA-adducts. Finally, autophagy constitutes a cell's major adaptive (survival) strategy in response to metabolic challenges such as glucose or amino acid deprivation, or starvation in general. Notably, the role of autophagy appears not to be restricted to nutrient recycling in order to maintain energy supply of cells and to adapt cell(organ) size to given physiological needs. For instance, using a newly established hepatoma cell line HCC-1.2, amino acid and glucose deprivation revealed a pro-apoptotic activity, additive to TGF-beta 1.The proapoptotic action of glucose deprivation was antagonized by 2-deoxyglucose, possibly by stabilizing the mitochondrial membrane involving the action of hexokinase II. These observations suggest that signaling cascades steering autophagy appear to provide links to those regulating cell number. Taken together, our data exemplify that a given cell may flexibly respond to type and degree of (micro)envir...

Actin filaments provide the internal scaffold of eukaryotic cells; they are involved in maintenance of cell shape, cytokinesis, organelle movement, and cell motility. The major component of these filaments, actin, is one of the most well-conserved eukaryotic proteins. Recently genes more distantly related to the conventional actins were cloned from several organisms. In the budding yeast, Saccharomyces cerevisiae, one conventional actin gene, ACT) (coding for the filament actin), and a so-caled actin-like gene, ACT2 (of unknown function), have so far been identified. We report here the discovery of a third member of the actin gene family from this organism, which we named ACT3. The latter gene is essential for viability and codes for a putative polypeptide, Act3, of 489 amino acids (Mr = 54,831). The deduced amino acid sequence of Act3 is less related to conventional actins than is the deduced amino acid sequence of Act2, mainly because of three unique hydrophobic segments. These segments are found inserted into a part of the sequence corresponding to a surface loop of the known three-dimensional structure of the actin molecule. According to sequence comparison, the basal core structure of conventional actin may well be conserved in Act3.Our findings demonstrate that, unexpectedly, there exist three members of the diverse actin protein family in buddin yeast that obviously provide different essential functions for survival.Actin, one of the most highly conserved eukaryotic proteins, is the major component of thin filaments, which provide the internal scaffold of eukaryotic cells. The actin filaments participate in vital cellular functions like cytokinesis, maintenance of cell shape, cell locomotion, and organelle transport (1, 2). Performance and regulation of these actin functions involve a number of actin-binding proteins (3-6). Actin proteins isolated from a broad range of phyla are about 90% identical to each other (7). Recently, however, the isolation of three genes coding for proteins more distantly related to "conventional" actins was reported: Saccharomyces cerevisiae ACT2 (8), Schizosaccharomyces pombe act2 (9), and a gene coding for vertebrate centractin/actin-RPV (10, 11) are, respectively, 47%, 35-40%, and 54% identical to conventional actins (reviewed in ref. 7). Furthermore it was shown that the functionally diverse protein molecules, the actins, the hsp70 heat shock proteins, and the sugar kinases, all of which bind and hydrolyze ATP, as well as several prokaryotic cell cycle proteins, have similar three-dimensional structures (12, 13), thus forming a large superfamily to which two phosphatases were recently added (14).Here we report the detection of a previously unknown essential gene from S. cerevisiae that codes for another actin-related protein and that we, therefore, call ACT3.1 It represents the most distantly related member of the actin family, indicating that this protein family is more divergent than previously thought. Furthermore, we show that, most probably, the actin-related p...

We purified Saccharomyces cerevisiae RNase H(70) to homogeneity, using an optimized chromatographic purification procedure. Renaturation gel assay assigned RNase H activity to a 70 kDa polypeptide. Sequencing of tryptic peptides identified the open reading frame YGR276c on chromosome VII of the S. cerevisiae genome as the corresponding gene, which encodes a putative polypeptide of molecular mass of 62849. We therefore renamed this gene RNH70. Immunofluorescence microscopy using a RNH70-EGFP fusion construct indicates nuclear localization of RNase H(70). Deletion of RNH70 from the yeast genome did not result in any serious phenotype under the conditions tested. Homology searches revealed striking similarity with a number of eukaryotic proteins and open reading frames, among them the chimpanzee GOR protein, a homolog of a human autoimmune antigen, found to elicit autoimmune response in patients infected with hepatitis C virus.

Pedigree analyses of individual yeast cells recovering from DNA damage were performed and time intervals between morphological landmark events during the cell cycle (bud emergence and cell separation), were recorded for three generations. The associated nuclear behavior was monitored with the aid of DAPI staining. The following observations were made: All agents tested (X-rays, MMS, EMS, MNNG, nitrous acid) delayed the first bud emergence after treatment, which indicates inhibition of the initiation of DNA replication. Cells that survived X-irradiation progressed further through the cell cycle in a similar way to control cells. Progress of chemically treated cells became extremely asynchronous because surviving cells stayed undivided for periods of varying length. Prolongation of the time between bud emergence and cell separation was most pronounced for cells treated with the alkylating agents MMS and EMS. This is interpreted as retardation of ongoing DNA synthesis by persisting DNA adducts. Cell cycle prolongation in the second and third generation after treatment was observed only with MMS treated cells. In all experiments, individual cells of uniformly treated populations exhibited highly variable responses.