Lack of lactate-proton symport activity in pck1 mutants of Saccharomyces cerevisiae

Abstract Mutants of Saccharomyces cerevisiae without phosphoenolpyruvate carboxykinase activity showed no measurable lactate proton symport, while mutants without fructose-1,6-bisphosphatase had normal transport activity. Incubation of a pck1 mutant, under derepression conditions in the presence of glycerol, restored the activity of the lactate-proton symport, with identical kinetic characteristics to that in the wild-type. For efficient lactate-proton symport activity, not only is an external inducer such as lactic acid needed, but also a molecule derived from the acid metabolism may be necessary.     

The NADH diphosphatase encoded by the Saccharomyces cerevisiae NPY1 nudix hydrolase gene is located in peroxisomes

The NPY1 nudix hydrolase gene of Saccharomyces cerevisiae has been cloned and shown to encode a diphosphatase (pyrophosphatase) with NADH as the preferred substrate, giving NMNH and AMP as products. NADPH, diadenosine diphosphate, NAD+, NADP+, and ADP-ribose were also utilized efficiently. Km values for NADH, NAD+, and ADP-ribose were 0.17, 0.5, and 1.3 mM and kcat values 1.5, 0.6, and 0.6 s(-1), respectively. NPY1 has a potential C-terminal tripeptide PTS1 peroxisomal targeting signal (SHL). By fusing NPY1 to the C-terminus of yeast-enhanced green fluorescent protein, the enzyme was found to be targeted to peroxisomes. Colocalization with peroxisomal thiolase was also shown by indirect immunofluorescence. Related sequences in other organisms also have potential PTS1 signals, suggesting an important peroxisomal function for this protein. This function may be the regulation of nicotinamide coenzyme concentrations independently of those in other compartments or the elimination of oxidized nucleotide derivatives from the peroxisomal environment.     

Kinetics and pH-dependence of glycine-proton symport in Saccharomyces cerevisiae

Interactions between intracellular pH (pHi) and H+-coupled transmembrane transport of glycine have been studied by means of 31P-NMR, using both aerobic and 'energy starved' cells of the yeast Saccharomyces cerevisiae. The general features of glycine transport in the yeast strain used (NCYC 239) are similar to those already reported for Saccharomyces carlsbergensis and S. cerevisiae, there being two kinetically distinct glycine uptake systems, with pH-independent K1/2 values near 14 and 0.4mM, respectively, but pH-dependent maximal velocities. Glycine transport itself has no measurable effect on pHi in aerobic cells, and only a marginal effect in energy-starved cells, but changes of pHi, imposed by extracellular addition of butyric acid, strongly influence glycine transport. Indeed, the dependence of glycine influx (in energy-starved cells) upon cytoplasmic H+ concentration appears to be third order, showing Hill slopes of 2.7-3.0. A crucial kinetic role for cytoplasmic pH in glycine transport is further indicated by a proportionality between the decline of flux and the decline of pHi produced by various metabolic inhibitors and uncouplers. Extracellular pH (pHo), by contrast, has only a weak effect on glycine influx, showing a Hill slope of 0.5. The major observations can be accommodated by a simple cyclic carrier scheme, in which 2 or more protons are transported along with glycine, but only one extracellular proton binding site dissociates in the testing range, with a pK near 5.5. The model requires a finite membrane potential, which must be somewhat sensitive to both pHi and pHo, and accommodates the discrepancy between measured net proton flux (one per glycine) and the kinetically required proton flux (two or more per glycine) by shunting through other proton-conducting pathways in the yeast membrane.     

Citrate synthase encoded by the CIT2 gene of Saccharomyces cerevisiae is peroxisomal.

