Turnover of canavanine-containing proteins inSaccharomyces cerevisiae

Saccharomyces cerevisiae grown for 2 h in the presence of 0.5 mmol/L canavanine in a synthetic medium with ethanol as the sole carbon source (OEC) exhibited a slowing down of protein synthesis for 3–4 h after a shift to fresh ethanolbased medium containing 1.0 mmol/L arginine (OEA) in comparison with untreated cells grown on OEA. The change of carbon source from ethanol to glucose (OGA) after growth in the OEC medium resulted in an even deeper decline of protein synthesis. The degradation of canavanine-containing proteins in cells pregrown and labelled in an OEC medium after transfer to OEA was more rapid than in the OGA medium. The initial rate of protein degradation during the first hour in the OGA medium was less than 1%/h whereas in the OEA medium it reached almost 10%/h. The fraction of proteins with high turnover (half-life 0.46 h) constituted 8.3% on OEA, while during subsequent growth on OGA it was only 0.75% with a half-life of 0.12 h.     

Apparent half-lives of sugar transport proteins inSaccharomyces cerevisiae

Using incubation in the presence of 0.4 mM cycloheximide the half-lives of the principal membrane transport proteins in baker's yeast were found to be: more than 24 h for the constitutive glucose carrier, 2.2 h for the inducible galactose carrier, 1.2 h for the inducible maltose carrier and 0.8 h for the inducible alpha-methyl-D-glucoside carrier. The distinct nature of the two last-named carriers was thus supported. De-induction of the galactose carrier was enhanced in the presence of glucose plus cycloheximide but not of either substance alone. Chloramphenicol suppressed all effects of cycloheximide. In contrast to the enzymes of galactose metabolism, the induction of the glactose carrier was not under the control of a mitochondrial factor and took place in a rho-mutant. The system induced by maltose but not the one induced by alpha-methyl-D-glucoside was de-induced rapidly by the intervention of a cytoplasm-synthesized protein.     

NMCP/LINC proteins: Putative lamin analogs in plants?

Lamins are the main components of the metazoan lamina, and while the organization of the nuclear lamina of metazoans and plants is similar, there are apparently no genes encoding lamins or most lamin-binding proteins in plants. Thus, the plant lamina is not lamin-based and the proteins that form this structure are still to be characterized. Members of the plant NMCP/LINC/CRWN protein family share the typical tripartite structure of lamins, although the 2 exhibit no sequence similarity. However, given the many similarities between NMCP/LINC/CRWN proteins and lamins (structural organization, position of conserved regions, sub-nuclear distribution, solubility, and pattern of expression), these proteins are good candidates to carry out the functions of lamins in plants. Moreover, functional analysis of NMCP/LINC mutants has revealed their involvement in maintaining nuclear size and shape, another activity fulfilled by lamins. This review summarizes the current understanding of NMCP/LINC proteins and discusses future studies that will be required to demonstrate definitively that these proteins are plant analogs of lamins.     

The production of anti-hexapeptide antibodies which recognize the S7, L6 and L13 ribosomal proteins of Escherichia coli.

Here we report the synthesis of the N-terminal hexapeptide H-Pro-Arg-Arg-Arg-Val-Ile-OH of the E. coli ribosomal protein S7. the C-terminal hexapeptide H-Lys-Glu-Ala-Lys-Lys-Lys-OH of L6 and the C-terminal hexapeptide H-Pro-Gln-Val-Leu-Asp-Ile-OH of L13. All peptides were prepared by SPPS following the Fmoc-strategy, using DIC/HOBt and/or HBTU as coupling reagents and 2-chlorotrityl chloride resin as the solid support. The carrier linked synthetic peptides were injected into rabbits and elicited an anti-peptide response. These anti-hexapeptide antibodies were found to recognize the corresponding peptides and proteins.     

