Cortical Num1p interacts with the dynein intermediate chain Pac11p and cytoplasmic microtubules in budding yeast.

Num1p, a cortical 313-kD protein, controls cytoplasmic microtubule (cMT) functions and nuclear migration through the bud neck in anaphase cells. A green fluorescent protein (GFP)-Num1p fusion protein localizes at the bud tip and the distal mother pole of living cells, apparently forming cMT capture sites at late anaphase. In addition, galactose-induced GFP-Num1p is seen at the bud neck and in lateral regions of the mother cortex. The bud tip location of Num1p depends on Bni1p but does not require Kar9p, Dyn1p, or cMTs, whereas cMT contacts with polar Num1p dots are reduced in cells lacking Dyn1p. Num1p associates with the dynein intermediate chain Pac11p in the presence of Dyn1p, and with the alpha-tubulin Tub3p, as shown by coimmune precipitation of tagged proteins. Num1p also forms a complex with Bni1p and Kar9p, although Num1p is not required for Bni1p- and Kar9p-dependent nuclear migration to the bud neck in preanaphase cells. Our data suggest that Num1p controls nuclear migration during late anaphase by forming dynein-interacting cortical cMT capture sites at both cellular poles. In addition, Num1p may transiently cooperate with an associated Bni1p-Kar9p complex at the bud tip of early anaphase cells.

Phospholipase C binds to the receptor-like GPR1 protein and controls pseudohyphal differentiation in Saccharomyces cerevisiae.

The hormone receptor-like protein Gpr1p physically interacts with phosphatidylinositol-specific phospholipase C (Plc1p) and with the Galpha protein Gpa2p, as shown by two-hybrid assays and co-immune precipitation of epitope-tagged proteins. Plc1p binds to Gpr1p in either the presence or absence of Gpa2, whereas the Gpr1p/Gpa2p association depends on the presence of Plc1p. Genetic interactions between the null mutations plc1Delta, gpr1Delta, gpa2Delta, and ras2Delta suggest that Plc1p acts together with Gpr1p and Gpa2p in a growth control pathway operating in parallel to the Ras2p function. Diploid cells lacking Gpr1p, Plc1p, or Gpa2p fail to form pseudohyphae upon nitrogen depletion, and the filamentation defect of gpr1Delta and plc1Delta strains is rescued by activating a mitogen-activated protein kinase pathway via STE11-4 or by activating a cAMP pathway via overexpressed Tpk2p. Plc1p is also required for efficient expression of the FG(TyA)::lacZ reporter gene under nitrogen depletion. In conclusion, we have identified two physically interacting proteins, Gpr1p and Plc1p, as novel components of a nitrogen signaling pathway controlling the developmental switch from yeast-like to pseudohyphal growth. Our data suggest that phospholipase C modulates the interaction of the putative nutrient sensor Gpr1p with the Galpha protein Gpa2p as a downstream effector of filamentation control.

[Drinking water supply with reference to geogenic arsenic contamination]. / Trinkwassergewinnung unter Berücksichtigung geogener Arsenbelastungen.

Geogenic Arsenic in Drinking Water. Drinking water production of surface spring water in southern Lower Saxony (Niedersachsen, Germany) was reduced because of microbiological contaminations and unreliably variable water reserves. Surface spring water in general has a low arsenic content. As a consequence ground water has been increasingly used for drinking water. Thus, high geogenic concentrations of arsenic in the central "Buntsandstein" in southern Lower Saxony caused high arsenic contents in the groundwater. Under the regulation of the German Drinking Water Ordinance (1986) the limit for total arsenic (40 micrograms/l) was exceeded in 2% of 150 fountains, wells and sources in southern Lower Saxony. Because of the well-known cancerogenic potential of arsenic the limit for total arsenic in drinking water was reduced from 40 micrograms/l to 10 micrograms/l suspending the new standard value until January 1996. This regulation based on new calculations revealing a skin cancer risk of roughly 6:10,000 and a mortality risk of roughly 1:10(6) in respect of lifetime in case of arsenic concentrations of 10 micrograms As/l drinking water. After that limit change 40% of 150 wells and sources in southern Lower Saxony exceeded the arsenic limit of 10 micrograms/l drinking water. As a matter of fact, it became necessary for a large number of water supply works to eliminate arsenic from the drinking water by technical means or to dilute drinking water with high concentrations of arsenic.

