Regulation of phosphatidylglycerolphosphate synthase in Saccharomyces cerevisiae by factors affecting mitochondrial development.

Phosphatidylglycerolphosphate synthase (PGPS; CDP-diacylglycerol glycerol 3-phosphate 3-phosphatidyltransferase; EC 2.7.8.5) catalyzes the first step in the synthesis of cardiolipin, an acidic phospholipid found in the mitochondrial inner membrane. In the yeast Saccharomyces cerevisiae, PGPS expression is coordinately regulated with general phospholipid synthesis and is repressed when cells are grown in the presence of the phospholipid precursor inositol (M. L. Greenberg, S. Hubbell, and C. Lam, Mol. Cell. Biol. 8:4773-4779, 1988). In this study, we examined the regulation of PGPS in growth conditions affecting mitochondrial development (carbon source, growth stage, and oxygen availability) and in strains with genetic lesions affecting mitochondrial function. PGPS derepressed two- to threefold when cells were grown in a nonfermentable carbon source (glycerol-ethanol), and this derepression was independent of the presence of inositol. PGPS derepressed two- to fourfold as cells entered the stationary phase of growth. Stationary-phase derepression occurred in both glucose- and glycerol-ethanol-grown cells and was slightly greater in cells grown in the presence of inositol and choline. PGPS expression in mitochondria was not affected when cells were grown in the absence of oxygen. In mutants lacking mitochondrial DNA [( rho0] mutants), PGPS activity was 30 to 70% less than in isogenic [rho+] strains. PGPS activity in [rho0] strains was subject to inositol-mediated repression. PGPS activity in [rho0] cell extracts was derepressed twofold as the [rho0] cells entered the stationary phase of growth. No growth phase derepression was observed in mitochondrial extracts of the [rho0] cells. Relative cardiolipin content increased in glycerol-ethanol-grown cells but was not affected by growth stage or by growth in the presence of the phospholipid precursors inositol and choline. These results demonstrate that (i) PGPS expression is regulated by factors affecting mitochondrial development; (ii) regulation of PGPS by these factors is independent of cross-pathway control; and (iii) PGPS expression is never fully repressed, even during anaerobic growth.     

Nuclear DNA synthesis in vitro is mediated via stable replication forks assembled in a temporally specific fashion in vivo.

A cell-free nuclear replication system that is S-phase specific, that requires the activity of DNA polymerase alpha, and that is stimulated three- to eightfold by cytoplasmic factors from S-phase cells was used to examine the temporal specificity of chromosomal DNA synthesis in vitro. Temporal specificity of DNA synthesis in isolated nuclei was assessed directly by examining the replication of restriction fragments derived from the amplified 200-kilobase dihydrofolate reductase domain of methotrexate-resistant CHOC 400 cells as a function of the cell cycle. In nuclei prepared from cells collected at the G1/S boundary of the cell cycle, synthesis of amplified sequences commenced within the immediate dihydrofolate reductase origin region and elongation continued for 60 to 80 min. The order of synthesis of amplified restriction fragments in nuclei from early S-phase cells in vitro appeared to be indistinguishable from that in vivo. Nuclei prepared from CHOC 400 cells poised at later times in the S phase synthesized characteristic subsets of other amplified fragments. The specificity of fragment labeling patterns was stable to short-term storage at 4 degrees C. The occurrence of stimulatory factors in cytosol extracts was cell cycle dependent in that minimal stimulation was observed with early G1-phase extracts, whereas maximal stimulation was observed with cytosol extracts from S-phase cells. Chromosomal synthesis was not observed in nuclei from G1 cells, nor did cytosol extracts from S-phase cells induce chromosomal replication in G1 nuclei. In contrast to chromosomal DNA synthesis, mitochondrial DNA replication in vitro was not stimulated by cytoplasmic factors and occurred at equivalent rates throughout the G1 and S phases. These studies show that chromosomal DNA replication in isolated nuclei is mediated by stable replication forks that are assembled in a temporally specific fashion in vivo and indicate that the synthetic mechanisms observed in vitro accurately reflect those operative in vivo.     

