scholarly journals The STK2 gene, which encodes a putative Ser/Thr protein kinase, is required for high-affinity spermidine transport in Saccharomyces cerevisiae.

1997 ◽  
Vol 17 (6) ◽  
pp. 2994-3004 ◽  
Author(s):  
M Kaouass ◽  
M Audette ◽  
D Ramotar ◽  
S Verma ◽  
D De Montigny ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Protein Kinase ◽  
Fusion Gene ◽  
Transport Systems ◽  
Coding Region ◽  
Kinase Gene ◽  
Lower Affinity ◽  
High Affinity ◽  
Polyamine Transport ◽  
Exogenous Spermidine

Eukaryotic polyamine transport systems have not yet been characterized at the molecular level. We have used transposon mutagenesis to identify genes controlling polyamine transport in Saccharomyces cerevisiae. A haploid yeast strain was transformed with a genomic minitransposon- and lacZ-tagged library, and positive clones were selected for growth resistance to methylglyoxal bis(guanylhydrazone) (MGBG), a toxic polyamine analog. A 747-bp DNA fragment adjacent to the lacZ fusion gene rescued from one MGBG-resistant clone mapped to chromosome X within the coding region of a putative Ser/Thr protein kinase gene of previously unknown function (YJR059w, or STK2). A 304-amino-acid stretch comprising 11 of the 12 catalytic subdomains of Stk2p is approximately 83% homologous to the putative Pot1p/Kkt8p (Stk1p) protein kinase, a recently described activator of low-affinity spermine uptake in yeast. Saturable spermidine transport in stk2::lacZ mutants had an approximately fivefold-lower affinity and twofold-lower Vmax than in the parental strain. Transformation of stk2::lacZ cells with the STK2 gene cloned into a single-copy expression vector restored spermidine transport to wild-type levels. Single mutants lacking the catalytic kinase subdomains of STK1 exhibited normal parameters for the initial rate of spermidine transport but showed a time-dependent decrease in total polyamine accumulation and a low-level resistance to toxic polyamine analogs. Spermidine transport was repressed by prior incubation with exogenous spermidine. Exogenous polyamine deprivation also derepressed residual spermidine transport in stk2::lacZ mutants, but simultaneous disruption of STK1 and STK2 virtually abolished high-affinity spermidine transport under both repressed and derepressed conditions. On the other hand, putrescine uptake was also deficient in stk2::lacZ mutants but was not repressed by exogenous spermidine. Interestingly, stk2::lacZ mutants showed increased growth resistance to Li+ and Na+, suggesting a regulatory relationship between polyamine and monovalent inorganic cation transport. These results indicate that the putative STK2 Ser/Thr kinase gene is an essential determinant of high-affinity polyamine transport in yeast whereas its close homolog STK1 mostly affects a lower-affinity, low-capacity polyamine transport activity.

Efficient expression of the Saccharomyces cerevisiae PGK gene depends on an upstream activation sequence but does not require TATA sequences

1986 ◽  
Vol 6 (12) ◽  
pp. 4335-4343
Author(s):  
J E Ogden ◽  
C Stanway ◽  
S Kim ◽  
J Mellor ◽  
A J Kingsman ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Transcription Initiation ◽  
Fusion Gene ◽  
Phosphoglycerate Kinase ◽  
Human Interferon ◽  
Coding Region ◽  
Kinase Gene ◽  
Upstream Activation Sequence ◽  
Efficient Expression ◽  
Activation Sequence

The Saccharomyces cerevisiae PGK (phosphoglycerate kinase) gene encodes one of the most abundant mRNA and protein species in the cell. To identify the promoter sequences required for the efficient expression of PGK, we undertook a detailed internal deletion analysis of the 5' noncoding region of the gene. Our analysis revealed that PGK has an upstream activation sequence (UASPGK) located between 402 and 479 nucleotides upstream from the initiating ATG sequence which is required for full transcriptional activity. Deletion of this sequence caused a marked reduction in the levels of PGK transcription. We showed that PGK has no requirement for TATA sequences; deletion of one or both potential TATA sequences had no effect on either the levels of PGK expression or the accuracy of transcription initiation. We also showed that the UASPGK functions as efficiently when in the inverted orientation and that it can enhance transcription when placed upstream of a TRP1-IFN fusion gene comprising the promoter of TRP1 fused to the coding region of human interferon alpha-2.

scholarly journals Efficient expression of the Saccharomyces cerevisiae PGK gene depends on an upstream activation sequence but does not require TATA sequences.