Effects of growth conditions on mitochondrial morphology inSaccharomyces cerevisiae

Effects of growth conditions on mitochondrial morphology were studied in livingSaccharomyces cerevisiae cells by vital staining with the fluorescent dye dimethyl-aminostyryl-methylpyridinium iodine (DASPMI), fluorescence microscopy, and confocal-scanning laser microscopy. Cells from respiratory, ethanol-grown batch cultures contained a large number of small mitochondria. Conversely, cells from glucose-grown batch cultures, in which metabolism was respiro-fermentative, contained small numbers of large, branched mitochondria. These changes did not significantly affect the fraction of the cellular volume occupied by the mitochondria. Similar differences in mitochondrial morphology were observed in glucose-limited chemostat cultures. In aerobic chemostat cultures, glucose metabolism was strictly respiratory and cells contained a large number of small mitochondria. Anaerobic, fermentative chemostat cultivation resulted in the large, branched mitochondrial structures also seen in glucose-grown batch cultures. Upon aeration of a previously anaerobic chemostat culture, the maximum respiratory capacity increased from 10 to 70 µmole.min^–1.g weight^–1 within 10 h. This transition resulted in drastic changes of mitochondrial number, morphology and, consequently, mitochondrial surface area. These changes continued for several hours after the respiratory capacity had reached its maximum. Cyanide-insensitive oxygen consumption contributed ca. 50% of the total respiratory capacity in anaerobic cultures, but was virtually absent in aerobic cultures. The response of aerobic cultures to oxygen deprivation was qualitatively the reverse of the response of anaerobic cultures to aeration. The results indicate that mitochondrial morphology inS. cerevisiae is closely linked to the metabolic activity of this yeast: conditions that result in repression of respiratory enzymes generally lead to the mitochondrial morphology observed in anaerobically grown, fermenting cells.     

The RAS-adenylate cyclase pathway and cell cycle control inSaccharomyces cerevisiae

The cell cycle ofSaccharomyces cerevisiae contains a decision point in G₁ called start, which is composed of two specific sites. Nutrient-starved cells arrest at the first site while pheromone-treated cells arrest at the second site. Functioning of the RAS-adenylate cyclase pathway is required for progression over the nutrient-starvation site while overactivation of the pathway renders the cells unable to arrest at this site. However, progression of cycling cells over the nutrient-starvation site does not appear to be triggered by the RAS-adenylate cyclase pathway in response to a specific stimulus, such as an exogenous nutrient. The essential function of the pathway appears to be limited to provision of a basal level of cAMP. cAMP-dependent protein kinase rather than cAMP might be the universal integrator of nutrient availability in yeast. On the other hand stimulation of the pathway in glucose-derepressed yeast cells by rapidly-fermented sugars, such as glucose, is well documented and might play a role in the control of the transition from gluconeogenic growth to fermentative growth. The initial trigger of this signalling pathway is proposed to reside in a glucose sensing complex which has both a function in controlling the influx of glucose into the cell and in activating in addition to the RAS-adenylate cyclase pathway all other glucose-induced regulatory pathways in yeast. Two crucial problems remaining to be solved with respect to cell cycle control are the nature of the connection between the RAS-adenylate cyclase pathway and nitrogen-source induced progression over the nutrient-starvation site of start and second the nature of the downstream processes linking the RAS-adenylate cyclase pathway to Cyclin/CDC28 controlled progression over the pheromone site of start.Abbreviations cAMP-PK cAMP-dependent protein kinase     

Immunological relationships between Artemia RNA polymerases and between RNA polymerases II from different eukaryotic organisms

Summary Rabbit antibodies against Artemia RNA polymerase II have been raised and utilized to study the immunological relationships between the subunits from RNA polymerases I, II and III from this organism and RNA polymerase II from other eukaryotes. We describe here for the first time the subunit structure of Artemia RNA polymerases I and III. These enzymes have 9 and 13 subunits respectively. The anti-RNA polymerase II antibodies recognize two subunits of 19.4 and 18 kDa common to the three enzymes, and another subunit of 25.6 kDa common to RNA polymerases II and III. The antibodies against Artemia RNA polymerase II also react with the subunits of high molecular weight and with subunits of around 25 and 33 kDa of RNA polymerase II from other eukaryotes (Drosophila melanogaster, Chironomus thummi, triticum (wheat) and Rattus (rat)). This interspecies relatedness is a common feature of eukaryotic RNA polymerases.Abbreviations RNAp RNA polymerase - DPT diazophenylthioether - SDS sodium dodecylsulfate     

Expression of nutritionally well-balanced protein, AmA1, inSaccharomyces cerevisiae

