Yeast cell cycle protein CDC48p shows full-length homology to the mammalian protein VCP and is a member of a protein family involved in secretion, peroxisome formation, and gene expression.

Yeast mutants of cell cycle gene cdc48-1 arrest as large budded cells with microtubules spreading aberrantly throughout the cytoplasm from a single spindle plaque. The gene was cloned and disruption proved it to be essential. The CDC48 sequence encodes a protein of 92 kD that has an internal duplication of 200 amino acids and includes a nucleotide binding consensus sequence. Vertebrate VCP has a 70% identity over the entire length of the protein. Yeast Sec18p and mammalian N-ethylmaleimide-sensitive fusion protein, which are involved in intracellular transport, yeast Pas1p, which is essential for peroxisome assembly, and mammalian TBP-1, which influences HIV gene expression, are 40% identical in the duplicated region. Antibodies against CDC48 recognize a yeast protein of apparently 115 kD and a mammalian protein of 100 kD. Both proteins are bound loosely to components of the microsomal fraction as described for Sec18p and N-ethylmaleimide-sensitive fusion protein. This similarity suggests that CDC48p participates in a cell cycle function related to that of N-ethylmaleimide-sensitive fusion protein/Sec18p in Golgi transport.

Cloning of hexokinase structural genes from Saccharomyces cerevisiae mutants with regulatory mutations responsible for glucose repression.

The regulatory hexokinase PII mutants isolated previously (K.-D. Entian and K.-U. Fröhlich, J. Bacteriol. 158:29-35, 1984) were characterized further. These mutants were defective in glucose repression. The mutation was thought to be in the hexokinase PII structural gene, but it did not affect the catalytic activity of the enzyme. Hence, a regulatory domain for glucose repression was postulated. For further understanding of this regulatory system, the mutationally altered hexokinase PII proteins were isolated from five mutants obtained independently and characterized by their catalytic constants and bisubstrate kinetics. None of these characteristics differed from those of the wild type, so the catalytic center of the mutant enzymes remained unchanged. The only noticeable difference observed was that the in vivo modified form of hexokinase PII, PIIM, which has been described recently (K.-D. Entian and E. Kopetzki, Eur. J. Biochem. 146:657-662, 1985), was absent from one of these mutants. It is possible that the PIIM modification is directly connected with the triggering of glucose repression. To establish with certainty that the mutation is located in the hexokinase PII structural gene, the genes of these mutants were isolated after transforming a hexokinaseless mutant strain and selecting for concomitant complementation of the nuclear function. Unlike hexokinase PII wild-type transformants, glucose repression was not restored in the hexokinase PII mutant transformants. In addition mating experiments with these transformants followed by tetrad analysis of sporulated diploids gave clear evidence of allelism to the hexokinase PII structural gene.

Tyrosine phosphorylation regulates cell cycle-dependent nuclear localization of Cdc48p.

Cdc48p from Saccharomyces cerevisiae and its highly conserved mammalian homologue VCP (valosin-containing protein) are ATPases with essential functions in cell division and homotypic fusion of endoplasmic reticulum vesicles. Both are mainly attached to the endoplasmic reticulum, but relocalize in a cell cycle-dependent manner: Cdc48p enters the nucleus during late G1; VCP aggregates at the centrosome during mitosis. The nuclear import signal sequence of Cdc48p was localized near the amino terminus and its function demonstrated by mutagenesis. The nuclear import is regulated by a cell cycle-dependent phosphorylation of a tyrosine residue near the carboxy terminus. Two-hybrid studies indicate that the phosphorylation results in a conformational change of the protein, exposing the nuclear import signal sequence previously masked by a stretch of acidic residues.

The isolation of Saccharomyces cerevisiae nuclear membranes with nuclease and high-salt treatment.

Saccharomyces cerevisiae nuclear membranes were prepared from isolated nuclei by digesting chromatin with deoxyribonuclease and ribonuclease, washing of residual nuclei with 0.5 M MgCl2, and discontinuous gradient centrifugation in buffered Ficoll solutions. Electron microscopic examination of the preparations showed single membrane and double membrane vesicles and membrane sheets. Pores or residual pores were often visible. In double membrane profiles the two unit membranes were often separated by the remains of the perinuclear cistern. The nuclear membrane fragments contained 58% protein, 23.8% phospholipid, 6% sterols, 7.1% neutral acylglycerols, 4.8% RNA, and 0.3% DNA. The phospholipid content of the membrane preparations was influenced by a phospholipase activity with acidic pH optimum.