Modulation of collagen synthesis by a growth factor and by the extracellular matrix: comparison of cellular response to two different stimuli

Cultured bovine corneal endothelial cells can be grown in three ways: on plastic, on plastic with fibroblast growth factor present in the media, and on their own preformed extracellular matrix. On plastic alone, cells grow in a disorderly fashion and secrete matrix on all cell surfaces. Cells grown on plastic with growth factor or on a matrix, at confluence, have matrix deposition only on the basal surface of the cells and an orderly contact-inhibited pattern of growth. This correlates with the polarity they demonstrate histologically. This cell-matrix pattern resembles the pattern observed in vivo. Both the soluble growth factor and the extracellular matrix are able to modulate the pattern of collagen synthesis and deposition by cells, but they do so in two entirely different ways. In cells grown on the extracellular matrix, total collagen synthesis is lower but more efficient. Collagen is deposited primarily into the cell layer even at the early sparse stage of culture. In cells grown on plastic with growth factor in the media, collagen is initially secreted into the media and does not become incorporated into the matrix. The deposition of collagen on the basal surface of cell occurs only late in the culture, and is achieved by increments in a stepwise manner. The in vivo-like pattern is not manifest until confluence has been reached. Thus, the extracellular matrix functions not only as a structural support, but is also instructional to the cells plated on it. In this case, the matrix regulates the level of collagen synthesis in the cells and modulates the pattern of collagen deposition. Soluble growth factors may act in part by enhancing a cell's ability to elaborate an appropriate matrix pattern necessary for the cell's own growth and accurate function.     

Specific changes in the synthesis of mitochondrial DNA in chick embryo fibroblasts transformed by Rous sarcoma viruses

In chick-embryo fibroblasts infected with the Schmidt-Ruppin strain of Rous sarcoma virus, subgroup A (wild type), or with a thermosensitive mutant of this virus, T5, the rates of mitochondrial DNA synthesis differ in cells that exhibit normal and malignant phenotypes. In wild type virus-infected cells grown at 36 or 41 degrees C, morphological transformation is expressed, the rate of 2-deoxy-D-[3H]glucose uptake is stimulated, and mitochondrial DNA synthesis in vivo is stimulated three- to fivefold over that in uninfected cells. In T5-infected cells these changes occur only at the permissive temperature (36 degrees C); a shift to the nonpermissive temperature (41 degrees C) causes the reversal of these effects, and the specific activity of purified mitochondrial DNA is characteristic of that from uninfected cells. In contrast, the specific activities of nuclear DNA purified from cells maximally transformed under the permissive conditions do not differ between wild type-infected and uninfected with the T5 virus. In parallel experiments with isolated mitochondria, the rate of mtDNA synthesis in vitro is again greater in mitochondria isolated from transformed cells. In addition, mitochondrial DNA synthesis in vitro in mitochondria from nontransformed and virus-transformed cells exhibits differential sensitivity to inhibition by mercaptoethanol. Furthermore, the ntDNAP polymerase activity in mitochondrial extracts prepared from cells with transformed phenotypes is about sevenfold higher than in extracts from cells with nontransformed phenotypes.     

Amino acids Thr56 and Thr58 are not essential for elongation factor 2 function in yeast

Yeast elongation factor 2 is an essential protein that contains two highly conserved threonine residues, T56 and T58, that could potentially be phosphorylated by the Rck2 kinase in response to environmental stress. The importance of residues T56 and T58 for elongation factor 2 function in yeast was studied using site directed mutagenesis and functional complementation. Mutations T56D, T56G, T56K, T56N and T56V resulted in nonfunctional elongation factor 2 whereas mutated factor carrying point mutations T56M, T56C, T56S, T58S and T58V was functional. Expression of mutants T56C, T56S and T58S was associated with reduced growth rate. The double mutants T56M/T58W and T56M/T58V were also functional but the latter mutant caused increased cell death and considerably reduced growth rate. The results suggest that the physiological role of T56 and T58 as phosphorylation targets is of little importance in yeast under standard growth conditions. Yeast cells expressing mutants T56C and T56S were less able to cope with environmental stress induced by increased growth temperatures. Similarly, cells expressing mutants T56M and T56M/T58W were less capable of adapting to increased osmolarity whereas cells expressing mutant T58V behaved normally. All mutants tested were retained their ability to bind to ribosomes in vivo. However, mutants T56D, T56G and T56K were under-represented on the ribosome, suggesting that these nonfunctional forms of elongation factor 2 were less capable of competing with wild-type elongation factor 2 in ribosome binding. The presence of nonfunctional but ribosome binding forms of elongation factor 2 did not affect the growth rate of yeast cells also expressing wild-type elongation factor 2.     