Food yeast.Saccharomyces cerevisiae, is a safe organism with a long history of use for the production of biomass rich in high quality proteins and vitamins. AmA1, a seed storage albumin fromAmaranthus hypochondriacus, has a well-balanced amino acid composition and high levels of essential amino acids and offers the possibility of further improving food and animal feed additives. In order to find an effective means of expressingAmA1 in yeast, the gene was cloned into an episomal shuttle vector. Four different promoters were tested: the glyceraldehyde-3-phosphate dehydrogenase promoter, galactose dehydrogenase 10 promoter, alcohol dehydrogenase II promoter, and a hybrid ADH2-GPD promoter. The recombinantAmA1 genes were then introduced into the yeastSaccharomyces cerevisiae 2805. Northern and Western blot analyses of the yeast under appropriate conditions revealed thatAmA1 was expressed by all four promoters at varying levels. An enzyme-linked immunosorbent assay demonstrated that the amount of AmA1 protein in the recombinant yeast was 1.3–4.3% of the total soluble proteins. The highest expression level was obtained from the hybrid ADH2-GPD promoter.     

A method for rapid mapping of mutations by plasmid rescue strategy inSaccharomyces cerevisiae

The products ofPRP17 andPRP18 genes are required for the second step of pre-mRNA splicing reactions inSaccharomyces cerevisiae. Temperature-sensitive mutants at either of these loci accumulate products of the first splicing reaction at nonpermissive temperature. To characterize functional regions in these proteins the mutations in three temperature-sensitive alleles ofPRP17 and two temperature-sensitive alleles ofPRP18 were mapped by the plasmid rescue strategy, One of the procedures adopted in the past is plasmid rescue of the mutant allele followed by sequencing of the entire gene. In this work we describe an adaptation of the above procedure that allows, first, rapid mapping of chromosomal segments bearing the mutations, followed by sequence characterization of the minimal segment. The strategy adopted was to integrate a wild-type copy of the gene at the homologous mutant chromosomal locus, followed by recovery of the chromosomal fragments from these integrants as plasmids inE. coli. The recovered plasmids were screened by a complementation assay for those that contained in them the chromosomal mutation. The mutations in all the three alleles ofPRP17 map to a small region in the N-terminal half of the protein, whereas the temperature-sensitive mutations in the two alleles ofPRP18 map to different regions of the PRP18 protein. The recovered mutant plasmids from all five alleles at the two loci were sequenced and the nucleotide changes were found to result in missense mutations in each case. Our strategy is therefore a rapid method to map chromosomal mutations and is of general use in structure-function analysis of cloned genes.     

Saccharomyces cerevisiae Dma proteins participate in cytokinesis by controlling two different pathways

Cytokinesis completion in the budding yeast S. cerevisiae is driven by tightly regulated pathways, leading to actomyosin ring contraction coupled to plasma membrane constriction and to centripetal growth of the primary septum, respectively. These pathways can partially substitute for each other, but their concomitant inactivation leads to cytokinesis block and cell death. Here we show that both the lack of the functionally redundant FHA-RING ubiquitin ligases Dma1 and Dma2 and moderate Dma2 overproduction affect actomyosin ring contraction as well as primary septum deposition, although they do not apparently alter cell cycle progression of otherwise wild-type cells. In addition, overproduction of Dma2 impairs the interaction between Tem1 and Iqg1, which is thought to be required for AMR contraction, and causes asymmetric primary septum deposition as well as mislocalization of the Cyk3-positive regulator of this process. In agreement with these multiple inhibitory effects, a Dma2 excess that does not cause any apparent defect in wild-type cells leads to lethal cytokinesis block in cells lacking the Hof1 protein, which is essential for primary septum formation in the absence of Cyk3. Altogether, these findings suggest that the Dma proteins act as negative regulators of cytokinesis.     

Genome-wide identification of genes involved in tolerance to various environmental stresses inSaccharomyces cerevisiae

