Regulation of yeast phosphorylase by phosphorylase kinase and cAMP‐dependent protein kinase

An RGRI gene product is required to repress expression of glucose-regulated genes in Saccharomyces cerevisiae. The abnormal morphology of rgrl cells was studied. Scanning and transmission electron microscopic observations revealed that the cell wail of the daughter cell remained attached to that of mother cell. We cloned the RGRI gene by complementation and showed that the cloned DNA was tightly linked to the chromosomal RGRI locus. The cloned RGRI gene suppressed all of the phenotypes caused by the mutation and encoded a 3.6-kilobase poly(A)' RNA. The RGRI gene is located on chromosome XII, as determined by pulsed-field gel electrophoresis, and we mapped rgrl between gal2 and pep3 by genetic analysis. rgrl was shown to be a new locus. We also determined the nucleotide sequence of RGRI, which was predicted to encode a 123-kilodalton protein. The null mutation resulted in lethality, indicating that the RGRI gene is essential for growth. On the other hand, a carboxy-terminal deletion of the gene caused phenotypes similar to but more severe than those caused by the original mutation. The amount of reserve carbohydrates was reduced in rgrl cells. Possible functions of the RGRI product are discussed.Glucose regulates the expression of many genes in Saccharomyces cerevisiae (for a review, see reference 5). One of them, the SUC2 gene, which encodes invertase, is repressed by glucose (2, 3). Recently, we reported the isolation of a new mutation, rgrl, which affects expression of the SUC2 gene (21). A recessive rgrl-l mutation which caused overexpression of mouse a-amylase under the control of the SUC2 promoter was isolated, and the RGRI gene was found to be required for glucose repression. The rgrl mutation affected several cellular functions. Cells were resistant to glucose repression, temperature sensitive for cell growth, and sporulation deficient and showed abnormal cell morphology. Expression of the SUC2 gene in rgrl strains was resistant to glucose repression, and SUC2 expression was increased under glucose-derepressing conditions. In this report, we describe studies of the morphology of rgrl cells, the cloning and molecular analysis of the RGRI gene, and meiotic linkage analysis of rgrl. We constructed deletion mutations to determine the phenotypes of strains lacking a functional RGRI gene product and determined the nucleotide sequence of the gene. The RGRI gene affected accumulation of reserve carbohydrates. MATERIALS AND METHODSStrains and genetic methods. The strains of S. cerevisiae used in this study are listed in Table 1. All strains were derived from S288C. Crossing, sporulation, and tetrad analysis were carried out by standard genetic methods (23). The permissive and restrictive temperatures were 24 and 37°C, respectively. The phenotype of pep3 strains was scored as described elsewhere (12). The transformation of yeast was performed by the LiOAc-method of Ito et al. (11). Escherichia coli HB101 and JM109 were employed as hosts for * Corresponding author.constructing and propagating plasmids. The transform...

Section: Discussionmentioning

confidence: 99%

Structure and molecular analysis of RGR1, a gene required for glucose repression of Saccharomyces cerevisiae.

Sakai¹,

Shimizu²,

Kondou³

et al. 1990

“…One trait expected for such mutants is reduced glycogen accumulation. This is because the enzymes glycogen phosphorylase and glycogen synthase are two substrates for cAMP-dependent protein kinase that are activated and inhibited, respectively, by phosphorylation (20,21,54). Conceivably, the additional traits associated with sral deletions reflect the modulation of additional cAMP-dependent protein kinase substrates.…”

Section: Discussionmentioning

confidence: 99%

Characterization of Saccharomyces cerevisiae genes encoding subunits of cyclic AMP-dependent protein kinase.

