Sulfur metabolism has been studied extensively and is, to a large degree, universal for eukaryotes [1][2][3][4]. However, there are still predicted pathways in which all enzymes ⁄ genes have not been identified. Sulfur is needed in the form of the amino acids cysteine and methionine, as well as S-adenosylmethionine, the adenosylated form of methionine. S-adenosylmethionine has many different functions in the cell, including as a starting point for the synthesis of polyamines: putrescine, spermine and spermidine. Polyamines are important for growth, and yeast has an absolute requirement for putrescine and spermidine, as studied in deletion mutants of biosynthetic enzymes [5,6]. During the formation of spermidine and spermine, the metabolite 5¢-methylthioadenosine (MTA) is formed, which contains the sulfur atom of methionine.As the assimilation of sulfur is strongly energy consuming in the form of redox equivalents [2], most organisms from bacteria to mammals and plants have evolved recycling pathways to reuse sulfur and regenerate methionine: the 'methionine salvage pathway' (MTA cycle) [7][8][9][10][11]. The cycle of mammals and yeast consists of six enzymatic steps and one spontaneous reaction. The active enzymes are MTA phosphorylase The methionine salvage pathway is universally used to regenerate methionine from 5¢-methylthioadenosine, a byproduct of certain reactions involving S-adenosylmethionine. We identified and verified the genes encoding the enzymes of all steps in this cycle in a commonly used eukaryotic model system: the yeast Saccharomyces cerevisiae. The genes encoding 5¢-methylthioribose-1-phosphate isomerase and 5¢-methylthioribulose-1-phosphate dehydratase are herein named MRI1 and MDE1, respectively. The 5¢-methylthioadenosine phosphorylase was verified as Meu1p, the 2,3-dioxomethiopentane-1-phosphate enolase ⁄ phosphatase as Utr4p and the aci-reductone dioxygenase as Adi1p. The homologue of the enolase ⁄ phosphatase gene, YNL010w, was excluded from its candidate role in the cycle. The methodology used involved auxotrophic growth tests and analysis of intracellular 5¢-methylthioadenosine in deletion mutants. The last step, a transamination of 4-methylthio-2-oxobutyrate to yield methionine, was found to be a highly redundant step. It was catalysed by amino acid transaminases, mainly coupled with aromatic and branched chain amino acids as amino donors, but also with proline, lysine and glutamate ⁄ glutamine. The aromatic amino acid transaminases, Aro8p and Aro9p, and the branched chain amino acid transaminases, Bat1p and Bat2p, seemed to be the main enzymes exhibiting 4-methylthio-2-oxobutyrate transaminase activity. Bat2p was found to be less specific and used proline, lysine, tyrosine and glutamate as amino donors in addition to the branched chain amino acids. Thus, for the first time, all enzymes of the methionine salvage pathway were identified in a eukaryote.Abbreviations MOB, 4-methylthio-2-oxobutyrate; MTA, 5¢-methylthioadenosine; SGD, Saccharomyces Genome Database

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BackgroundSpore germination of the yeast Saccharomyces cerevisiae is a multi-step developmental path on which dormant spores re-enter the mitotic cell cycle and resume vegetative growth. Upon addition of a fermentable carbon source and nutrients, the outer layers of the protective spore wall are locally degraded, the tightly packed spore gains volume and an elongated shape, and eventually the germinating spore re-enters the cell cycle. The regulatory pathways driving this process are still largely unknown. Here we characterize the global gene expression profiles of germinating spores and identify potential transcriptional regulators of this process with the aim to increase our understanding of the mechanisms that control the transition from cellular dormancy to proliferation.ResultsEmploying detailed gene expression time course data we have analysed the reprogramming of dormant spores during the transition to proliferation stimulated by a rich growth medium or pure glucose. Exit from dormancy results in rapid and global changes consisting of different sequential gene expression subprograms. The regulated genes reflect the transition towards glucose metabolism, the resumption of growth and the release of stress, similar to cells exiting a stationary growth phase. High resolution time course analysis during the onset of germination allowed us to identify a transient up-regulation of genes involved in protein folding and transport. We also identified a network of transcription factors that may be regulating the global response. While the expression outputs following stimulation by rich glucose medium or by glucose alone are qualitatively similar, the response to rich medium is stronger. Moreover, spores sense and react to amino acid starvation within the first 30 min after germination initiation, and this response can be linked to specific transcription factors.ConclusionsResumption of growth in germinating spores is characterized by a highly synchronized temporal organisation of up- and down-regulated genes which reflects the metabolic reshaping of the quickening spores.