Nutrient-induced signal transduction through the protein kinase A pathway and its role in the control of metabolism, stress resistance, and growth in yeast.

Yeast cells growing in the presence of glucose or a related rapidly-fermented sugar differ strongly in a variety of physiological properties compared to cells growing in the absence of glucose. Part of these differences appear to be caused by the protein kinase A (PKA) and related signal transduction pathways. Addition of glucose to cells previously deprived of glucose triggers cAMP accumulation, which is apparently mediated by the Gpr1-Gpa2 G-protein coupled receptor system. However, the resulting effect on PKA-controlled properties is only transient when there is no complete growth medium present. When an essential nutrient is lacking, the cells arrest in the stationary phase G0. At the same time they acquire all characteristics of cells with low PKA activity, even if there is ample glucose present. When the essential nutrient is added again, a similar PKA-dependent protein phosphorylation cascade is triggered as observed after addition of glucose to glucose-deprived cells, but which is not cAMP-mediated. Because the pathway involved requires a fermentable carbon source and a complete growth medium, at least for its sustained activation, it has been called "fermentable growth medium (FGM)-induced pathway."

Structure-function analysis of yeast hexokinase: structural requirements for triggering cAMP signalling and catabolite repression.

In baker's yeast (Saccharomyces cerevisiae) the hexokinases PI (Hxk1) and PII (Hxk2) are required for triggering of the activation of the Ras-cAMP pathway and catabolite repression. Specifically, Hxk2 is essential for the establishment of glucose repression, whereas either Hxk1 or Hxk2 can sustain fructose repression. Previous studies have suggested that the extent of glucose repression is inversely correlated with hexokinase catalytic activity and hence with an adequate elevation of intracellular sugar phosphate levels. However, several lines of evidence indicate that glucose 6-phosphate is not the trigger of catabolite repression in yeast. In the present study we employed site-directed mutagenesis of amino acids important for the binding of sugar and ATP, for efficient phosphoryl transfer and for the closure of the substrate-binding cleft, to obtain an insight into the structural requirements of Hxk2 for sugar-induced signalling. We show that the ATP-binding Lys-111 is not essential for catalysis in vivo or for signal triggering. Substitution of the catalytic-centre Asp-211 caused loss of catalytic activity, but high-affinity sugar binding was retained. However, this was not sufficient to cause cAMP activation nor catabolite repression. Mutation of Ser-158 abrogated glucose-induced, but not fructose-induced, repression. Moreover, 2-deoxyglucose sustained repression despite an extremely low catalytic activity. We conclude that the establishment of catabolite repression is dependent on the onset of the phosphoryl transfer reaction on hexokinase and is probably related to the stable formation of a transition intermediate and concomitant conformational changes within the enzyme. In contrast, the role of Hxk2 in Ras-cAMP activation seems to be directly connected to its catalytic function. The implications of this model are discussed.

A Saccharomyces cerevisiae G-protein coupled receptor, Gpr1, is specifically required for glucose activation of the cAMP pathway during the transition to growth on glucose.

In the yeast Saccharomyces cerevisiae the accumulation of cAMP is controlled by an elaborate pathway. Only two triggers of the Ras adenylate cyclase pathway are known. Intracellular acidification induces a Ras-mediated long-lasting cAMP increase. Addition of glucose to cells grown on a non-fermentable carbon source or to stationary-phase cells triggers a transient burst in the intracellular cAMP level. This glucose-induced cAMP signal is dependent on the G alpha-protein Gpa2. We show that the G-protein coupled receptor (GPCR) Gpr1 interacts with Gpa2 and is required for stimulation of cAMP synthesis by glucose. Gpr1 displays sequence homology to GPCRs of higher organisms. The absence of Gpr1 is rescued by the constitutively activated Gpa2Val-132 allele. In addition, we isolated a mutant allele of GPR1, named fil2, in a screen for mutants deficient in glucose-induced loss of heat resistance, which is consistent with its lack of glucose-induced cAMP activation. Apparently, Gpr1 together with Gpa2 constitute a glucose-sensing system for activation of the cAMP pathway. Deletion of Gpr1 and/or Gpa2 affected cAPK-controlled features (levels of trehalose, glycogen, heat resistance, expression of STRE-controlled genes and ribosomal protein genes) specifically during the transition to growth on glucose. Hence, an alternative glucose-sensing system must signal glucose availability for the Sch9-dependent pathway during growth on glucose. This appears to be the first example of a GPCR system activated by a nutrient in eukaryotic cells. Hence, a subfamily of GPCRs might be involved in nutrient sensing.

Transcription activation of yeast ribosomal protein genes requires additional elements apart from binding sites for Abf1p or Rap1p.

All ribosomal protein (rp) gene promoters from Saccharomyces cerevisiae studied so far contain either (usually two) binding sites for the global gene regulator Rap1p or one binding site for another global factor, Abf1p. Previous analysis of the rpS33 and rpL45 gene promoters suggested that apart from the Abf1 binding site, additional cis-acting elements play a part in transcription activation of these genes. We designed a promoter reconstruction system based on the beta-glucuronidase reporter gene to examine the role of the Abf1 binding site and other putative cis-acting elements in promoting transcription. An isolated Abf1 binding site turned out to be a weak activating element. A T-rich sequence derived from the rpS33 proximal promoter was found to be stronger, but full transcription activation was only achieved by a combination of these elements. Both in the natural rpL45 promoter and in the reconstituted promoter, a Rap1 binding site could functionally replace the Abf1 binding site. Characteristic rp gene nutritional control of transcription, evoked by a carbon source upshift or by nitrogen re-feeding to nitrogen starved cells, could only be mediated by the combined Abf1 (or Rap1) binding site and T-rich element and not by the individual elements. These results demonstrate that Abf1p and Rap1p do not activate rp genes in a prototypical fashion, but rather may serve to potentiate transcription activation through the T-rich element.