Cannon¹,

Tatchell²

1987

239

157

Mutations in the SRA1 or SRA3 gene eliminate the requirement for either RAS gene (RAS] (4,7).In this study we cloned and sequenced SRAI and SRA3 and characterized mutations constructed in them. We demonstrate that SRAI encodes the regulatory subunit and that SRA3 likely encodes the catalytic subunit of cAMPdependent protein kinase. MATERIALS AND METHODSYeast strains, plasmids, and media. The genotypes of yeast strains and plasmids are given in Table 1. The inserts of plasmids are shown in Fig. 1 and 3B. The ras2-530 mutation is a LEU2 disruption of RAS2 (45 Cloning SRAJ. KT293 was transformed (2) with a yeast genomic library constructed in YCp5O, kindly provided by Mark Rose. The approximately 5,000 Ura+ transformants were incubated in nitrogen-deficient medium (7) for 3 days at 30°C. DNA was prepared from surviving transformants (less than 0.1%) that reverted two additional sral traits: lack of glycogen accumulation and ability to allow ras2-530 mutants to grow on nonfermentable carbon sources. The plasmid p713 which reverts all these traits of sral-l was recovered.Cloning SRA3. A 10-pLg sample of EcoRI-digested JC303-54 (ras2-530 SRA4-6) chromosomal DNA was ligated to 2 ,ug of EcoRI-digested YCp50 and used to transform JC302-26B spheroplasts to Ura+ (2). After transformant colonies had grown to a diameter of 2 mm (4 days at 30°C), they were extracted from the agar, diluted, and spread on YEP-acetate plates at 37°C. DNA was prepared from transformants that grew on YEP-acetate after 4 days at 37°C. The plasmid p1017 was recovered; it allowed JC302-26B (ras2-530) transformants to grow on YEP-acetate and other nonfermentable carbon sources.DNA sequence analysis. DNA restriction fragments were cloned into M13 vectors mplO and mpll (31) and used as templates for DNA sequence determination with avian myeloblastosis virus reverse transcriptase (Bio-Rad Laboratories, Richmond, Calif., or Boehringer Mannheim Biochemicals, Indianapolis, Ind.) or the large fragment of DNA polymerase I (P-L Biochemicals, Inc., Milwaukee, Wis.) (37). We used primers complementary to the mp

“…Previous studies have shown that levels of glycogen and trehalose in yeast cells change dramatically in response to a variety of different physiological situations (29,31,48). The enzymes involved in synthesizing and breaking down these carbohydrates are regulated through the cyclic AMP (cAMP)-dependent kinase system in yeasts (22,36,49,50,52,53). Glycogen and trehalose levels, in fact, have been useful phenotypic indicators for studying various mutants in the RAS, adenylate cyclase, and cAMP kinase genes (16,46,47).…”

mentioning

confidence: 99%

Molecular analysis of GPH1, the gene encoding glycogen phosphorylase in Saccharomyces cerevisiae.

Hwang¹,

Tugendreich²,

Fletterick³

1989

In yeast cells, the activity of glycogen phosphorylase is regulated by cyclic AMP-mediated phosphorylation of the enzyme. We have previously cloned the gene for glycogen phosphorylase (GPH1) in Saccharomyces cerevisiae. To assess the role of glycogen and phosphorylase-catalyzed glycogenolysis in the yeast life cycle, yeast strains lacking a functional GPHI gene or containing multiple copies of the gene were constructed. GPHI was found not to be an essential gene in yeast cells. Haploid cells disrupted in GPHI lacked phosphorylase activity and attained higher levels of intracellular glycogen but otherwise were similar to wild-type cells. Diploid cells homozygous for the disruption were able to sporulate and give rise to viable ascospores. Absence of functional GPHI did not impair cells from synthesizing and storing trehalose. Increases in phosphorylase activity of 10-to 40-fold were detected in cells carrying multiple copies of GPHI-containing 2,um plasmid. Northern (RNA) analysis indicated that GPHI transcription was induced at the late exponential growth phase, almost simultaneous with the onset of intracellular glycogen accumulation. Thus, the low level of glycogen in exponential cells was not primarily maintained through regulating the phosphorylation state of a constitutive amount of phosphorylase. GPHI did not appear to be under formal glucose repression, since transcriptional induction occurred well in advance of glucose depletion from the medium.