1986 ◽  
Vol 6 (12) ◽  
pp. 4335-4343 ◽  
Author(s):  
J E Ogden ◽  
C Stanway ◽  
S Kim ◽  
J Mellor ◽  
A J Kingsman ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Transcription Initiation ◽  
Fusion Gene ◽  
Phosphoglycerate Kinase ◽  
Human Interferon ◽  
Coding Region ◽  
Kinase Gene ◽  
Upstream Activation Sequence ◽  
Efficient Expression ◽  
Activation Sequence

The Saccharomyces cerevisiae PGK (phosphoglycerate kinase) gene encodes one of the most abundant mRNA and protein species in the cell. To identify the promoter sequences required for the efficient expression of PGK, we undertook a detailed internal deletion analysis of the 5' noncoding region of the gene. Our analysis revealed that PGK has an upstream activation sequence (UASPGK) located between 402 and 479 nucleotides upstream from the initiating ATG sequence which is required for full transcriptional activity. Deletion of this sequence caused a marked reduction in the levels of PGK transcription. We showed that PGK has no requirement for TATA sequences; deletion of one or both potential TATA sequences had no effect on either the levels of PGK expression or the accuracy of transcription initiation. We also showed that the UASPGK functions as efficiently when in the inverted orientation and that it can enhance transcription when placed upstream of a TRP1-IFN fusion gene comprising the promoter of TRP1 fused to the coding region of human interferon alpha-2.

scholarly journals Screening for Modulators of Spermine Tolerance Identifies Sky1, the SR Protein Kinase of Saccharomyces cerevisiae, as a Regulator of Polyamine Transport and Ion Homeostasis

2001 ◽  
Vol 21 (1) ◽  
pp. 175-184 ◽  
Author(s):  
Omri Erez ◽  
Chaim Kahana
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Protein Kinase ◽  
Osmotic Shock ◽  
Ion Homeostasis ◽  
Inorganic Ions ◽  
Yeast Cells ◽  
Wild Type ◽  
Sr Protein ◽  
Polyamine Transport ◽  
Increased Sensitivity

ABSTRACT Although most cells are capable of transporting polyamines, the mechanism that regulates polyamine transport in eukaryotes is still largely unknown. Using a genetic screen for clones capable of restoring spermine sensitivity to spermine-tolerant mutants ofSaccharomyces cerevisiae, we have demonstrated that Sky1p, a recently identified SR protein kinase, is a key regulator of polyamine transport. Yeast cells deleted for SKY1 developed tolerance to toxic levels of spermine, while overexpression of Sky1p in wild-type cells increased their sensitivity to spermine. Expression of the wild-type Sky1p but not of a catalytically inactive mutant restored sensitivity to spermine. SKY1 disruption results in dramatically reduced uptake of spermine, spermidine, and putrescine. In addition to spermine tolerance, sky1Δ cells exhibit increased tolerance to lithium and sodium ions but somewhat increased sensitivity to osmotic shock. The observed halotolerance suggests potential regulatory interaction between the transport of polyamines and inorganic ions, as suggested in the case of the Ptk2p, a recently described regulator of polyamine transport. We demonstrate that these two kinases act in two different signaling pathways. While deletion or overexpression of SKY1 did not significantly affect Pma1p activity, the ability of overexpressed Sky1p, Ptk1p, and Ptk2p to increase sensitivity to LiCl depends on the integrity ofPPZ1 but not of ENA1.

scholarly journals Mutational Analysis of the Structure and Localization of the Nucleolus in the Yeast Saccharomyces cerevisiae

1998 ◽  
Vol 143 (1) ◽  
pp. 23-34 ◽  
Author(s):  
M. Oakes ◽  
J.P. Aris ◽  
J.S. Brockenbrough ◽  
H. Wai ◽  
L. Vu ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Nuclear Envelope ◽  
Mutational Analysis ◽  
Fusion Gene ◽  
Nuclear Periphery ◽  
Rdna Locus ◽  
Coding Region ◽  
Yeast Saccharomyces Cerevisiae ◽  
Pol I ◽  
Rdna Expression