Cellular basis of hypocotyl growth in Arabidopsis thaliana.

The Arabidopsis thaliana hypocotyl is widely used to study the effects of light and plant growth factors on cell elongation. To provide a framework for the molecular-genetic analysis of cell elongation in this organ, here we describe, at the cellular level, its morphology and growth and identify a number of characteristic, developmental differences between light-grown and dark-grown hypocotyls. First, in the light epidermal cells show a characteristic differentiation that is not observed in the dark. Second, elongation growth of this organ does not involve significant cortical or epidermal cell divisions. However, endoreduplication occurs, as revealed by the presence of 4C and 8C nuclei. In addition, 16C nuclei were found specifically in dark-grown seedlings. Third, in the dark epidermal cells elongate along a steep, acropetal spatial and temporal gradient along the hypocotyl. In contrast, in the light all epidermal cells elongated continuously during the entire growth period. These morphological and physiological differences, in combination with previously reported genetic data (T. Desnos, V. Orbovic, C. Bellini, J. Kronenberger, M. Caboche, J. Traas, H. Höfte [1996] Development 122: 683-693), illustrate that light does not simply inhibit hypocotyl growth in a cell-autonomous fashion, but that the observed growth response to light is a part of an integrated developmental change throughout the elongating organ.     

Effects of Oxygen Tension and Glucose Repression on Mitochondrial Protein Synthesis in Continuous Cultures of Saccharomyces cerevisiae

Continuous cultures of Saccharomyces cerevisiae grown over a range of oxygen and glucose concentrations were used to determine the effects of these two physiological regulators on mitochondrial protein synthesis in vivo. Quantitative estimates were obtained of the contribution of the mitochondrial protein-synthesizing system to the formation of mitochondrial membranes in cells grown over a wide range of dissolved oxygen concentrations, under conditions of glucose limitation or glucose excess in the cultures. The nature of the products of the mitochondrial system formed under these conditions was examined by selectively labeling membrane proteins in vivo in the presence of cycloheximide, and fractionating the products by gel electrophoresis under dissociating conditions. These results have been correlated with changes in the lipid composition of the cells and with the synthesis and assembly of components of the mitochondrial adenosine 5'-triphosphatase complex.     

Similarities between fibroblast-derived growth factor and platelet-derived growth factor

Platelet-derived growth factor (PDGF), one of the most potent mitogens in serum for non-transformed cells, shares many biological and physical properties with fibroblast-derived growth factor (FDGF), a polypeptide produced by BHK cells transformed by SV40. Thus FDGF and PDGF have biological activity which is recoverable from sodium dodecyl sulphate (SDS) polyacrylamide gel electrophoresis, at positions indicating similar molecular weights. Further, the biological activity of both factors is heat-stable but sensitive to mercaptoethanol. FDGF and PDGF have similar abilities to induce DNA synthesis synergistically in the presence of either insulin, epidermal growth factor (EGF), vasopressin or colchicine. In contrast to other growth factors, (i) either FDGF or PDGF can induce DNA synthesis in the absence of other mitogens in 3T3 cells maintained in serum-free medium and (ii) a transient exposure of cultures to FDGF or PDGF causes a persistent stimulation of DNA synthesis. Either FDGF or PDGF enhances colony formation of non-transformed cells cultured in suspension in the presence of EGF and serum. FDGF is not PDGF adsorbed by SV40-BHK cells from serum, since SV40-BHK cells plated and grown in the absence of serum still produce FDGF. In view of the similarities between PDGF and FDGF, we suggest that they may belong to the same family of growth factors.     