During fermentation, yeast cells are exposed to a number of stresses — such as high alcohol concentration, high osmotic pressure, and temperature fluctuation — so some overlap of mechanisms involved in the response to these stresses has been suggested. To identify the genes required for tolerance to alcohol (ethanol, methanol, and 1-propanol), heat, osmotic stress, and oxidative stress, we performed genome-wide screening by using 4828 yeast deletion mutants. Our screens identified 95, 54, 125, 178, 42, and 30 deletion mutants sensitive to ethanol, methanol, 1-propanol, heat, NaCl, and H₂O₂, respectively. These deleted genes were then classified based on their cellular functions, and cross-sensitivities between stresses were determined. A large number of genes involved in vacuolar H⁺-ATPase (V-ATPase) function, cytoskeleton biogenesis, and cell wall integrity, were required for tolerance to alcohol, suggesting their protective role against alcohol stress. Our results revealed a partial overlap between genes required for alcohol tolerance and those required for thermotolerance. Genes involved in cell wall integrity and the actin cytoskeleton are required for both alcohol tolerance and thermotolerance, whereas the RNA polymerase II mediator complex seems to be specific to heat tolerance. However, no significant overlap of genes required for osmotic stress and oxidative stress with those required for other stresses was observed. Interestingly, although mitochondrial function is likely involved in tolerance to several stresses, it was found to be less important for thermotolerance. The genes identified in this study should be helpful for future research into the molecular mechanisms of stress response.     

Crosslinking of DNA to nuclear lamina proteins by UV irradiation in vivo

We have been able to demonstrate that a fraction of DNA becomes crosslinked to nuclear lamina shells isolated from Ehrlich ascites tumour cells irradiated with UV light. Terminal labeling of short DNA fragments covalently attached to proteins reveals that DNA has become crosslinked to all three lamins and to a protein comigrating with vimentin.     

Cell cycle-related proteins: a flow cytofluorometric study in human tumors

We used 2-parameter flow cytometry (FCM) to investigate the relationship between the cell cycle phases and 3 proteins whose expression is known to increase in proliferating cells: the surface antigen transferrin receptor (Trf-r), the "cyclin" (a proliferating cell nuclear antigen, PCNA), and the nuclear antigen recognized by the monoclonal antibody (MoAb) Ki-67. FITC-labeled antibodies against Trf-r, PCNA, and the Ki-67-reactive antigen, as well as propidium iodide-DNA distribution, were simultaneously measured on human leukemia HL-60 and K562, and breast carcinoma MCF-7 cell lines and on fresh human leukemic and glioblastoma cells. The 70% ethanol fixation for Trf-r and PCNA and the 95% acetone fixation for Ki-67 plus permeabilization (with 0.1% and 1% Triton X100, respectively, for the surface and the nuclear antigens) produced cell suspensions with negligible cell clumping, high-quality DNA profiles, and bright specific immunofluorescent staining. The investigated proteins have different relationships with the proliferative state of the cell. Trf-r is expressed mainly at the transition from G0/G1 to S-phase. PCNA expression is prominent in late G1 and through S-phase and decreases in G2-M. The Ki-67-reactive antigen is widely distributed in G1, S, and G2-M phases. Knowledge regarding the relationships between proliferation-associated antigens and cell cycle phase in normal and neoplastic cells could improve our understanding of the mechanisms underlying growth regulation and neoplastic transformation. Bivariate FCM is an easy method for obtaining these data from large numbers of cells.     

High mobility group-like proteins of the insect Plodia interpunctella

Nuclei from Plodia interpunctella larvae contain four major proteins, which are extracted by 5% perchloric acid and 0.35 M NaCl. The proteins have been designated PL1, PL2, PL3, and PL4. The amino acid analyses of these proteins show that they have high proportions of acidic and basic amino acid residues, a property characteristic of the high mobility group (HMG) proteins isolated from vertebrate tissues. Immunological characterization of these proteins clearly shows that PL1, PL2, and PL4 are more closely related to HMG1 dipteran proteins, while PL3 is more closely related to HMGI dipteran proteins. The possible relatedness of these proteins to HMG proteins is discussed.     

Immunological characterization of rat cardiac gap junctions: Presence of common antigenic determinants in heart of other vertebrate species and in various organs

Summary Antibodies to the following synthetic peptide, SALGKLLDKVQAY, were purified by affinity chromatography and characterized by ELISA and immunoblotting. These antibodies, shown to be specific to the major protein constitutent of isolated rat heart junctions: connexin 43, cross-reacted with a homologous protein in immunoreplicas of whole heart fractions of trout, frog, chicken, guinea pig, mouse and rat, suggesting a phylogenic conservation of connexin 43 in vertebrates. By immunoblotting of whole organ fractions it was also demonstrated that these antibodies cross-reacted with major proteins ofM _r 32 and 22 kD in rat and mouse liver, ofM _r 41 kD in rat cerebellum, ofM _r 43 kD in uterus, stomach and kidney of rat, ofM _r 46 and 70 kD in rat lens, suggesting that these proteins share common or related epitopes with the synthetic peptide and connexin 43.     