Specific inactivation of glucose metabolism from eucaryotic cells by pentalenolactone.

Pentalenolactone, an antibiotic related to the class of the sesquiterpene-lactones and produced by the strain Streptomyces arenae Tü-469, inhibits specifically the glucose metabolism by inactivation of the enzyme glyceraldehyde-3-phosphate dehydrogenase (D-glyceraldehyde-3-phosphate: NAD oxidoreductase (phosphorylating) ED 1.2.1.1.2). The sensitivity of several eucaryotic cell-systems for pentalenolactone was shown under in vivo conditions. The glycolytic as well as the gluconeogenetic pathway of mammalian cells can be completely inhibited with low concentrations of the antibiotic. In all cases, the minimum inhibitory concentration is dependent on cell density. The inhibitory effect in vivo and in vitro does not seem to be species-specific. In erythrocytes from rats, in Ehrlich-ascites tumor cells and in Plasmodium vinckei infected erythrocytes from mice glycolysis can be inhibited with concentrations of 18--90 micrometers pentalenolactone. In hepatocytes, glycolysis as well as gluconeogenesis in prevented by the same concentrations. In contrast to these results, in yeast the inhibition depends on growth conditions. The inhibition in glucose medium is cancelled by precultivation on acetate-containing medium.

Activation of glutaminase by phosphoribosyl-pyrophosphate and its interference with the assay of phosphoribosylpyrophosphate amidotransferase.

Phosphate-dependent glutaminase (L-glutamine amidohydrolase, EC 3.5.1.2) from rat liver was found to be strongly activated by phosphoribosylpyrophosphate (P-rib-PP), the substrate of amidophosphoribosyltransferase (EC 2.4.2.14). Since the assay of the latter is based on the P-rib-PP-dependent conversion of glutamine to glutamate, the amidotransferase activities determined in crude tissue preparations were found to be too high. The interference of glutaminase, however, could be completely eliminated by its inactivation at 50 degrees C. Amidotransferase was not affected by the heat treatment. Because of the increased rate of the glutamate formation at this temperature, the incubation time of the assay could be significantly reduced.

Regulation of enzymes and isoenzymes of carbohydrate metabolism in the yeast Saccharomyces cerevisiae.

An electrophoretic method has been devised to investigate the changes in the enzymes and isoenzymes of carbohydrate metabolism, upon adding glucose to derepressed yeast cells. (i) Of the glycolytic enzymes tested, enolase II, pyruvate kinase and pyruvate decarboxylase were markedly increased. This increase was accompanied by an overall increase in glycolytic activity and was prevented by cycloheximide, an inhibitor of protein synthesis. (ii) In contrast, respiratory activity decreased after adding glucose. This decrease was clearly shown to be the result of repression of respiratory enzymes. A rapid decrease within a few minutes of adding glucose, by analogy with the so-called ' Crabtree effect', was not observed in yeast. (iii) The gluconeogenic enzymes, fructose-1,6-bisphosphatase and malate dehydrogenase, which are inactivated after adding glucose, showed no significant changes in electrophoretic mobilities. Hence, there was no evidence of enzyme modifications, which were postulated as initiating degradation. However, it was possible to investigate cytoplasmic and mitochondrial malate dehydrogenase isoenzymes separately. Synthesis of the mitochondrial isoenzyme was repressed, whereas only cytoplasmic malate dehydrogenase was subject to glucose inactivation.

The ATPase activity of purified CDC48p from Saccharomyces cerevisiae shows complex dependence on ATP-, ADP-, and NADH-concentrations and is completely inhibited by NEM.

The cell cycle protein CDC48p from Saccharomyces cerevisiae is a member of a protein superfamily (AAA superfamily) characterized by a common region of approximately 200 amino-acid residues including an ATP binding consensus. CDC48p purified to homogeneity showed considerable ATPase activity which could be completely abolished by preincubation with NEM in the absence of ATP. ATP protects the protein from NEM and stabilizes the otherwise labile enzyme. The ATPase activity is reversibly inhibited by NADH and shows cooperativity with its substrate ATP. The application of the in vitro ATPase activity to the identification of physiologically interacting molecules is discussed. By electron microscopy, the enzyme was shown to consist of hexameric ring structures similar to its vertebrate homologue.

