NMR analysis of a cell division cycle mutant of Saccharomyces cerevisiae

cdc 19.1 is a temperature-sensitive lesion in the genome of Saccharomyces cerevisiae. The phenotype of this mutant is a cell cycle specific arrest in G1, which is expressed at 37 degrees C. In the present study, 31P- and 13C-NMR spectroscopy were used to analyze the metabolism of the mutant at the permissive and restrictive temperatures. Our results confirm previous findings which have indicated that cdc 19.1 contains temperature-sensitive pyruvate kinase activity. In contrast to previous findings, however, the present investigation demonstrates that restriction of pyruvate kinase activity in vivo takes as long as 24 h to be fully expressed. In addition, analysis by NMR has allowed us to assess the metabolic consequences of pyruvate kinase restriction which may contribute to the arrest of cell growth in the early G1 phase of the cell division cycle.     

Glucose regulation of Saccharomyces cerevisiae cell cycle genes

Nutrient-limited Saccharomyces cerevisiae cells rapidly resume proliferative growth when transferred into glucose medium. This is preceded by a rapid increase in CLN3, BCK2, and CDC28 mRNAs encoding cell cycle regulatory proteins that promote progress through Start. We have tested the ability of mutations in known glucose signaling pathways to block glucose induction of CLN3, BCK2, and CDC28. We find that loss of the Snf3 and Rgt2 glucose sensors does not block glucose induction, nor does deletion of HXK2, encoding the hexokinase isoenzyme involved in glucose repression signaling. Rapamycin blockade of the Tor nutrient sensing pathway does not block the glucose response. Addition of 2-deoxy glucose to the medium will not substitute for glucose. These results indicate that glucose metabolism generates the signal required for induction of CLN3, BCK2, and CDC28. In support of this conclusion, we find that addition of iodoacetate, an inhibitor of the glyceraldehyde-3-phosphate dehydrogenase step in yeast glycolysis, strongly downregulates the levels CLN3, BCK2, and CDC28 mRNAs. Furthermore, mutations in PFK1 and PFK2, which encode phosphofructokinase isoforms, inhibit glucose induction of CLN3, BCK2, and CDC28. These results indicate a link between the rate of glycolysis and the expression of genes that are critical for passage through G₁.     

Fluphenazine-resistant Saccharomyces cerevisiae mutants defective in the cell division cycle.

An fls1 mutant of Saccharomyces cerevisiae, which did not grow in the presence of 30 micrograms of fluphenazine per ml, was isolated. Mutants that were resistant to 90 micrograms of fluphenazine per ml and temperature sensitive for growth were obtained from the fls1 mutant. One fluphenazine-resistance mutation, fsr1, was located near the his7 locus on chromosome II. Growth of the fsr1 mutants at 35 degrees C was arrested after nuclear division. The other group of fluphenazine-resistant mutants, carrying fsr2 mutations, showed Ca2+-dependent growth at 35 degrees C. Growth of the fsr2 mutants at 35 degrees C was arrested at the G2 stage of the cell cycle in Ca2+-poor medium.     

Meiotic effects of DNA-defective cell division cycle mutations of Saccharomyces cerevisiae

The meiotic effects of several cell division cycle (cdc) mutations of Saccharomyces cerevisiae have been investigated by electron microscopy and by genetic and biochemical methods. Diploid strains homozygous for cdc mutations known to confer defects on vegetative DNA synthesis were subjected to restrictive conditions during meiosis. Electron microscopy revealed that all four mutants were conditionally arrested in meiosis after duplication of the spindle pole bodies but before spindle formation for the first meiotic division. None of these mutants became committed to recombination or contained synaptonemal complex at the meiotic arrest. — The mutants differed in their ability to undergo premeiotic DNA synthesis under restrictive conditions. Both cdc8 and cdc21, which are defective in the propagation of vegetative DNA synthesis, also failed to undergo premeiotic DNA synthesis. The arrest of these mutants at the stage before meiosis I spindle formation could be attributed to the failure of DNA synthesis because inhibition of synthesis by hydroxyurea also caused arrest at this stage. — Premeiotic DNA synthesis occurred before the arrest of cdc7, which is defective in the initiation of vegetative DNA synthesis, and of cdc2, which synthesizes vegetative DNA but does so defectively. The meiotic arrest of cdc7 homozygotes was partially reversible. Even if further semiconservative DNA replication was inhibited by the addition of hydroxyurea, released cells rapidly underwent commitment to recombination and formation of synaptonemal complexes. The cdc7 homozygote is therefore reversibly arrested in meiosis after DNA replication, whereas vegetative cultures have previously been shown to be defective only in the initiation of DNA synthesis.     