Cell cycle control and initiation of DNA replication in Saccharomyces cerevisiae.

The cell cycle of budding yeast is controlled by a single cyclin-dependent protein kinase (Cdc28/Cdk1) which successively associates with the stage-specific activators Cln1-3 (G1 phase), Clb5,6 (S phase), Clb3,4 (S/G2 phase) and Clb1,2 (mitosis). The temporal pattern of these Cdk1/cyclin activities imposes a strict order of DNA replication events during the cycle: formation of a pre-replication complex at late M phase, initiation of replication at the G1/s transition, support of replication during S phase, and the prevention of re-replication at G2. A key component involved in S phase initiation is the Cdc6 protein, a transiently expressed DNA-independent ATP/GTPase interacting at late mitosis with an origin-recognizing complex.

Yeast Num1p associates with the mother cell cortex during S/G2 phase and affects microtubular functions.

The NUM1 gene is involved in the control of nuclear migration in Saccharomyces cerevisiae. The content of NUM1 mRNA fluctuates during the cell cycle, reaching a maximum at S/G2 phase, and the translation product Num1p associates with the cortex of mother cells mainly during S, G2, and mitosis, as seen by indirect immunofluorescence. The nuclear spindle in NUM1-deficient large-budded cells often fails to align along the mother/bud axis, while abnormally elongated astral microtubules emanate from both spindle pole bodies. A num1 null mutation confers temperature sensitivity to the cold-sensitive alpha-tubulin mutant tub1-1, and shows synthetic lethality with the beta-tubulin mutant alleles tub2-402, tub2-403, tub2-404, and tub2-405. Deletion mapping has defined three functionally important Num1p regions: a potential EF hand Ca2+ binding site, a cluster of potential phosphorylation sites and a pleckstrin homology domain. The latter domain appears to be involved in targeting Num1p to the mother cell cortex. Our data suggest that the periodically expressed NUM1 gene product controls nuclear migration by affecting astral microtubule functions.

The Saccharomyces cerevisiae CDC6 gene is transcribed at late mitosis and encodes a ATP/GTPase controlling S phase initiation.

We have compared the periodic fluctuation of mRNAs encoded by CDC6, a cell cycle gene controlling initiation of DNA replication, and CLN1, a G1 cyclin gene expressed at late G1. The maxima of CDC6 mRNA precede those of CLN1 mRNA by about 15 min in cells synchronized by release from pheromone arrest or from a cdc15 thermal arrest. CDC6 mRNA accumulates in cdc15-arrested telophase cells, decays around cell separation, and reappears during telophase and nuclear division of the next cycle. CDC6 transcription at late mitosis is not affected by the pheromone signaling pathway. The CDC6 mRNA fluctuation pattern is imposed to a CLN1-derived reporter gene if fused to the CDC6 promoter. The CDC6 gene was expressed in Escherichia coli as a glutathione-S-transferase fusion protein and purified by affinity chromatography. The Cdc6 protein binds rATP and rGTP upon UV cross-linking and catalyzes the DNA-independent hydrolysis of purine nucleoside triphosphates, but does not appear to interact directly with DNA. The Cdc6 protein may control the ATP-dependent initiation of replication by conferring ATPase activity to an origin-recognizing complex.

Functional replacement of the Saccharomyces cerevisiae Trg1/Pdi1 protein by members of the mammalian protein disulfide isomerase family.

The TRG1/PDI1 gene of Saccharomyces cerevisiae is essential for growth and encodes a lumenal endoplasmic reticulum (ER) glycoprotein that is structurally related to thioredoxin and is involved in the secretory pathway. We have tested whether the yeast Trg1/Pdi1 protein can be replaced in vivo by three members of the mammalian thioredoxin-related protein family, protein disulfide isomerase (PDI), ERp72, and ERp61. Multicopy plasmids containing galactose-inducible rodent PDI and ERp72 genes support germination and growth of haploid trg1 null mutants in galactose-containing media, whereas the ERp61 gene is inactive. Strains expressing PDI or ERp72 instead of Trg1 are thermosensitive. An overproduced mutant Trg1 protein lacking the HDEL retention signal supports growth, whereas a truncated version of the protein containing only one thioredoxin-like domain is inactive. The mammalian proteins were localized to both the soluble and microsomal membrane fraction of yeast cells. Our observations indicate that the two unglycosylated mammalian proteins PDI and ERp72 are capable of replacing at least some of the critical functions of Trg1, in spite of the fact that the three proteins diverge considerably in sequences surrounding the thioredoxin-related domains.