The yeast counterparts of human 'MELAS' mutations cause mitochondrial dysfunction that can be rescued by overexpression of the mitochondrial translation factor EF-Tu

We have taken advantage of the similarity between human and yeast (Saccharomyces cerevisiae) mitochondrial tRNA^Leu(UUR), and of the possibility of transforming yeast mitochondria, to construct yeast mitochondrial mutations in the gene encoding tRNA^Leu(UUR) equivalent to the human A3243G, C3256T and T3291C mutations that have been found in patients with the neurodegenerative disease MELAS (for mitochondrial 'myopathy, encephalopathy, lactic acidosis and stroke-like episodes'). The resulting yeast cells (bearing the equivalent mutations A14G, C26T and T69C) were defective for growth on respiratory substrates, exhibited an abnormal mitochondrial morphology, and accumulated mitochondrial DNA deletions at a very high rate, a trait characteristic of severe mitochondrial defects in protein synthesis. This effect was specific at least in the pathogenic mutation T69C, because when we introduced A or G instead of C, the respiratory defect was absent or very mild. All defective phenotypes returned to normal when the mutant cells were transformed by multicopy plasmids carrying the gene encoding the mitochondrial elongation factor EF-Tu. The ability to create and analyse such mutated strains and to select correcting genes should make yeast a good model for the study of tRNAs and their interacting partners and a practical tool for the study of pathological mutations and of tRNA sequence polymorphisms.     

Identification of elongation factor g as the conserved cellular target of argyrin B

Argyrins, produced by myxobacteria and actinomycetes, are cyclic octapeptides with antibacterial and antitumor activity. Here, we identify elongation factor G (EF-G) as the cellular target of argyrin B in bacteria, via resistant mutant selection and whole genome sequencing, biophysical binding studies and crystallography. Argyrin B binds a novel allosteric pocket in EF-G, distinct from the known EF-G inhibitor antibiotic fusidic acid, revealing a new mode of protein synthesis inhibition. In eukaryotic cells, argyrin B was found to target mitochondrial elongation factor G1 (EF-G1), the closest homologue of bacterial EF-G. By blocking mitochondrial translation, argyrin B depletes electron transport components and inhibits the growth of yeast and tumor cells. Further supporting direct inhibition of EF-G1, expression of an argyrin B-binding deficient EF-G1 L693Q variant partially rescued argyrin B-sensitivity in tumor cells. In summary, we show that argyrin B is an antibacterial and cytotoxic agent that inhibits the evolutionarily conserved target EF-G, blocking protein synthesis in bacteria and mitochondrial translation in yeast and mammalian cells.     

Significance of the number of embryonic cells and the state of the zona pellucida for hatching of mouse blastocysts in vitro versus in vivo

We investigated the course of mouse blastocyst hatching in vitro after experimental modulation of the hatching process by growth hormone or by laser treatment and compared it to embryos grown in vivo. When embryos were grown in vitro, successful hatching was dependent on blastocyst expansion and was based on a minimum number of embryonic cells. Embryos grown in the presence of growth hormone were more advanced in their development and hatched earlier. When an artificial opening was laser-drilled into the zona pellucida, hatching occurred at lower numbers of embryonic cells. In vivo, escape from the zona pellucida occurred earlier and independent of blastocyst expansion. However, when we isolated in vivo-grown blastocysts with intact zonae that had developed in vivo and then cultured them in vitro, blastocysts started to expand and hatched the following day when a sufficiently high number of embryonic cells was present. Our data show that successful hatching in vitro is dependent on a sufficiently high number of embryonic cells, which enables blastocyst expansion and zona shedding. In vivo, the lower number of embryonic cells detected in zona-free blastocysts indicates that the underlying mechanism of zona escape is different, does not depend on blastocyst expansion, and presumably involves lytic factors from the uterus.     