Studies on the regulation of enolases and compartmentation of cytosolic enzymes in Saccharomyces cerevisiae.

Three enolase isoenzymes can be distinguished after electrophoresis of yeast crude extracts. After adding glucose to derepressed cells, there was a coordinated increase in the activity of enolase I and decrease in enolase II activity. Enolase I was found to be repressed and enolase II simultaneously induced by glucose. The third enolase activity remained unchanged and was identified as that of a hybrid enzyme. Enolase catalyses the first common step of glycolysis and gluconeogenesis. Gluconeogenic enolase I shows substrate inhibition for 2-phosphoglycerate (glycolytic substrate) and glycolytic enolase II is substrate-inhibited by phosphoenolpyruvate (gluconeogenic substrate). The gluconeogenic reaction was inhibited up to 45% by physiological concentrations of fructose 1,6-bisphosphate. To test for cytological compartmentation, a method was developed for isolating microsomes. Effective enrichment of rough and smooth endoplasmic reticulum was demonstrated by electron microscopy. No evidence was obtained for any compartmentation of either enolases or other glycolytic enzymes.

Purification procedure and N-terminal amino acid sequence of yeast malate dehydrogenase isoenzymes.

A method has been devised for the rapid isolation of malate dehydrogenase isoenzymes. First, anionic proteins were precipitated with polyethyleneimine, whilst hydrophobic malate dehydrogenase remained in the supernatant fluid. Secondly, the supernatant was 30% saturated with ammonium sulfate and the two isoenzymes were separated by hydrophobic phenyl-Sepharose CL-4B chromatography. For further purification the enzymes were chromatofocused, and polybuffer was removed by hydrophobic chromatography. Affinity chromatography with blue Sepharose CL-6B [1] was used as final purification step. The purified isoenzymes were homogeneous as shown by isoelectric focusing and could be used for N-terminal sequencing. 34 amino acid residues could be identified for the cytoplasmic isoenzyme and 56 amino acid residues for the mitochondrial isoenzyme. Although there are regions of strong homology between both isoenzymes, the sequence differences clearly showed support that both isoenzymes are coded by different genes. Sequence comparison clearly indicated that the N-terminus of the cytoplasmic enzyme extended that of the mitochondrial enzyme by 12 amino acid residues. The amino acid sequence of the extending sequence resembled that of leading sequences known for enzymes which are transported into the mitochondria. The assumed leading sequence is discussed with respect to its possible role in glucose inactivation.

Induction of glutamine synthetase in periportal hepatocytes by cocultivation with a liver epithelial cell line.

Cocultures of periportal, glutamine synthetase-negative (GS-) hepatocytes with endothelial cells of human veins or epithelial cells of rat liver (clone RL-ET-14) were established for testing whether GS could be induced in the hepatocytes by interactions between the different cell types. While GS activity in endothelial cells was below detection level that of RL-ET-14 cells decreased from 62 mU/mg (24 h) to 38 mU/mg (168 h). During cocultivation with endothelial cells no change in the low GS activity could be detected. In contrast, when periportal hepatocytes were cocultured with RL-ET-14 cells, GS activity of the cocultures increased continuously from 26 mU/mg (24 h) to 56 mU/mg during cultivation for 168 h. Immunocytochemical staining of the cocultures for GS showed that this rise of GS activity was associated with an increase of GS level in the periportal hepatocytes and a decrease in the RL-ET-14 cells. Correspondingly, cultivation of periportal hepatocytes with media conditioned by the RL-ET-14 cells led to an increase in GS activity which, however, remained below that of cocultures, while conditioned medium of hepatocytes resulted in a decrease of GS activity in pure cultures of RL-ET-14 cells. "Separated" cocultures, where hepatocytes and RL-ET-14 cells reached each other only at the border of a circular area, demonstrated that induction of GS was highest in the marginal hepatocytes and lowest in those located in the center indicating that besides (a) soluble factor(s) other kinds of cell-cell interactions might be responsible for full induction of GS expression in periportal hepatocytes.

Alterations in activity and ultrastructural localization of several phosphatases on the surface of adult rat hepatocytes in primary monolayer culture.