The product of the CIT2 gene has the tripeptide SKL at its carboxyl terminus. This amino acid sequence has been shown to act as a peroxisomal targeting signal in mammalian cells. We examined the subcellular site of this extramitochondrial citrate synthase. Cells of Saccharomyces cerevisiae were grown on oleate medium to induce peroxisome proliferation. A fraction containing membrane-enclosed vesicles and organelles was analyzed by sedimentation on density gradients. In wild-type cells, the major peak of citrate synthase activity was recovered in the mitochondrial fraction, but a second peak of activity cosedimented with peroxisomes. The peroxisomal activity, but not the mitochondrial activity, was inhibited by incubation at pH 8.1, a characteristic of the extramitochondrial citrate synthase encoded by the CIT2 gene. In a strain in which the CIT1 gene encoding mitochondrial citrate synthase had been disrupted, the major peak of citrate synthase activity was peroxisomal, and all of the activity was sensitive to incubation at pH 8.1. Yeast cells bearing a cit2 disruption were unable to mobilize stored lipids and did not form stable peroxisomes in oleate. We conclude that citrate synthase encoded by CIT2 is peroxisomal and participates in the glyoxylate cycle.     

Nuclear exopolyphosphatase of Saccharomyces cerevisiae is not encoded by the PPX1 gene encoding the major yeast exopolyphosphatase

Intact nuclei from a parental strain CRY and a PPX1-mutant CRX of Saccharomyces cerevisiae were isolated and found to be essentially free of cytoplasmic, mitochondrial and vacuolar marker enzymes. The protein-to-DNA ratios of the nuclei were 22 and 30 for CRY and CRX nuclei, respectively. An exopolyphosphatase (exopolyPase) with molecular mass of approximately 57 kDa and a pyrophosphatase (PPase) of approximately 41 kDa were detected in the parental strain CRY. Inactivation of PPX1 encoding a major exopolyPase (PPX1) in S. cerevisiae did not result in considerable changes in the content and properties of nuclear exopolyPase as compared to the parental strain of S. cerevisiae. Consequently, the nuclear exopolyPase was not encoded by PPX1. In the CRX strain, the exopolyPase was stimulated by bivalent metal cations. Co2+, the best activator, stimulated it by approximately 2.5-fold. The exopolyPase activity was nearly the same with polyphosphate (polyP) chain lengths ranging from 3 to 208 orthophosphate when measured with Mg2+. With Co 2+, the exopolyPase activity increased along with the increase in polymerization degree of the substrate.     

Expression of proteins encoded by foreign genes in Saccharomyces cerevisiae

The yeast, Saccharomyces cerevisiae is currently used for the production of recombinant DNA-generated proteins derived from a variety of eukaryotic organisms. The applications of a yeast-based technology in the production of proteins for pharmaceutical and industrial purposes is discussed including current methods for introducing recombinant genes into yeast and strategies for maximizing their expression.     

High molecular mass exopolyphosphatase from the cytosol of the yeast Saccharomyces cerevisiae is encoded by the PPN1 gene

It has been shown that the high molecular mass exopolyphosphatase localized in cytosol of the yeast Saccharomyces cerevisiae is encoded by the PPN1 gene. This enzyme is expressed under special culture conditions when stationary phase cells are passing on to new budding on glucose addition and phosphate excess. The enzyme under study releases orthophosphate from the very beginning of polyphosphate hydrolysis.     

ADP ribosylation factor is an essential protein in Saccharomyces cerevisiae and is encoded by two genes.

ADP ribosylation factor (ARF) is a ubiquitous 21-kDa GTP-binding protein in eucaryotes. ARF was first identified in animal cells as the protein factor required for the efficient ADP-ribosylation of the mammalian G protein Gs by cholera toxin in vitro. A gene (ARF1) encoding a protein homologous to mammalian ARF was recently cloned from Saccharomyces cerevisiae (Sewell and Kahn, Proc. Natl. Acad. Sci. USA, 85:4620-4624, 1988). We have found a second gene encoding ARF in S. cerevisiae, ARF2. The two ARF genes are within 28 centimorgans of each other on chromosome IV, and the proteins encoded by them are 96% identical. Disruption of ARF1 causes slow growth, cold sensitivity, and sensitivity to normally sublethal concentrations of fluoride ion in the medium. Disruption of ARF2 causes no detectable phenotype. Disruption of both genes is lethal; thus, ARF is essential for mitotic growth. The ARF1 and ARF2 proteins are functionally homologous, and the phenotypic differences between mutations in the two genes can be accounted for by the level of expression; ARF1 produces approximately 90% of total ARF. Among revertants of the fluoride sensitivity of an arf1 null mutation were ARF1-ARF2 fusion genes created by a gene conversion event in which the deleted ARF1 sequences were repaired by recombination with ARF2.     