Decreased mitochondrial biogenesis in temperature-sensitive cell division cycle mutants of Saccharomyces cerevisiae

The temperature-sensitive cell division cycle (cdc) G1 mutants cdc28 and cdc35 show decreased mitochondrial volumes with respect to the wild type strain A364A (WT) at the restrictive temperature. Of the three criteria of mitochondrial biogenesis studied, that is, number of mitochondria per cell, relative area of the cell occupied by mitochondria, or relative area of mitochondria occupied by inner membranes, only the second indicator was significantly lower in cdc mutants than in the WT. The mitochondrial inner membranes development did not compensate for the decrease in the organelles volume. Apparently, the reduced mitochondrial biogenesis was not due to the temperature shift because the relative area of the cell occupied by mitochondria was already significantly lower at 25°C in cdc mutants. The specific fluxes of oxygen consumption confirmed that the respiratory capacity of cdc mutants is largely impaired in respect to the WT. Cdc28 and cdc35 mutants of Saccharomyces cerevisiae had been previously shown to exhibit high respiratory quotients (from 3 to 7) in respect to the WT (RQ 1.0), which correlated with carbon and energy uncoupling probably the result of glucose-induced catabolite repression [Aon MA, Mónaco ME, Cortassa S (1995) Exp Cell Res 217, 42–51; Mónaco ME, Valdecantos PA, Aon MA (1995) Exp Cell Res 217, 52–56].     

In vivo analysis of chromosome condensation in Saccharomyces cerevisiae

Although chromosome condensation in the yeast Saccharomyces cerevisiae has been widely studied, visualization of this process in vivo has not been achieved. Using Lac operator sequences integrated at two loci on the right arm of chromosome IV and a Lac repressor-GFP fusion protein, we were able to visualize linear condensation of this chromosome arm during G2/M phase. As previously determined in fixed cells, condensation in yeast required the condensin complex. Not seen after fixation of cells, we found that topoisomerase II is required for linear condensation. Further analysis of perturbed mitoses unexpectedly revealed that condensation is a transient state that occurs before anaphase in budding yeast. Blocking anaphase progression by activation of the spindle assembly checkpoint caused a loss of condensation that was dependent on Mad2, followed by a delayed loss of cohesion between sister chromatids. Release of cells from spindle checkpoint arrest resulted in recondensation before anaphase onset. The loss of condensation in preanaphase-arrested cells was abrogated by overproduction of the aurora B kinase, Ipl1, whereas in ipl1-321 mutant cells condensation was prematurely lost in anaphase/telophase. In vivo analysis of chromosome condensation has therefore revealed unsuspected relationships between higher order chromatin structure and cell cycle control.     

Chitin synthesis and localization in cell division cycle mutants of Saccharomyces cerevisiae.

Growth of Saccharomyces cerevisiae cell cycle mutants cdc3, cdc4, cdc7, cdc24, and cdc28 at a nonpermissive temperature (37 degrees C) resulted in increased accumulation of chitin relative to other cell wall components, as compared with that observed at a permissive temperature (25 degrees C). Wild-type cells showed the same chitin/carbohydrate ratio at both temperatures, whereas mutants cdc13 and cdc21 yielded only a small increase in the ratio at 37 degrees C. These results confirm and extend those reported by B. F. Sloat and J. R. Pringle (Science 200:1171-1173, 1978) for mutant cdc24. The distribution of chitin in the cell wall was studied by electron microscopy, by specific staining with wheat germ agglutinin-colloidal gold complexes. At the permissive temperature, chitin was restricted to the septal region in all strains, whereas at 37 degrees C a generalized distribution of chitin in the cell wall was observed in all mutants. These results do not support a unique interdependence between the product of the cdc24 gene and localization of chitin deposition; they suggest that unbalanced conditions created in the cell by arresting the cycle at different stages result in generalized activation of the chitin synthetase zymogen. Thus, blockage of an event in the cell cycle may lead to consequences that are not functionally related to that event under normal conditions.     

Killer double-stranded ribonucleic acid synthesis in cell division cycle mutants of Saccharomyces cerevisiae.

The synthesis of killer double-stranded ribonucleic acid (dsRNA) in Saccharomyces cerevisiae was examined in seven different cell division cycle mutants (cdc) that are defective in nuclear deoxyribonucleic acid replication and contain the "killer character." In cdc28, cdc4, and cdc7, which are defective in the initiation of nuclear deoxyribonucleic acid synthesis, and in cdc23 or in cdc14, defective in medial or late nuclear division, an overproduction of dsRNA at the restrictive temperature was observed. In contrast to the above mutants, the synthesis of killer dsRNA is not enhanced at the restrictive temperature in either cdc8 or cdc21, which are defective in deoxyribonucleic acid chain elongation. Examination of killer sensitive strains (cdc7 K- and cdc4 K-) has shown that the complete killer dsRNA genome is essential for the overproduction of dsRNA at the restrictive temperature.     