Several enzymes associated with the hepatocyte cell surface, alkaline phosphatase (AP), 5'-nucleotidase (5'N), Mg++- and total Na+K+Mg++-ATpase, were assayed and localized cytochemically in order to gain insight into alterations of the plasma membrane components during reassociation of hepatocytes in primary monolayer culture. During a period of 4 days the activities of 5'nucleotidase and alkaline phosphatase increased spontaneously up to three- and four-fold, respectively. Dexamethasone reinforce the rise of alkaline phosphatase activity but retarded the increase of that of 5'nucleotidase. However, after the third day the level of 5'nucleotidase activity converged with the untreated controls. The activities of Mg++- and Na+K+Mg++-ATPase, which closely paralleled each other, remained essentially unchanged throughout cultivation and were not affected by dexamethasone. Cytochemical demonstration of alkaline phosphatase, 5'nucleotidase and Mg++-ATPase, using the lead salt method, revealed the potential presence of reaction product on the whole cell surface. However, the cells did not react uniformly, particularly on bile canalicular membranes. This heterogeneity seems to be due to different stages of canalicular development and to different functional states of the cultured hepatocytes.

Complete nucleotide sequence of the hexokinase PI gene (HXK1) of Saccharomyces cerevisiae.

The nucleotide sequence of the yeast glycolytic hexokinase isoenzyme PI-gene, HXK1, has been determined by sequencing the yeast DNA insert of the previously isolated plasmid HXK1 clone [Entian et al., Mol. Gen. Genet. 198 (1984) 50-54]. The structural gene sequence included 1452 bp coding for 484 amino acid (aa) residues corresponding to the Mr of 153 605 for the HXK1 monomer. Several initiation regions and termination points were located using nuclease S1 mapping. The HXK1 sequence was 76% homologous with that of HXK2, which is responsible for triggering glucose repression in yeasts. Since HXK1 is not involved in this regulatory system, the regulatory function of HXK2 must correspond to one or more of the differences between both isoenzymes. Most changes in the amino acid sequence were statistically distributed; however, four clustered regions with more than five altered aa residues were identified.

The primary structure of the yeast hexokinase PII gene (HXK2) which is responsible for glucose repression.

The nucleotide sequence of the Saccharomyces cerevisiae gene encoding the glycolytic isoenzyme hexokinase PII (HXK2), which is responsible for triggering glucose repression, has been determined. The reading frame was identified by comparison with the N-terminal undecameric amino acid (aa) sequence, determined previously [Schmidt and Colowick, Arch. Biochem. Biophys. 158 (1973) 458-470]. The codon sequence was not random, with 82.1% of the aa specified by only 25 codons. The structural gene sequence corresponded to 1455 bp, coding for 485 aa residues, corresponding to the Mr of 53 800 for the HXK2 monomer. Five initiation regions spanning 162 bp and three termination sites spanning 29 bp were detected. Sequences with similarities to a 5'-TATAAA-3' sequence were located 24-39 bp upstream of each initiation region. The most pronounced initiation region corresponded to the 5'-TATAAA-3' sequence at position -152. Two of the minor initiation sites were inside the coding sequence in front of two ATG codons.

Gene cloning and sequencing, and enzyme purification of the malate synthase of Streptomyces arenae.

Streptomyces arenae is able to grow on acetate or ethanol as the sole carbon source. The metabolic pathway used for gluconeogenesis from C2 compounds in streptomycetes has not yet been characterized. In the course of a sequencing project we identified the gene for malate synthase (aceB), a key enzyme in the glyoxylate cycle in S. arenae. The gene was cloned and sequenced. The open reading frame of 1632 bp codes for a potential protein of 61.360 kDa. A comparison with the sequences of malate synthase from other organisms shows that the phylogenetic distance to the E. coli aceB gene is no closer than that to genes from plants or fungi. Malate synthase activity was detected in cell extracts from S. arenae. Its dependence on media conditions and on the growth phase was investigated. A purification procedure was established which allows a 188-fold enrichment of the enzyme. The molecular weight of the monomer determined by SDS PAGE confirms the weight calculated from the gene sequence. However, the holoenzyme appears to be dimeric as shown by gel filtration. All other known malate synthases from eubacteria are monomeric, while those of fungi or plants are oligomeric (di-, tri-, tetra- or octameric). The apparent Km value for glyoxylate is significantly higher than that of the malate synthases of all other species published so far. The enzyme is inactive at pH values of 7 and below; the strain cannot grow on ethanol or acetate as the sole carbon source at media pH values of 7 or below.

