Growth-related expression of ribosomal protein genes in Saccharomyces cerevisiae

Nutritional upshift response of ribosomal protein gene transcription in Saccharomyces cerevisiae

“…Addition of glucose to a culture of Saccharomyees ceret,tstae growing on a medium with a non-fermentable carbon source (throughout this paper designated as nutritional upshIft) leads to a transient arrest of cell growth During this transl-139 tlon phase, transcription of rp-genes is strongly induced, in order to meet the greater need for protein-biosynthetic capacity in the new growth conditions [2][3][4] We performed a nutritional upshift in the presence of the protein synthesis inhibitor cyclohexlmtde and analyzed the level of rp-mRNA by Northern analysis The results ( Fig 1) demonstrate the typically rapid coordinate Increase of rp-gene transcription upon addition of glucose Under normal conditions, rp-gene transcription, after the initial nutritional upshlft effect, is subsequently adjusted precisely to the new cellular growth rate However, in the presence of cyclohexlmlde the elevated rp-mRNA productton is maintained over a pertod of at least 3 h, despite cell growth being arrested These data strongly suggest that the glucose-induced initial increase of transcription of yeast rp-genes is independent of de novo protein synthesis, whereas the subsequent adjustment of transcription is not We hypothesize that the initial shift-up effect IS triggered by post-translational modification, of either Raplp and Abflp themselves or of auxiliary factors recruited by them…”

“…In a first analysis of the signal transduction pathway that IS involved in the glucose-induced transcriptional activation of yeast rp-genes, we obtained evidence that cAMP does not act as the principal second messenger in this pathway [4] In these experiments we made use of the Icrl mu- Pka actwlty and with pkcl deletion strata DL376 (B) The growth medmm of DL376 was supplemented with 1 M sorbltol [6] tant [11], in which, due to a defect in the adenylate cyclase gene, the level of mtracellutar cAMP does not increase upon addition of glucose We extended these studies by investigating the nutritional response in a mutant lacking the regulatory subunIt Bcylp of protem kmase A (Pka) and hawng low constitutwe protein kinase A actwity due to deletions of the two catalytic subunit genes TPK2 and TPK3 and a TPK1 w mutation In the third [8] Consistent with recent findings of Struhl and Klein (12), no upshIft occurred in this mutant (Fig 2A) Apparently, control of Pka actwity, probably through Bcylp, Is a major determmant m the upshtft response, although not dependmg on the intracellular cAMP concentration Similar observations have been made for other processes, which are induced by glucose but nevertheless are also cAMP-independent, like activation of trehalase [13] or control of glycolytlc flux [14] In addition, actwatlon of Pka, independent of changes in cAMP levels, has been reported to occur after exposing yeast to heat shock [15] To our knowledge, our data are the first evidence that regulation of transcription, too, may be mediated through protein klnase A but in a cAMP-Independent fashion…”

“…Nutrients are major determinants of yeast cell cycle control and therefore are of crucial importance for the cellular growth rate How nutritional components are sensed by the cell and how the resulting signal(s) are subsequently transmitted to evoke a cellular response, however, is so far poorly understood In particular the effect of adding glucose in fermentable amounts to yeast cultured on a non-fermentable carbon source has drawn a lot of attention m recent years [1] This * Corresponding author Tel (+31) 20-4447548, Fax (+31) or 4447509 so-called nutritional upshlft lmtlates a series of molecular events such as the rapid (posttranslatlonal) Inhibition of gluconeogenesis and uptake of alternative carbon subsWates, the rapid stimulation of glycolysts and moblhzatlon of trehalose and, in the long term, glucose repression of a large number of genes involved m, for instance, gluconeogenesls, galactose and maltose transport and mltochondrlal function [1] Earlier studies have shown the RAS-adenylate cyclase pathway to be tmportant for maintaining the nutrient slgnalhng capacity of yeast Exposure of yeast to a high glucose concentration leads to a sudden, transient elevation of cAMP levels, which subsequently triggers a protein phosphorylatlon cascade The RAS-adenylate cyclase pathway, on the other hand, does not medaate the nutnUonal regulation of cell growth, sance this pathway as eventually repressed by glucose Therefore the nutrient stgnalhng role of the RAS-adenylate cyclase pathway may be restricted to the transataon period between resparo-gluconeogenlc growth, when glucose ~s absent or present only m low amounts, and fermentatave growth induced by hagh glucose concentrations [1] Ribosome formation represents a prime example of a process, the rate of which is accurately tuned to cellular growth, in order to prowde the cell w~th the protean-b~osynthetlc capaoty reqmred [2][3][4] Therefore ribosome blogenesas provades an attractave model for mvestagatmg nutntaonal effects on growth Indeed, ammedmtely upon a nutritional upshfft, when growth of yeast cells as transaently arrested, ribosomal RNA and protein synthes~s display a rap~d increase, m order to efficaently adjust the cellular ribosome content to the new growth demands [2,3] The coordinate control of the synthesas of the rabosomal constituents an Saccharomyces cereL,tstae, upon a swatch to a glucose-containing medmm, has been shown to occur at the level of transcraptlon of the respective genes [2,3] As far as the ribosomal protein genes (rp-genes) are concerned, studaes have been concentrated on the major potentmtors of transcriptional actavat~on of these genes, vaz Raplp and Abflp From various promoter deletion and reconstructaon analyses ~t has become evadent that the nutrltaonal upshlft response of rp-gene transcnptaon as medmted, directly or mdarectly, through these factors [2,3] However, bandshfft analyses using protean extracts prepared from yeast grown at different condmons d~d not reveal major changes m the cellular amounts of Raplp or Abflp or their affinmes for the respectave bmdang sites [4] Apparently, upshfft regulataon of rp-gene transcnpta...…”

“…Northern analysis of rp-mRNA levels upon a nutritional upshfft performed with strain CH2881-1D Cyclohexlmlde (100 mg ml -l) was added to exponentmlly growing yeast cells 20 mm before the addition of glucose (time = 0') Time samples were taken as indicated Probes are described m Matermls and Methods part of the actln gene, which was used as internal control for the amount of RNA loaded on gel Probes were labelled using random primers and Klenow polymerase[4] …”

Cell Cycle, Differentiation and Tissue-Independent Expression of Ribosomal Protein L37

“…After 2 days, the serum-reduced medium was replaced by 1 0 %~ fetal bovine serum growth medium. Cells were harvested for RNA isolation and ['Hlthymidine incorporation assays were performed at 0, 10 and 30 min, and 1, 2, 3,5,7,9,12 and 15 h after serum induction. Analysis of acid-precipitable [ 'Hlthymidine incorporation was performed by pulse-labeling each cell fraction with 1 pCi/rnl [3H]thymidine in complete growth medium for 30 min.…”