CDC25-dependent induction of inositol 1,4,5-trisphosphate and diacylglycerol in Saccharomyces cerevisiae by nitrogen.

The addition of ammonium sulfate to starved yeast cells leads to a 3- to 4-fold rapid increase of the second messengers inositol 1,4,5-trisphosphate (IP3) and diacylglycerol (DAG), the products of phosphoinositide-specific phospholipase C (PI-PLC). This response is reduced by dissecting the RAS-activating Cdc25 protein, and is completely abolished by the cdc25-1 mutation even at permissive temperature. Starved cdc25-1 mutant cells have a strongly reduced IP3 content, but an at least 10-fold increased DAG level compared to the isogenic wild-type strain. NH4 does not stimulate cAMP synthesis, and glucose does not induce IP3 and DAG. Our data suggest that the Cdc25 protein controls a nitrogen-specific signalling pathway involving the effector PI-PLC, in addition to the glucose-induced activation of adenylyl cyclase (AC).

The Saccharomyces cerevisiae TRG1 gene is essential for growth and encodes a lumenal endoplasmic reticulum glycoprotein involved in the maturation of vacuolar carboxypeptidase.

We have isolated and mapped to the left end of chromosome III a single-copy gene (TRG1) encoding a 72-kDa glycoprotein, by screening a yeast genomic library with a DNA probe specifying the catalytic center (APWCGHCK) of thioredoxin-related proteins. the TRG1 gene sequence predicts an amino-terminal leader peptide, two thioredoxin-like domains, five N-glycosylation sites and a carboxyl-terminal HDEL retention signal. The TRG1 protein shows about equal sequence similarity to a mammalian multifunctional protein family residing in the lumen of the endoplasmic reticulum (ER), and to a putative cytosolic alpha form of phosphoinositide-specific phospholipase C. Haploid cells do not survive TRG1 gene disruptions, unless an additional wild-type copy is generated by interchromosomal transposition. Antibodies raised against synthetic amino- and carboxyl-terminal epitopes recognize a pair of lumenl ER glycoproteins (gp70/72) and a cytosolic 48-kDa protein. A 1.8-kilobase TRG1 transcript was translated by a reticulocyte lysate into a 60-kDa protein, which was translocated and processed to a 72-kDa glycoprotein in the presence of ER membrane vesicles. The TRG1 gene was placed under the control of the galactose-inducible and glucose-repressible GAL1 promoter, leading to growth arrest in glucose media. Glucose repression of the TRG1 gene caused the disappearance of gp72 and the accumulation of procarboxypeptidase. Our data indicate that the TRG1 gene encodes a growth essential lumenal ER glycoprotein involved the maturation of vacuolar carboxypeptidase.

Nuclear migration in Saccharomyces cerevisiae is controlled by the highly repetitive 313 kDa NUM1 protein.

We have isolated a novel gene (NUM1) with unusual internal periodicity. The NUM1 gene encodes a 313 kDa protein with a potential Ca2+ binding site and a central domain containing 12 almost identical tandem repeats of a 64 amino acid polypeptide. num1-disrupted strains grow normally, but contain many budded cells with two nuclei in the mother cell instead of a single nucleus at the bud neck, while all unbudded cells are uninucleate. This indicates that most G2 nuclei divide in the mother before migrating to the neck, followed by the migration of one of the two daughter nuclei into the bud. Furthermore, haploid num1 strains tend to diploidize during mitosis, and homozygous num1 diploid or tetraploid cells sporulate to form many budded asci with up to eight haploid or diploid spores, respectively, indicating that meiosis starts before nuclear redistribution and cytokinesis. Our data suggest that the NUM1 protein is involved in the interaction of the G2 nucleus with the bud neck.

Site-directed mutagenesis of the Saccharomyces cerevisiae CDC25 gene: effects on mitotic growth and cAMP signalling.