Mitochondrial translational-initiation and elongation factors in Saccharomyces cerevisiae

C155 and E252 are respiratory-defective mutants of Saccharomyces cerevisiae, previously assigned to complementation groups G37 and G142, respectively. The following evidence suggested that both mutants were likely to have lesions in components of the mitochondrial translational machinery: C155 and E252 display a pleiotropic deficiency in cytochromes a, a3 and b; both strains are severly limited in their ability to incorporate radioactive methionine into the mitochondrial translation products and, in addition, display a tendency to loose wild-type mitochondrial DNA. This set of characteristics is commonly found in strains affected in mitochondrial protein synthesis. To identify the biochemical lesions, each mutant was transformed with a wild-type yeast genomic library and clones complemented for the respiratory defect were selected for growth on a non-fermentable substrate. Analysis of the cloned genes revealed that C155 has a mutation in a protein which has high sequence similarity to bacterial elongation factor G and that E252 has a mutation in a protein homologous to bacterial initiation factor 2. Disruption of the chromosomal copy of each gene in a wild-type haploid yeast induced a phenotype analogous to that of the original mutants, but does not affect cell viability. These results indicate that both gene products function exclusively in mitochondrial protein synthesis. Subcloning of the IFM1 gene, coding for the mitochondrial initiation factor, indicates that the amino-terminal 423 residues of the protein are sufficient to promote peptide-chain initiation in vivo.     

Aminolaevulinate synthetase of Micrococcus denitrificans. Purification and properties of the enzyme, and the effects of growth conditions on the enzyme activity in cells

1. 5-Aminolaevulinate synthetase was detected in extracts of the non-photosynthetic bacterium Micrococcus denitrificans. 2. Activity is high in cells grown anaerobically in a defined nitrate medium, but is low in cells grown in an iron-deficient medium, and in cells grown aerobically. 3. Aminolaevulinate synthetase was purified extensively; it has a molecular weight of about 68000; apparent K(m) values for glycine, succinyl-CoA and pyridoxal phosphate are 12mm, 10mum and 11mum respectively; 2mum-haemin and 14mum-protoporphyrin inhibit by 50%. 4. The low activity of aminolaevulinate synthetase in iron-deficient cells increases on adding iron salts to cells only under conditions where protein synthesis can occur. 5. In defined nitrate medium with a high Ca(2+) concentration anaerobic growth yield is higher, but aminolaevulinate synthetase activity is lower than in cells grown in the medium with a low Ca(2+) concentration. In medium made from AnalaR constituents, growth yield is low and aminolaevulinate synthetase activity is high even in the presence of high concentrations of Ca(2+); on adding Cu(2+) (0.1mum) to the medium growth yield and aminolaevulinate synthetase activity become the same as in non-AnalaR medium. 6. Cells incubated under conditions where protein synthesis does not occur but where electron transport does, lose their aminolaevulinate synthetase activity rapidly. 7. The activities of aminolaevulinate dehydratase and succinic thiokinase do not change under any of the conditions of growth examined. 8. The possible mechanisms controlling aminolaevulinate synthetase activity and the role of this enzyme in controlling the synthesis of haem in this organism are discussed.     

Cytochrome c1 of bakers' yeast. II. Synthesis on cytoplasmic robosomes and influence of oxygen and heme on accumulation of the apoprotein.

