Alternative topogenic signals in peroxisomal citrate synthase of Saccharomyces cerevisiae.

The tripeptide serine-lysine-leucine (SKL) occurs at the carboxyl terminus of many peroxisomal proteins and serves as a peroxisomal targeting signal. Saccharomyces cerevisiae has two isozymes of citrate synthase. The peroxisomal form, encoded by CIT2, terminates in SKL, while the mitochondrial form, encoded by CIT1, begins with an amino-terminal mitochondrial signal sequence and ends in SKN. We analyzed the importance of SKL as a topogenic signal for citrate synthase, using oleate to induce peroxisomes and density gradients to fractionate organelles. Our experiments revealed that SKL was necessary for directing citrate synthase to peroxisomes. C-terminal SKL was also sufficient to target a leaderless version of mitochondrial citrate synthase to peroxisomes. Deleting this tripeptide from the CIT2 protein caused peroxisomal citrate synthase to be missorted to mitochondria. These experiments suggest that the CIT2 protein contains a cryptic mitochondrial targeting signal.     

The CIT3 gene of Saccharomyces cerevisiae encodes a second mitochondrial isoform of citrate synthase

We have identified a third citrate synthase gene in Saccharomyces cerevisiae which we have called CIT3 Complementation of a citrate synthase-deficient strain of Escherichia coli by lacZ  :: CIT3 gene fusions demonstrated that the CIT3 gene encodes an active citrate synthase. The CIT3 gene seems to be regulated in the same way as CIT1 , which encodes the mitochondrial isoform of citrate synthase. Deletion of the CIT3 gene in a Δ cit1 background severely reduced growth on the respiratory substrate glycerol, whilst multiple copies of the CIT3 gene in a Δ cit1 background significantly improved growth on acetate. In vitro import experiments showed that cit3p is transported into the mitochondria. Taken together, these data show that the CIT3 gene encodes a second mitochondrial isoform of citrate synthase.     

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.     

The lactate-proton symport of Saccharomyces cerevisiae is encoded by JEN1

A mutant of Saccharomyces cerevisiae deficient in the lactate-proton symport was isolated. Transformation of the mutant with a yeast genomic library allowed the isolation of the gene JEN1 that restored lactate transport. Disruption of JEN1 abolished uptake of lactate. The results indicate that, under the experimental conditions tested, no other monocarboxylate permease is able to efficiently transport lactate in S. cerevisiae.     

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.     

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.     

Transposed LEU2 gene of Saccharomyces cerevisiae is regulated normally.

The repression of beta-isopropylmalate dehydrogenase, the LEU2 gene product, by leucine and leucine plus threonine was unaffected by the transposition of LEU2 from its original locus on chromosome III to a new locus within the ribosomal deoxyribonucleic acid gene cluster on chromosome XII. Since the expression of the LEU2 gene is probably controlled at a pretranslational level, we conclude that the recombinant plasmid used for transformation carries regulatory information in addition to LEU2 structural information.     

Mechanism of the aromatic aminotransferase encoded by the Aro8 gene from Saccharomyces cerevisiae

The amino acid l-lysine is synthesized in Saccharomyces cerevisiae via the α-aminoadipate pathway. An as yet unidentified PLP-containing aminotransferase is thought to catalyze the formation of α-aminoadipate from α-ketoadipate in the l-lysine biosynthetic pathway that could be the yeast Aro8 gene product. A screen of several different amino acids and keto-acids showed that the enzyme uses l-tyrosine, l-phenylalanine, α-ketoadipate, and l-α-aminoadipate as substrates. The UV–visible spectrum of the aminotransferase exhibits maxima at 280 and 343 nm at pH 7.5. As the pH is decreased the peak at 343 nm (the unprotonated internal aldimine) disappears and two new peaks at 328 and 400 nm are observed representing the enolimine and ketoenamine tautomers of the protonated aldimine, respectively. Addition, at pH 7.1, of α-ketoadipate to free enzyme leads to disappearance of the absorbance at 343 nm and appearance of peaks at 328 and 424 nm. The V/E_t and V/K_{α-ketoadipate}E_t pH profiles are pH independent from pH 6.5 to 9.6, while the V/K_l-tyrosine pH-rate profile decreases below a single pK_a of 7.0 ± 0.1. Data suggest the active enzyme form is with the internal aldimine unprotonated. We conclude the enzyme should be categorized as a α-aminoadipate aminotransferase.     

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.     

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.     

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.