A potential membrane-interacting site within the essential growth-controlling carboxy-terminal region of the CDC25 protein was interrupted by a lethal mutation (1461 Tyr----Asp and 1462 Leu----Arg). The elimination of two potential phosphorylation sites found in the same region (1489 Thr----Pro and 1584 Ser----Pro) does not affect growth but completely prevents glucose-induced cAMP signalling in the double mutant, whereas the single mutants produce normal or slightly retarded cAMP signals. A cluster of five potential targets for cAMP-dependent phosphorylation at the amino-terminal region could be deleted without affecting phenotypic properties. It is concluded that the carboxy-terminal 137 residues of the CDC25 protein are involved in three different functions: control of mitotic growth, glucose-induced hyperactivation of adenylate cyclase, and feed-back inhibition of cAMP synthesis.

Restoration of the yeast LEU2 gene by transcriptionally controlled recombination between tandem repeats.

The LEU2 gene of a his3 strain was inactivated by inserting the HIS3 gene between two overlapping inactive leu2 gene fragments, and mitotic stability of the resulting leu2:HIS3::leu2 sequence was measured under leucine repression and derepression. Both inactive leu2 regions were transcribed under derepressing conditions (growth in low leucine), and the LEU2 gene was completely restored by illegitimate recombination between the overlapping tandem repeats, leading to the loss of the intervening HIS3 gene. In contrast, only the downstream leu2 fragment was transcribed upon leucine repression, and the HIS3 insert in the leu2 region remained intact. The reciprocal experiment (inactivation of the HIS3 gene by inserting the marker gene LEU2) revealed a moderate rate of HIS3 restoration and LEU2 excision, reflecting transcriptional activity of the HIS3 region intermediate between that of LEU2 transcription in the repressed and derepressed state.

Glucose-induced cAMP signaling in Saccharomyces cerevisiae is mediated by the CDC25 protein.

Functional mapping of the cell cycle START gene CDC25 has revealed two domains which are dispensable for viability (germination and growth in glucose media), but are essential for sporulation and differentially involved in glucose-induced cAMP signaling. The transient rise of cAMP is completely prevented by various deletions within the amino-terminal half (alpha domain) of the CDC25 gene product. In contrast, the deletion of the carboxy-terminal 38 residues (beta 2 domain) results in a rapid, but persisting, rise of cAMP. Our data suggest that the alpha domain of the CDC25 protein is involved in glucose signal transduction, whereas the beta 2 domain is required for downregulating the cAMP control chain.

Cloning and characterization of the Saccharomyces cerevisiae CDC6 gene.

The yeast cell division cycle gene CDC6 was isolated by complementation of a temperature-sensitive cdc6 mutant with a genomic library. The amino acid sequence of the 48 kDalton CDC6 gene product, as deduced from DNA sequence data, includes the three consensus peptide motifs involved in guanine nucleotide binding and GTPase activity, a target site for cAMP-dependent protein kinase and a carboxy-terminal domain related to metallothionein sequences. A plasmid-encoded CDC6-beta-galactosidase hybrid protein was located at the plasma membrane by indirect immunofluorescence. Disruption experiments indicate that the CDC6 gene product is essential for mitotic growth.

Domains of the Saccharomyces cerevisiae CDC25 gene controlling mitosis and meiosis.

The cell division cycle gene CDC25 was replaced by various disrupted and deleted mutant copies. Mutants disrupted at a central position of the gene, or lacking 532 residues within the amino-terminal half of the gene product grow normally in glucose, but not in acetate media, and they fail to sporulate as homozygous diploids. Disruptions or deletions within the carboxy-terminal half are lethal, except for the deletion of the 38 carboxy-terminal residues, which are required for sporulation but not for growth in glucose or acetate media. It is concluded that distinct domains of the CDC25 gene product are involved in the control of mitosis and/or meiosis.

Purification of a yeast protein kinase sharing properties with type I and type II casein kinases.