In the preceding paper (Ross, E., and Schatz, G. (1976) J. Biol. Chem. 251, 1991-1996) yeast cytochrome c1 was characterized as a 31,000 dalton polypeptide with a covalently bound heme group. In order to determine the site of translation of this heme-carrying polypeptide, yeast cells were labeled with [H]leu(be under the following conditions: (a) in the absence of inhibitors, (b) in the presence of acriflavin (an inhibitor of mitochondrial translation), or (c) in the presence of cycloheximide (an inhibitor of cytoplasmic translation). The incorporation of radioactivity into the hemeprotein was measured by immunoprecipitating it from mitochondrial extracts and analyzing it by dodecyl sulfate-polyacrylamide gel electrophoresis. Label was incorporated into the cytochrome c1 apoprotein only in the presence of acriflavin or in the absence of inhibitor, but not in the presence of cycloheximide. Cytochrome c1 is thus a cytoplasmic translation product. This conclusion was further supported by the demonstration that a cytolasmic petite mutant lacking mitochondrial protein synthesis still contained holocytochrome c1 that was indistinguishable from cytochrome c1 of wild type yeast with respect to molecular weight, absorption spectru, the presence of a covalently bound heme group, and antigenic properties. Cytochrome c1 in the mitochondria of the cytoplasmic petite mutant is firmly bound to the membrane, and its concentration approaches that typical of wild type mitochondria. However, its lability to proteolysis appeared to be increased. A mitochondrial translation product may thus be necessary for the correct conformation or orientation of cytochrome c1 in the mitochondrial inner membrane. Accumulation of cytochrome c1 protein in mitochondria is dependent on the abailability of heme. This was shown with a delta-aminolevulinic acid synthetase-deficient yeast mutant which lacks heme and any light-absorbing peaks attributable to cytochromes. Mitochondria from mutant cells grown without added delta-aminolevulinic acid contained at least 20 times less protein immunoprecipitable by cytochrome c1-antisera than mitochondria from cells grown in the presence of the heme precursor. Similarly, the respiration-deficient promitochondria of anaerobically grown wild type cells are almost completely devoid of material cross-reacting with cytochrome c1-antisera. A 105,000 X g supernatant of aerobically grown wild type cells contains a 29,000 dalton polypeptide that is precipitated by cytochrome c1-antiserum but not by nonimmune serum. This polypeptide is also present in high speed supernatants from the heme-deficient mutant or from anaerobically gorwn wild type cells. The possible identity of this polypeptide with soluble apocytochrome c1 is being investigated.     

Lymphoma models for B cell activation and tolerance. VI. Reversal of anti-Ig-mediated negative signaling by T cell-derived lymphokines

We have recently described three "immature" B cell lymphomas which are exquisitely sensitive to growth inhibition by anti-Ig reagents and may serve as models for tolerance induction in normal B cells. These cells are inhibited from cell cycle progression into S after receiving a negative signal in early G1. In this paper, we demonstrate that the growth inhibition by anti-Ig can be prevented and reversed by the addition of supernatants from T cell lines. One such line, called Tova, produces factors which restore normal levels of DNA synthesis in the presence of concentrations of anti-Fab or anti-kappa immunoglobulins which cause up to a 90% inhibition of thymidine incorporation in a 2- to 3-day culture period. This factor is at least partially effective when added up to 24 hr after anti-Ig to unsynchronized lymphoma cells and it does not alter the growth of control cultures. Studies using synchronized lymphoma cells indicated that the T cell factor permitted cycle progression into S when added during the early G1 exposure to anti-kappa and was less effective when added late in G1. Preliminary characterization suggests that both B cell growth factor II (interleukin 5) and B cell stimulatory factor 1 (interleukin 4) have additive activity in this system, although another unidentified lymphokine may also be involved. The relevance of T cell reversal of Ig receptor-mediated negative signaling to neonatal B cell tolerance is emphasized.     

Biogenesis of mitochondrial membranes in Neurospora crassa during cellular differentiation: changes in oxidative phosphorylation and synthesis of mitochondrial phospholipids.

Changes in the capacity of mitochondria to carry out oxidative phosphorylation and in the rate of synthesis and incorporation of phospholipids into mitochondria were measured during the germination of conidiospores of Neurospora crassa. The competence of isolated mitochondria to carry out coupled respiration was very low during the first 3 h growth, but it increased rapidly, reaching maximal levels at 5 to 6 h growth. Changes in mitochondrial function were the same in cells grown in 2% sucrose- or 15% glucose-supplemented medium. The rate of synthesis of mitochondrial phospholipids was very low during the first 2 h growth and increased to maximal levels between 3 and 5 h. The rate of synthesis of mitochondrial phospholipids was approximately three times higher in cells grown in 15% glucose than in those grown in 2% sucrose. The maximal rate of synthesis of mitochondrial phospholipids occurred during spore germination and preceded attainment of full competence for oxidative phosphorylation. The lipid-rich condition of the mitochondrial resulting from the high rate of synthesis of phospholipids in glucose-grown cells is postulated to be related to the whorled inclusions observed in thin sections of Neurospora cells.