Mutations in cell division cycle genes CDC36 and CDC39 activate the Saccharomyces cerevisiae mating pheromone response pathway.

Conditional mutations in the genes CDC36 and CDC39 cause arrest in the G1 phase of the Saccharomyces cerevisiae cell cycle at the restrictive temperature. We present evidence that this arrest is a consequence of a mutational activation of the mating pheromone response. cdc36 and cdc39 mutants expressed pheromone-inducible genes in the absence of pheromone and conjugated in the absence of a mating pheromone receptor. On the other hand, cells lacking the G beta subunit or overproducing the G alpha subunit of the transducing G protein that couples the receptor to the pheromone response pathway prevented constitutive activation of the pathway in cdc36 and cdc39 mutants. These epistasis relationships imply that the CDC36 and CDC39 gene products act at the level of the transducing G protein. The CDC36 and CDC39 gene products have a role in cellular processes other than the mating pheromone response. A mating-type heterozygous diploid cell, homozygous for either the cdc36 or cdc39 mutation, does not exhibit the G1 arrest phenotype but arrests asynchronously with respect to the cell cycle. A similar asynchronous arrest was observed in cdc36 and cdc39 cells where the pheromone response pathway had been inactivated by mutations in the transducing G protein. Furthermore, cdc36 and cdc39 mutants, when grown on carbon catabolite-derepressing medium, did not arrest in G1 and did not induce pheromone-specific genes at the restrictive temperature.     

In vivo reconstitution of autophagy in Saccharomyces cerevisiae

Autophagy is a major intracellular degradative pathway that is involved in various human diseases. The role of autophagy, however, is complex; although the process is generally considered to be cytoprotective, it can also contribute to cellular dysfunction and disease progression. Much progress has been made in our understanding of autophagy, aided in large part by the identification of the autophagy-related (ATG) genes. Nonetheless, our understanding of the molecular mechanism remains limited. In this study, we generated a Saccharomyces cerevisiae multiple-knockout strain with 24 ATG genes deleted, and we used it to carry out an in vivo reconstitution of the autophagy pathway. We determined minimum requirements for different aspects of autophagy and studied the initial protein assembly steps at the phagophore assembly site. In vivo reconstitution enables the study of autophagy within the context of the complex regulatory networks that control this process, an analysis that is not possible with an in vitro system.     

Mutants of Saccharomyces cerevisiae cell division cycle defective in cytokinesis. Biosynthesis of the cell wall and morphology

The four temperature-sensitive mutants of Saccharomyces cerevisiae in the cell division cycle defective in cytokinesis (cdc, 3, 10, 11 and 12), have been analyzed with respect to the biosynthesis of the cell wall polymers. After 3 hours of incubation at the non-permissive temperature (37°C) these strains stop growing. The synthesis of glucan, mannan and chitin (wall polymers) level off in a similar time, but glucan, mannan and chitin synthases remained active for at least 4 hours.If the mutants are analyzed by transmission and scanning electron microscopy different pictures emerge. Two of the mutants cdc 10 and cdc 12, after 3 hours of incubation at 37°C present apparently normal cytoplasms and cell wall surfaces with multiple elongated buds. The other two mutants, cdc 3 and cdc 11, present a completely disarranged cytoplasmic content and damage at the level of the plasma membrane is evident.These and other observations, suggest that between the execution points of cdc 3 (0.27) and cdc 10 (0.58), essential processes in the assembly of cell membrane occur.This work was supported in part by a grant from la Comisión de Investigación Científica y Técnica of the Spanish Ministerio de Educación y Ciencia (Project no. 4593-1980).     

Characteristic alteration in the nuclear DNA polymerase activity during the cell division cycle of Saccharomyces cerevisiae

Specific activity of the intranuclear DNA polymerase in cdc-mutant cells of Saccharomyces cerevisiae was found to be characteristically changed by arrest in their specific stage of cell division cycle without a notable alteration in the total cellular activity. The activities were low in the nuclei of cdc 25, cdc 28 and cdc 4, which were arrested in early to mid G1 phase by temperature shift-up, and in the nuclei of wild-type cells (A364A), which were arrested in early G1 phase by alpha-factor treatment, while high level of the activity was found in the nuclei of cdc 7 and cdc 8, which were arrested at late G1 and S phase, respectively. Activity-gel analysis of DNA polymerase in the nuclear extracts revealed the presence of two active peptides (120K and 72K), and the characteristic decrease in both active peptides was induced by arrest in early to mid G1 phase. Consequently, it is strongly suggested that intranuclear DNA polymerase activity alters in a dependent fashion on progression of cell division cycle. Subunit analysis indicated that the purified DNA polymerase I is constructed from two subunit peptides of 120K and 62K, and the large subunit possesses catalytic activity.     