Cyclic nucleotide independent protein kinases preferring casein as in vitro substrates were resolved into four distinct species. Only one of the enzymes (CKII) was retained by DEAE-cellulose, whereas the three other enzymes (CKI-1, CKI-2, and CKI-3) were absorbed to CM-Sephadex, eluted with 250 and 600 mM NaCl, and fractionated by heparin-Sepharose chromatography. The casein kinase CKI-3 eluting at the highest NaCl concentration (550 mM) was purified to electrophoretic homogeneity by fast protein liquid chromatography. CKI-1 and CKI-2 correspond to mammalian type I casein kinase, because they bind to CM-Sephadex, they are monomeric enzymes of molecular weights below 50,000, they accept ATP exclusively (CKI-1) or predominantly (CKI-2) as phosphate donor, and they are either completely or relatively heparin insensitive. CKII corresponds to type II casein kinase due to its chromatographic properties, complex quaternary structure, nucleotide specificity (both ATP and GTP are phosphate donors), and heparin sensitivity. CKI-3 shares the following properties with type I casein kinases: it is retained by CM-Sephadex but not by DEAE-cellulose, and it consists of a monomeric protein having a molecular weight of 38,000. On the other hand, CKI-3 accepts both ATP and GTP with equal efficiency, and it is heparin sensitive (50% inhibition at 0.3 microgram/mL) like type II casein kinases. CKI-3 differs from the other three yeast casein kinases in requiring a low pH (5.5) and a high MgCl2 concentration (50 mM) for optimal activity. All four casein kinases phosphorylate their own catalytic protein at serine and threonine residues.

Isolation and nucleotide sequence of a Saccharomyces cerevisiae protein kinase gene suppressing the cell cycle start mutation cdc25.

We have isolated two unlinked yeast genes complementing the cell division cycle mutant cdc25-1, one containing the wild type allele CDC25 and the other acting as an extragenic suppressor of the cdc25-1 lesion if present on a multicopy plasmid. Nucleotide sequence analysis of the suppressor gene has revealed an open reading frame that encodes a 45,000-dalton protein belonging to the protein kinase family. The cdc25-suppressing protein kinase (PK-25) shows 48% sequence similarity to the catalytic subunit (CA) of mammalian cAMP-dependent protein kinase and 27-31% similarity to cyclic nucleotide-independent enzymes, including the yeast CDC28 gene product. The PK-25 gene was targeted by integrative transformation into a chromosomal region unlinked to the CYR2 site, the structural gene of CA. The cdc25-suppressing protein kinase is also functionally different from CA, since cyr2 strains deficient in the free catalytic subunit remain temperature sensitive if transformed with a multicopy plasmid containing the PK-25 gene. Furthermore, a deficiency of the cAMP-binding regulatory subunit (RA) caused by the bcy1 mutation fails to suppress the cdc25 mutation, indicating that PK-25 does not interact with the cAMP receptor protein. Our data suggest that the cdc25 suppressor gene encodes a cAMP-independent protein kinase involved in the control of the cell cycle start.

Mitochondrial polypeptide elongation factor EF-Tu of Saccharomyces cerevisiae. Functional and structural homologies to Escherichia coli EF-Tu.

The polypeptide elongation factor EF-Tu was isolated from a mitochondrial 100 000 x g supernatant of the yeast Saccharomyces cerevisiae and purified over 880-fold by DEAE-Sephadex chromatography and gel filtration. The factor efficiently replaces bacterial EF-Tu in a phenylalanine polymerizing cell-free system of Escherichia coli, it binds GDP and it protects phenylalanyl-tRNA against hydrolysis of the ester bond in the presence of 10 mM GTP. The polymerizing activity of the mitochondrial factor is inhibited to 90% by 50 microM N-ethylmaleimide and to 50% by 2.5 microM kirromycin. The purified factor contains two major polypeptides of apparent molecular weights 48 000 and 34 000. Antibodies raised against the 48 000-Mr protein react with EF-TuE. coli, as revealed by immune blotting and by the inhibition of phenylalanine polymerization. No reaction was observed between anti-(34 000-Mr) and 48 000-Mr protein or EF-TuE. coli. The 48 000-Mr protein has the same isoelectric point (pI = 6.2) and a content of cysteine and basic amino acids similar to the bacterial EF-Tu. It is concluded that the 48 000-Mr protein is the analogue to EF-TuE. coli, and that yeast mitochondrial EF-Tu is functionally and structurally more related to bacterial EF-Tu than cytosolic EF-1 of the same cell.