Peroxisome fission in Hansenula polymorpha requires Mdv1 and Fis1, two proteins also involved in mitochondrial fission

We show that Mdv1 and Caf4, two components of the mitochondrial fission machinery in Saccharomyces cerevisiae , also function in peroxisome proliferation. Deletion of MDV1 , CAF4 or both, however, had only a minor effect on peroxisome numbers at peroxisome-inducing growth conditions, most likely related to the fact that Vps1 – and not Dnm1 – is the key player in peroxisome fission in this organism. In contrast, in Hansenula polymorpha , which has only a Dnm1-dependent peroxisome fission machinery, deletion of MDV1 led to a drastic reduction of peroxisome numbers. This phenotype was accompanied by a strong defect in mitochondrial fission. The MDV1 paralog CAF4 is absent in H. polymorpha . In wild-type H. polymorpha , cells Dnm1–mCherry and green fluorescent protein (GFP)–Mdv1 colocalize in spots that associate with both peroxisomes and mitochondria. Furthermore, Fis1 is essential to recruit Mdv1 to the peroxisomal and mitochondrial membrane. However, formation of GFP–Mdv1 spots – and related to this normal organelle fission – is strictly dependent on the presence of Dnm1. In dnm1 cells, GFP–Mdv1 is dispersed over the surface of peroxisomes and mitochondria. Also, in H. polymorpha mdv1 or fis1 cells, the number of Dnm1–GFP spots is strongly reduced. These spots still associate to organelles but are functionally inactive.     

Immunological evidence for the involvement of cell wall proteins in phosphate uptake in the yeast Saccharomyces cerevisiae

Immunological cross-reactivity between cell wall proteins obtained from two yeast genera (Candida tropicalis and Saccharomyces cerevisiae) is reported. Specific retention of two cell wall proteins from Saccharomyces cerevisiae by an immunoabsorbent column coupled with antibodies against phosphate binding protein 2 (PiBP2) from Candida tropicalis allowed to generate antibodies against the proteins from S. cerevisiae. These antibodies were effective in inhibiting phosphate uptake by S. cerevisiae cells. The proteins from S. cerevisiae displayed a phosphate binding activity which was inhibited in the presence of the forementioned antibodies. These results and the observation that the amount of these proteins in the shock fluid was dependent of the growth conditions (i.e., in the presence or in the absence of phosphate) support the idea that these proteins are involved in the high affinity phosphate transport system.Abbreviations Pi inorganic phosphate - PiBP2 phosphate binding protein 2 obtained from Candida tropicalis - Tris Tris(hydroxymethyl)-aminoethane - MES [2-(N-Morpholino)] ethanesulfonic acid - EDTA ethylene diamine tetraacetic acid, disoldium salt - PMSF phenylmethyl sulfonyl fluoride - SDS sodium dodecyl sulfate - SDS-PAGE sodium dodecyl sulfate polyacrylamide gel electrophoresis     

Import and export of nuclear proteins: Focus on the nucleocytoplasmic movements of two different species of mammalian estrogen receptor

There is a wealth of information regarding the import and export of nuclear proteins in general. Nevertheless, the available data that deals with the nucleocytoplasmic movement of steroid hormone receptors remains highly limited. Some research findings reported during the past five years have succeeded in identifying proteins related to the movement of estrogen receptor from the cytoplasm to the nucleus. What is striking in these findings is the facilitatory role of estradiol in the transport process. A similar conclusion has been drawn from the studies on the plasma membrane-to nucleus movement of the alternative form of estrogen receptor, the non-activated estrogen receptor (naER). The internalization of naER from the plasma membrane takes place only in the presence of estradiol. While the gene regulatory functions of ER appear to get terminated following its ubiquitinization within the nucleus, the naER, through its deglycosylated form, the nuclear estrogen receptor II (nER II) continues to remain functional even beyond its existence within the nucleus. Recent studies have indicated the possibility that the estrogen receptor that regulates the nucleo cytoplasmic transport of m RNP is the nERII. This appears to be the result of the interaction between nERII and three proteins belonging to a group of small nuclear ribonucleo proteins (snRNP). The interaction of nERII with two of this protein appears to activate the inherent Mg²⁺ ATPase activity of the complex, which leads to the exit of the RNP through the nuclear pore complex.