The nucleolus in Saccharomyces cerevisiae is a crescent-shaped structure that makes extensive contact with the nuclear envelope. In different chromosomal rDNA deletion mutants that we have analyzed, the nucleolus is not organized into a crescent structure, as determined by immunofluorescence microscopy, fluorescence in situ hybridization, and electron microscopy. A strain carrying a plasmid with a single rDNA repeat transcribed by RNA polymerase I (Pol I) contained a fragmented nucleolus distributed throughout the nucleus, primarily localized at the nuclear periphery. A strain carrying a plasmid with the 35S rRNA coding region fused to the GAL7 promoter and transcribed by Pol II contained a rounded nucleolus that often lacked extensive contact with the nuclear envelope. Ultrastructurally distinct domains were observed within the round nucleolus. A similar rounded nucleolar morphology was also observed in strains carrying the Pol I plasmid in combination with mutations that affect Pol I function. In a Pol I–defective mutant strain that carried copies of the GAL7-35S rDNA fusion gene integrated into the chromosomal rDNA locus, the nucleolus exhibited a round morphology, but was more closely associated with the nuclear envelope in the form of a bulge. Thus, both the organization of the rDNA genes and the type of polymerase involved in rDNA expression strongly influence the organization and localization of the nucleolus.

scholarly journals Molecular analysis of SSN6, a gene functionally related to the SNF1 protein kinase of Saccharomyces cerevisiae.

1987 ◽  
Vol 7 (10) ◽  
pp. 3637-3645 ◽  
Author(s):  
J Schultz ◽  
M Carlson
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Protein Kinase ◽  
Nucleotide Sequence ◽  
Temperature Sensitive ◽  
Missense Mutations ◽  
Kinase Gene ◽  
Multiple Copies ◽  
Protein Kinase Gene ◽  
Rna Disruption ◽  
High Level

Mutations in the SSN6 gene suppress the invertase derepression defect caused by a lesion in the SNF1 protein kinase gene. We cloned the SSN6 gene of Saccharomyces cerevisiae and identified its 3.3-kilobase poly(A)-containing RNA. Disruption of the gene caused phenotypes similar to, but more severe than, those caused by missense mutations: high-level constitutivity for invertase, clumpiness, temperature-sensitive growth, alpha-specific mating defects, and failure to homozygous diploids to sporulate. In contrast, the presence of multiple copies of SSN6 interfered with derepression of invertase. An ssn6 mutation was also shown to cause glucose-insensitive expression of a GAL10-lacZ fusion and maltase. The mating defects of MAT alpha ssn6 strains were associated with production of two a-specific products, a-factor and barrier, and reduced levels of alpha-factor; no deficiency of MAT alpha 2 RNA was detected. We showed that ssn6 partially restored invertase expression in a cyr1-2 mutant, although ssn6 was clearly not epistatic to cyr1-2. We also determined the nucleotide sequence of SSN6, which is predicted to encode a 107-kilodalton protein with stretches of polyglutamine and poly(glutamine-alanine). Possible functions of the SSN6 product are discussed.

scholarly journals Characterization of putrescine- and spermidine-transport systems of a rat pancreatic acinar tumoral cell line (AR4-2J)

1990 ◽  
Vol 269 (3) ◽  
pp. 629-632 ◽  
Author(s):  
T G Nicolet ◽  
J L Scemama ◽  
L Pradayrol ◽  
C Seva ◽  
N Vaysse
Keyword(s):  
Cell Line ◽  
Choline Chloride ◽  
The Other ◽  
Transport Systems ◽  
Lower Affinity ◽  
Polyamine Transport ◽  
Tumoral Cell ◽  
Cell Growth And Differentiation ◽  
The Difference

Polyamines are polycationic molecules essential for cell growth and differentiation. Recent work has focused on cell polyamine-transport systems as a way to regulate intracellular polyamine levels. In this study, we demonstrate the presence of two different active transporters for putrescine and spermidine in a rat tumoral cell line (AR4-2J). The first has a Km of 3.1 microM and a Vmax of 3.7 pmol/15 min per micrograms of DNA for putrescine and the second a Km of 0.42 microM and a Vmax of 4.7 pmol/15 min per micrograms of DNA for spermidine. Competition studies performed between the polyamines confirm the difference between these two carriers; one has an equal affinity for the three main polyamines, and the other has a lower affinity for putrescine. Amino acids do not share this transport system, which is Na(+)-independent. Choline chloride inhibits selectively and in a dose-responsive manner the uptake of putrescine without affecting that of spermidine. These data demonstrate that AR4-2J cells possess two polyamine transporters; one is specific for aminopropyl groups (spermidine and spermine), and the other is choline-sensitive, but cannot discriminate between aminobutyl (putrescine) and aminopropyl groups.