Growth-related expression of ribosomal protein genes in Saccharomyces cerevisiae.

The rate of ribosomal protein gene (rp-gene) transcription in yeast is accurately adjusted to the cellular requirement for ribosomes under various growth conditions. However, the molecular mechanisms underlying this co-ordinated transcriptional control have not yet been elucidated. Transcriptional activation of rp-genes is mediated through two different multifunctional transacting factors, ABF1 and RAP1. In this report, we demonstrate that changes in cellular rp-mRNA levels during varying growth conditions are not parallelled by changes in the in vitro binding capacity of ABF1 or RAP1 for their cognate sequences. In addition, the nutritional upshift response of rp-genes observed after addition of glucose to a culture growing on a non-fermentative carbon source turns out not to be the result of increased expression of the ABF1 and RAP1 genes or of elevated DNA-binding activity of these factors. Therefore, growth rate-dependent transcription regulation of rp-genes is most probably not mediated by changes in the efficiency of binding of ABF1 and RAP1 to the upstream activation sites of these genes, but rather through other alterations in the efficiency of transcription activation. Furthermore, we tested the possibility that cAMP may play a role in elevating rp-gene expression during a nutritional shift-up. We found that the nutritional upshift response occurs normally in several mutants defective in cAMP metabolism.

Functional analysis of the promoter of the gene encoding the acidic ribosomal protein L45 in yeast.

The gene encoding the acidic ribosomal protein L45 in yeast is expressed coordinately with other rp-genes. The promoter region of this gene harbours binding sites for CP1 and ABF1. We demonstrate that the CP1-site is not involved in the transcription activation of the L45-gene. Rather, the ABF1-site, through deviating from the consensus sequence (RTARY3N3ACG), appears to be essential for efficient transcription. Replacement of this site by a consensus RAP1-binding site (an RPG box) did not alter the transcriptional yield of the L45-gene. An additional transcription activating region is present downstream of the ABF1-site. The relevant nucleotide sequence, which is repeated in the L45-gene promoter, gives rise to complex formation with a yeast protein extract in a bandshift assay. The results indicate that the L45-gene promoter has a complex architecture.

The divergently transcribed genes encoding yeast ribosomal proteins L46 and S24 are activated by shared RPG-boxes.

Transcription of the majority of the ribosomal protein (rp) genes in yeast is activated through common cis-acting elements, designated RPG-boxes. These elements have been shown to act as specific binding sites for the protein factor TUF/RAP1/GRF1 in vitro. Two such elements occur in the intergenic region separating the divergently transcribed genes encoding L46 and S24. To investigate whether the two RPG-boxes mediate transcription activation of both the L46 and S24 gene, two experimental strategies were followed: cloning of the respective genes on multicopy vectors and construction of fusion genes. Cloning of the L46 + S24 gene including the intergenic region in a multicopy yeast vector indicated that both genes are transcriptionally active. Using constructs in which only the S24 or the L46 gene is present, with or without the intergenic region, we obtained evidence that the intergenic region is indispensable for transcription activation of either gene. To demarcate the element(s) responsible for this activation, fusions of the intergenic region in either orientation to the galK reporter gene were made. Northern analysis of the levels of hybrid mRNA demonstrated that the intergenic region can serve as an heterologous promoter when it is in the 'S24-orientation'. Surprisingly, however, when fused in the reverse orientation the intergenic region did hardly confer transcription activity on the fusion gene. Furthermore, a 274 bp FnuDII-FnuDII fragment from the intergenic region that contains the RPG-boxes, could replace the naturally occurring upstream activation site (UASrpg) of the L25 rp-gene only when inserted in the 'S24-orientation'. Removal of 15 bp from the FnuDII fragment appeared to be sufficient to obtain transcription activation in the 'L46 orientation' as well. Analysis of a construct in which the RPG-boxes were selectively deleted from the promoter region of the L46 gene indicated that the RPG-boxes are needed for efficient transcriptional activation of the L46 gene. We conclude that all promoter elements for the S24 gene are located within the intergenic region, where the RPG-boxes are the most likely UAS-elements. However, the intergenic region (including the RPG-boxes) is required but not sufficient to confer transcription activity on the L46 gene.

Nucleotide sequence comparison of five human pepsinogen A (PGA) genes: evolution of the PGA multigene family.

To unravel the genetic basis for the pepsinogen A (PGA) protein polymorphism, we have isolated and characterized a number of PGA genes, distinguishable by polymorphic EcoRI fragments of 12.0, 15.0, and 16.6 kb. Using a HindIII or AvaII polymorphism, we can discriminate between different 15.0 (15.0 and 15.0*) and 12.0 (12.0s and 12.0l) genes, respectively. The coding sequences of a 15.0 and a 16.6 gene were determined, together with considerable stretches of the 5'- and 3'-flanking regions and introns. The genes were demonstrated to encode Pg5 and Pg4, respectively. Because substitutions in codons 43 and 207 appeared to be critical in the determination of the encoded proteins, we sequenced only these regions in the two 12.0 genes and the 15.0* gene. On the basis of these partial sequences, we assume that these genes encode Pg3. In the evolutionary model of the PGA gene cluster presented here, the 12.0 genes arose by an unequal, but homologous crossover. The results of sequence analysis of the second intron of the 12.0s, 12.0l, 15.0, and 16.6 genes suggest that the two 12.0 genes have arisen from two different crossover events.