Reversible loss of affinity induced by glucose in the maltose-H+ symport of Saccharomyces cerevisiae

Glucose represses and inactivates maltose transport in Saccharomyces cerevisiae. The inactivation has been described as an irreversible process involving proteolysis. We have studied the inactivation of the maltose-H+ symport in this yeast and have observed that the mechanism of inactivation depends on the physiological conditions. In resting cells there was a decrease in transport capacity. The rate of decrease was enhanced nonspecifically by the presence of a sugar, glucose being more effective than maltose. In growing cells, glucose induced a decrease in affinity of the H+-symport which could be recovered by starvation, even in the presence of cycloheximide; there was no loss in capacity or, if present, this loss could be explained fully by the dilution due to repression during growth on glucose. We submit that in growing cells inactivation consists in a reversible modification of the permease not involving proteolysis.     

Nis1 encoded by YNL078W: a new neck protein of Saccharomyces cerevisiae

An uncharacterized gene, YNL078W, was isolated by the two-hybrid screening method using SHS1 (one of the septin genes) as bait and designated NIS1 (Neck protein Interacting with Septins). Nis1 interacts with all septins in the two-hybrid assay system. Physical interaction between Nis1 and Shs1 in vivo was confirmed by a co-immunoprecipitation experiment. Neither disruption nor overexpression of NIS1 caused a prominent phenotypic change. NAP1 was isolated by two-hybrid screening using NIS1 as bait. We detected physical interaction between Nis1 and Nap1 in vivo by a co-immunoprecipitation experiment. Nis1 was found to bind Gin4 and Kcc4 in the two-hybrid assay. Thus, a number of the proteins interacting with Nis1 are members of the mitotic signaling network. The stable maintenance of Nis1 was dependent on Nap1. Nis1 was phosphorylated throughout the cell cycle and was less abundant in G2/M phase. GFP-Nis1 is localized in the nucleus throughout the cell cycle and in the bud neck at G2/M phase in a septin-dependent manner. Altogether, the findings suggest that Nis1 may play a non-essential role in the mitotic signaling network.     

Transposition of Saccharomyces cerevisiae Ty1 retrotransposon is activated by improper cryopreservation

Ty1 is a retrotransposon of the yeast Saccharomyces cerevisiae whose transposition at new locations in the host genome is activated by stress conditions, such as exposure to UV light, X-rays, nitrogen starvation. In this communication, we supply evidence that cooling for 2 h at +4 °C followed by freezing for 1 h at −10 °C and 16 h at −20 °C also increased Ty1 transposition. The mobility of Ty1 was induced by cooling at slow rates (3 °C/min) and the accumulation of trehalose inside cells or the cooling at high rates (100 °C/min) inhibited significantly the induction of the transposition. The freeze-induced Ty1 transposition did not occur in mitochondrial mutants (rho⁻) and in cells with disrupted SCO1 gene (Δsco1 cells) evidencing that the Ty1 transposition induced by cooling depends on the mitochondrial oxidative phosphorylation. We also found that the freeze induced Ty1 transposition is associated with increased synthesis and accumulation of superoxide anions (O⁻₂) into the cells. Accumulation of O⁻₂ and activation of Ty1 transposition were not observed after cooling of cells with compromised mitochondrial functions (rho⁻, Δsco1), or in cells pretreated with O⁻₂ scavengers. It is concluded that (i) elevated levels of reactive oxygen species (ROS) have a key role in activation the transposition of Ty1 retrotransposon in yeast cells undergoing freezing and (ii) given the deleterious effect of increased ROS levels on cells, special precautions should be taken to avoid ROS production and accumulation during cryopreservation procedures.     