scholarly journals Variable H+/substrate stoicheiometries in Rhodotorula gracilis are caused by a pH-dependent protonation of the carrier(s)

1982 ◽  
Vol 208 (2) ◽  
pp. 459-464 ◽  
Author(s):  
R Hauer ◽  
M Höfer
Keyword(s):  
Positive Charge ◽  
Low Ph ◽  
The Other ◽  
Transport Systems ◽  
Lower Affinity ◽  
High Affinity ◽  
High Sugar ◽  
Saturation Kinetics ◽  
Rhodotorula Gracilis ◽  
Ph Dependent

Two carrier-mediated systems transport sugars in the yeast Rhodotorula gracilis depending on the pH. One system, with higher affinity for sugars, catalyses a symport of protons with sugar, whereas the other system, having lower affinity, is independent of protons. This was shown in three different ways. (1) At low pH, where only the high-affinity system works, a H+/sugar stoicheiometry of 1 was found. An increase of the pH and of the sugar concentration, which allowed the low-affinity system to operate, brought about a drop of the stoicheiometry to values below 1. (2) During H+ symport the influx of positive charge was electrically compensated by an equivalent efflux of K+ from the cells. At high pH and high sugar concentrations this stoicheiometry of K+ and sugar decreased concomitant with the H+/sugar stoicheiometry. (3) At pH 7.5 both transport systems were operating, as shown by biphasic saturation kinetics. Under these conditions only the high-affinity transport was found to be electrogenic. These results agree with the theory of an electrogenic H+/sugar symport where changes in the affinity for substrate are brought about by reversible protonation and deprotonation of the carrier.

scholarly journals Identification of Escherichia coli ubiB, a Gene Required for the First Monooxygenase Step in Ubiquinone Biosynthesis

2000 ◽  
Vol 182 (18) ◽  
pp. 5139-5146 ◽  
Author(s):  
Wayne W. Poon ◽  
Diana E. Davis ◽  
Huan T. Ha ◽  
Tanya Jonassen ◽  
Philip N. Rather ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Saccharomyces Cerevisiae ◽  
Protein Kinase ◽  
Mutant Allele ◽  
Coenzyme Q ◽  
Low Frequency ◽  
Coding Region ◽  
E Coli ◽  
Mutant Strains ◽  
Providencia Stuartii

ABSTRACT It was recently discovered that the aarF gene inProvidencia stuartii is required for coenzyme Q (CoQ) biosynthesis. Here we report that yigR, theEscherichia coli homologue of aarF, isubiB, a gene required for the first monooxygenase step in CoQ biosynthesis. Both the P. stuartii aarF and E. coli ubiB (yigR) disruption mutant strains lack CoQ and accumulate octaprenylphenol. Octaprenylphenol is the CoQ biosynthetic intermediate found to accumulate in the E. coli strain AN59, which contains the ubiB409 mutant allele. Analysis of the mutation in the E. coli strain AN59 reveals no mutations within the ubiB gene, but instead shows the presence of an IS1 element at position +516 of the ubiE gene. The ubiE gene encodes aC-methyltransferase required for the synthesis of both CoQ and menaquinone, and it is the 5′ gene in an operon containingubiE, yigP, and ubiB. The data indicate that octaprenylphenol accumulates in AN59 as a result of a polar effect of the ubiE::IS1mutation on the downstream ubiB gene. AN59 is complemented by a DNA segment containing the contiguous ubiE,yigP, and ubiB genes. Although transformation of AN59 with a DNA segment containing the ubiB coding region fails to restore CoQ biosynthesis, transformation with theubiE coding region results in a low-frequency but significant rescue attributed to homologous recombination. In addition, the fre gene, previously considered to correspond toubiB, was found not to be involved in CoQ biosynthesis. TheubiB gene is a member of a predicted protein kinase family of which the Saccharomyces cerevisiae ABC1 gene is the prototypic member. The possible protein kinase function of UbiB and Abc1 and the role these polypeptides may play in CoQ biosynthesis are discussed.

Sign in / Sign up

Export Citation Format

Share Document