Glutamate uptake by cultured rat hepatocytes is mediated by hormonally inducible, sodium-dependent transport systems.

Glutamate uptake by rat hepatocytes in primary monolayer culture was found to be mediated by a Na+-independent and by two Na+-dependent transport systems of high and low affinity. Inhibition studies with cysteate and other model amino acids rules out the participation of the neutral amino acid transport systems A, ASC, and N and revealed that the Na+-dependent agencies represent unequivocally anionic transport systems. Na+-dependent uptake of glutamate in isolated hepatocytes was slow compared to the Na+-independent portion, but increased spontaneously during cultivation. In the presence of dexamethasone it was stimulated about 10-fold at the second day of cultivation.

Isolation and primary structure of the gene encoding fructose-1,6-bisphosphatase from Saccharomyces cerevisiae.

The gene encoding Saccharomyces cerevisiae fructose-1,6-bisphosphatase (FBP1) was isolated. Constructed fbp1::HIS3 null mutants were unable to grow with ethanol, and growth was restored after transformation with the cloned fbp gene. The gene codes for a protein of 347 amino acid residues with an Mr of 38131. Homology with the pig kidney cortex and the sheep liver enzyme is 47.7% and 46.6%, respectively, within a central core of 328 amino acid residues. The cloned promoter size was 318 bp and allowed only low level expression of the gene. This indicates a positive activation site (UAS) upstream of the cloned DNA fragment.

Irreversible inactivation of Saccharomyces cerevisiae fructose-1,6-bisphosphatase independent of protein phosphorylation at Ser11.

The fructose-1,6-bisphosphatase gene was used with multicopy plasmids to study rapid reversible and irreversible inactivation after addition of glucose to derepressed Saccharomyces cerevisiae cells. Both inactivation systems could inactivate the enzyme, even if 20-fold over-expressed. The putative serine residue, at which fructose-1,6-bisphosphatase is phosphorylated, was changed to an alanine residue without notably affecting the catalytic activity. No rapid reversible inactivation was observed with the mutated enzyme. Nonetheless, the modified enzyme was still irreversibly inactivated, clearly demonstrating that phosphorylation is an independent regulatory circuit that reduces fructose-1,6-bisphosphatase activity within seconds. Furthermore, irreversible glucose inactivation was not triggered by phosphorylation of the enzyme.

Inhibition of glyceraldehyde-3-phosphate dehydrogenase by pentalenolactone in Trypanosoma brucei.

Pentalenolactone (PL), an antibiotic produced by several strains of Streptomycetes, is a specific irreversible inhibitor of glyceraldehyde-3-phosphate dehydrogenase (EC 1.2.1.12). The effect of this antibiotic was studied in Trypanosoma brucei. In infected mice, due to the rapid metabolic inactivation of PL in vivo, trypanosomes were not affected by concentrations that were lethal to the host. Bloodstream trypanosomes in vitro were killed by low concentrations of PL (1.5 microgram ml-1), suggesting that there is no alternative to the glycolytic pathway for the generation of ATP in the bloodstream forms. In contrast, even high concentrations of PL (75 micrograms ml-1) were unable to inhibit growth of the procyclic form in vitro, presumably due to their ability to generate ATP independently of the glycolytic pathway.

Differentiation of Trypanosoma brucei bloodstream trypomastigotes from long slender to short stumpy-like forms in axenic culture.

An axenic cultivation system was used to study the differentiation of Trypanosoma brucei bloodstream forms from long slender to short stumpy-like forms. Trypanosomes in the logarithmic phase are similar to long slender bloodstream forms freshly isolated from infected mice, differing only in the rate of oxygen uptake. In contrast, trypanosomes in the stationary phase show a decreased level of glucose oxidation, express pyrroline-5-carboxylate reductase (proline oxidase), are inhibited in oxygen uptake to about 44% by KCN, undergo considerable morphological changes on the cellular and subcellular level, and have a significantly smaller cell volume. These results are comparable to those observed during the differentiation of long slender to short stumpy forms in infected animals, suggesting that the differentiation process towards insect procyclic forms can be initiated in culture at 37 degrees C. As judged from immunofluorescence and electron microscopy analysis, the surface coat remains intact.