Control of the cell division cycle and sporulation in Saccharomyces cerevisiae by the cyclic AMP system

This paper reviews recent data on the adenylate cyclase system of the yeast Saccharomyces cerevisiae. Since the discovery of yeast adenylate cyclase mutants and the possibility of molecular biological analysis, adenylate cyclase and the subsequent steps in the cAMP cascade have become subject of intense investigation. CYR1, the structural gene for the adenylate cyclase catalytic subunit is necessary for cell division and in diploid cells is involved in the choice between sporulation and cell division. The cell division cycle in yeast is initiated by a step called START, which has been defined by mutations causing an arrest of the cells in an unbudded state. One class of mutation causes the cell to arrest at the same stage of the cell division cycle as the pheromone implicated in conjugation. A second class causes cells to cease growth in a different manner, but one which is similar to the arrest brought about by nutient deprivation. The adenylate cyclase gene belongs to the second class and has been identified as CDC35. Two genes of the first class have been cloned and sequenced. CDC28 codes for a kinase which has homology with the src proto-oncogene family. CDC36 is partly homologous with the oncogene ets. Two genes related to the ras oncogene family have also been implicated in the control of START. START can be dissociated in two subsequent phases, the first being controlled by the AMPc system and the second including proto-oncogenes. A model in which cAMP is a positive indicator of available nutrients such as nitrogen has been constructed.(ABSTRACT TRUNCATED AT 250 WORDS)     

In vivo investigations of glucose transport in Saccharomyces cerevisiae

In the present study, the glucose transport into the yeast Saccharomyces cerevisiae has been investigated. The approach suggested is based on a rapid sampling technique for studying the dynamic response of the yeast to rapid changes in extracellular glucose concentrations. For this purpose a concentrated glucose solution has been injected into a continuous culture at steady state growth conditions resulting in a shift of the extracellular glucose level. Samples have been taken every 5 s for determination of extracellular glucose and intracellular glucose-6-phosphate concentrations. Attempts to fit the experimental observations with simulations from existing models failed. The mechanism then proposed is based on a facilitated diffusion of glucose superimposed by an inhibition of glucose-6-phosphate. The use of the so-called in vivo approach suggested in this article appears to be proper, because the investigations can be performed at defined physiological states of the microbial cultures. Furthermore, the experimental observations are not being corrupted by the preparation of the samples for the transport studies as it happens during radioactive measurements. (c) 1996 John Wiley & Sons, Inc.     

Glucose uptake in the cell cycle of Saccharomyces cerevisiae

Glucose uptake was determined in the cell cycle of the yeast Saccharomyces cerevisiae. It was observed that there are two periods per cell cycle at which cells utilize glucose. This finding could give an explanation for the known fact that yeast cells in the stationary phase of growth are of two size classes.     

Functions of microtubules in the Saccharomyces cerevisiae cell cycle

We used the inhibitor nocodazole in conjunction with immunofluorescence and electron microscopy to investigate microtubule function in the yeast cell cycle. Under appropriate conditions, this drug produced a rapid and essentially complete disassembly of cytoplasmic and intranuclear microtubules, accompanied by a rapid and essentially complete block of cellular and nuclear division. These effects were similar to, but more profound than, the effects of the related drug methyl benzimidazole carbamate (MBC). In the nocodazole-treated cells, the selection of nonrandom budding sites, the formation of chitin rings and rings of 10-nm filaments at those sites, bud emergence, differential bud enlargement, and apical bud growth appeared to proceed normally, and the intracellular distribution of actin was not detectably perturbed. Thus, the cytoplasmic microtubules are apparently not essential for the establishment of cell polarity and the localization of cell-surface growth. In contrast, nocodazole profoundly affected the behavior of the nucleus. Although spindle-pole bodies (SPBs) could duplicate in the absence of microtubules, SPB separation was blocked. Moreover, complete spindles present at the beginning of drug treatment appeared to collapse, drawing the opposed SPBs and associated nuclear envelope close together. Nuclei did not migrate to the mother-bud necks in nocodazole-treated cells, although nuclei that had reached the necks before drug treatment remained there. Moreover, the double SPBs in arrested cells were often not oriented toward the budding sites, in contrast to the situation in normal cells. Thus, microtubules (cytoplasmic, intranuclear, or both) appear to be necessary for the migration and proper orientation of the nucleus, as well as for SPB separation, spindle function, and nuclear division.