Metallothioneins constitute a class of low-molecular-weight, cysteine-rich metal-binding stress proteins which are biosynthetically regulated at the level of gene transcription in response to metals, hormones, cytokines, and other physiological and environmental stresses. In this report, we demonstrate that the Saccharomyces cerevisiae metallothionein gene, designated CUP), is transcriptionally activated in response to heat shock and glucose starvation through the action of heat shock transcription factor (HSF) and a heat shock element located within the CUP) promoter upstream regulatory region. CUP) gene activation in response to both stresses occurs rapidly; however, heat shock activates CUP) gene expression transiently, whereas glucose starvation activates CUP) gene expression in a sustained manner for at least 2.5 h. Although a carboxylterminal HSF transcriptional activation domain is critical for the activation of CUP) transcription in response to both heat shock stress and glucose starvation, this region is dispensable for transient heat shock activation of at least two genes encoding members of the S. cerevisiae hsp7O family. Furthermore, inactivation of the chromosomal SNFI gene, encoding a serine-threonine protein kinase, or the SNF4 gene, encoding a SNF1 cofactor, abolishes CUPI transcriptional activation in response to glucose starvation without altering heat shock-induced transcription. These studies demonstrate that the S. cerevisiae HSF responds to multiple, distinct stimuli to activate yeast metallothionein gene transcription and that these stimuli elicit responses through nonidentical, genetically separable signalling pathways.The activation of gene transcription plays a vital role in the ability of all organisms to mount a protective response to environmental stresses. Upon exposure to a specific stress, cells activate the transcription of genes encoding proteins which either offer protection from the stress or repair stressinduced cellular damage. Although the precise mechanisms by which cells sense and respond to a specific environmental stress to activate the transcription of appropriate target genes are unknown, a growing number of transcription factors which participate in stress signal transduction pathways have been identified (14,19,28,46). Metallothioneins (MTs) are a class of low-molecular-weight, cysteine-rich metal-binding stress-responsive proteins which are present in bacteria, fungi, plants, and animals (25). Since the discovery of MTs, a number of biological roles have been postulated, including protection from metal toxicity, metal homeostasis, free radical detoxification, and protection from ionizing radiation. Indeed, consistent with these possible roles, MT genes in higher eukaryotic species are transcriptionally induced by a variety of stresses such as metals, glucocorticoid hormones, interferon, interleukins, and other agents involved in the inflammatory response or which generate oxidative stress (6,46
Copper-zinc superoxide dismutase catalyzes the disproportionation of superoxide anion to hydrogen peroxide and dioxygen and is thought to play an important role in protecting cells from oxygen toxicity. Saccharomyces cerevisiae strains lacking copper-zinc superoxide dismutase, which is encoded by the SODI gene, are sensitive to oxidative stress and exhibit a variety of growth defects including hypersensitivity to dioxygen and to superoxide-generating drugs such as paraquat. We have found that in addition to these known phenotypes, SODl-deletion strains fail to grow on agar containing the respiratory carbon source lactate. We demonstrate here that expression of the yeast or monkey metallothionein proteins in the presence ofcopper suppresses the lactate growth defect and some other phenotypes associated with SODl-deletion strains, indicating that copper metaflothioneins substitute for copperzinc superoxide dismutase in vivo to protect cells from oxygen toxicity. Consistent with these results, we show that yeast metallothionein mRNA levels are dramatically elevated under conditions of oxidative stress. Furthermore, in vito assays demonstrate that yeast metallothionein, purified or from whole-cell extracts, exhibits copper-dependent antioxidant activity. Taken together, these data suggest that both yeast and mammalian metallothioneins may play a direct role in the cellular defense against oxidative stress by functioning as antioxidants.
Mutations of the orphan nuclear receptors, steroidogenic factor 1 (SF-1) and DAX-1, cause complex endocrine phenotypes that include impaired adrenal development and hypogonadotrophic hypogonadism. These similar phenotypes suggest that SF-1 and DAX-1 act in the same pathway(s) of endocrine development. To explore this model, we now compare directly their sites of expression. In mouse embryos, SF-1 expression in the urogenital ridge and brain either preceded or coincided with Dax-1 expression, with coordinate expression thereafter in the adrenal cortex, testis, ovary, hypothalamus, and anterior pituitary. The striking colocalization of SF-1 and Dax-1 supports the model that they are intimately linked in a common pathway of endocrine development. The slightly earlier onset of SF-1 expression and its ability to bind specifically to a conserved sequence in the Dax-1 5'-flanking region suggested that SF-1 may activate Dax-1 expression. However, promoter activity of Dax-1 5'-flanking sequences did not require this potential SF-1-responsive element, and Dax-1 expression was unimpaired in knockout mice lacking SF-1, establishing that SF-1 is not required for Dax-1 gene expression in these settings. Although the precise mechanisms remain to be established and may be multifactorial, our results strongly suggest that these two orphan nuclear receptors interact in a common pathway of endocrine development.
Cardiolipin (CL) synthase activity was characterized in mitochondrial extracts of the yeast Saccharomyces cerevisiae and was shown for the first time to utilize CDP-diacylglycerol as a substrate. CL synthase exhibited a pH optimum of 9.0. Maximal activity was obtained in the presence of 20 mM magnesium with a Triton X-100: phospholipid ratio of 1:1. The apparent Km values for phosphatidylglycerol and CDP-diacylglycerol were 1 mM and 36 microM, respectively. CL synthase activity was maximal at 45 degrees C and heat inactivation studies showed that the enzyme retained greater than 75% of its activity at temperatures up to 55 degrees C. To study the regulation of CL synthase, the enzyme was assayed in cells grown under conditions known to affect general phospholipid synthesis. Unlike many phospholipid biosynthetic enzymes including PGP synthase, which catalyzes the initial step in CL biosynthesis, CL synthase was not repressed in cells grown in the presence of the phospholipid precursor inositol. Detailed procedures for the enzymatic synthesis of 32P-labelled substrates are described.
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