The kinesin-related protein Kip1p of Saccharomyces cerevisiae is bipolar.

Kip1p is a mitotic spindle-associated kinesin-related protein in Saccharomyces cerevisiae that participates in spindle pole separation. Here, we define the domain arrangement and polypeptide composition of the Kip1p holoenzyme. Electron microscopy of rotary shadowed Kip1p molecules revealed two globular domains 14 nm in diameter connected by a 73-nm long stalk. When the Kip1p domain homologous to the kinesin motor domain was decorated with an unrelated protein, the diameter of the globular domains at both ends of the stalk increased, indicating that Kip1p is bipolar. Soluble Kip1p isolated from S. cerevisiae cells was homomeric, based on the similarity of the sedimentation coefficients of native Kip1p from S. cerevisiae and Kip1p which was purified after expression in insect cells. The holoenzyme molecular weight was estimated using the sedimentation coefficient and Stokes radius, and was most consistent with a tetrameric composition. Kip1p exhibited an ionic strength-dependent transition in its sedimentation coefficient, revealing a potential regulatory mechanism. The position of kinesin motor-related domains at each end of the stalk may allow Kip1p to cross-link either parallel or antiparallel microtubules during mitotic spindle assembly and pole separation.     

Identification of two proteins encoded by the Saccharomyces cerevisiae GAL4 gene.

We placed the Saccharomyces cerevisiae GAL4 gene under control of the galactose regulatory system by fusing it to the S. cerevisiae GAL1 promoter. After induction with galactose, GAL4 is now transcribed at about 1,000-fold higher levels than in wild-type S. cerevisiae. This regulated high-level expression has enabled us to tentatively identify two GAL4-encoded proteins.     

The second subunit of DNA polymerase III (delta) is encoded by the HYS2 gene in Saccharomyces cerevisiae.

DNA polymerase III (delta) of Saccharomyces cerevisiae is purified as a complex of at least two polypeptides with molecular masses of 125 and 55 kDa as judged by SDS-PAGE. In this paper we determine partial amino acid sequences of the 125 and 55 kDa polypeptides and find that they match parts of the amino acid sequences predicted from the nucleotide sequence of the CDC2 and HYS2 genes respectively. We also show by Western blotting that Hys2 protein co-purifies with DNA polymerase III activity as well as Cdc2 polypeptide. The complex form of DNA polymerase III activity could not be detected in thermosensitive hys2 mutant cell extracts, although another form of DNA polymerase III was found. This form of DNA polymerase III, which could also be detected in wild-type extracts, was not associated with Hys2 protein and was not stimulated by addition of proliferating cell nuclear antigen (PCNA), replication factor A (RF-A) or replication factor C (RF-C). The temperature-sensitive growth phenotype of hys2-1 and hys2-2 mutations could be suppressed by the CDC2 gene on a multicopy plasmid. These data suggest that the 55 kDa polypeptide encoded by the HYS2 gene is one of the subunits of DNA polymerase III complex in S.cerevisiae and is required for highly processive DNA synthesis catalyzed by DNA polymerase III in the presence of PCNA, RF-A and RF-C.     

Saccharomyces cerevisiae protein phosphatase 2A performs an essential cellular function and is encoded by two genes.

Two genes (PPH21 and PPH22) encoding the yeast homologues of protein serine-threonine phosphatase 2A have been cloned from a Saccharomyces cerevisiae genomic library using a rabbit protein phosphatase 2A cDNA as a hybridization probe. The PPH genes are genetically linked on chromosome IV and are predicted to encode polypeptides each with 74% amino acid sequence identity to rabbit type 2A protein phosphatase, indicating once again the extraordinarily high degree of sequence conservation shown by protein-phosphatases from different species. The two PPH genes show less than 10% amino acid sequence divergence from each other and while disruption of either PPH gene alone is without any major effect, the double disruption is lethal. This indicates that protein phosphatase 2A activity is an essential cellular function in yeast. Measurement of type 2A protein phosphatase activity in yeast strains lacking one or other of the genes indicates that they account for most, if not all, protein phosphatase 2A activity in the cell.