A genome-wide screen for Saccharomyces cerevisiae iron-sulfur (Fe/S) cluster assembly mutants identified the gene IBA57. The encoded protein Iba57p is located in the mitochondrial matrix and is essential for mitochondrial DNA maintenance. The growth phenotypes of an iba57⌬ mutant and extensive functional studies in vivo and in vitro indicate a specific role for Iba57p in the maturation of mitochondrial aconitase-type and radical SAM Fe/S proteins (biotin and lipoic acid synthases). Maturation of other Fe/S proteins occurred normally in the absence of Iba57p. These observations identify Iba57p as a novel dedicated maturation factor with specificity for a subset of Fe/S proteins. The Iba57p primary sequence is distinct from any known Fe/S assembly factor but is similar to certain tetrahydrofolate-binding enzymes, adding a surprising new function to this protein family. Iba57p physically interacts with the mitochondrial ISC assembly components Isa1p and Isa2p. Since all three proteins are conserved in eukaryotes and bacteria, the specificity of the Iba57/Isa complex may represent a biosynthetic concept that is universally used in nature. In keeping with this idea, the human IBA57 homolog C1orf69 complements the iba57⌬ growth defects, demonstrating its conserved function throughout the eukaryotic kingdom.Iron-sulfur (Fe/S) cluster-containing proteins perform central tasks in electron transport, catalysis, and the regulation of environmental responses (1). The complex bacterial biosynthetic systems that assist in the assembly of Fe/S clusters and their transfer into apo-proteins fall into three classes: the house-keeping ISC system, which is widely distributed across taxa; the NIF machinery dedicated to the assembly of the Fe/S clusters of nitrogenase from nitrogen-fixing bacteria; and the SUF machinery, which is required under oxidative stress and iron-limiting conditions (17,30).In eukaryotes mitochondria are crucial for Fe/S protein biogenesis and contain an Fe/S cluster assembly machinery that is closely related to the bacterial ISC system. This mitochondrial ISC machinery appears to be essential for maturation of all cellular Fe/S proteins, whether located in the mitochondria, cytosol, or nucleus (37, 38). Biosynthesis of extramitochondrial Fe/S proteins further depends on the mitochondrial "ISC export machinery" that exports an unknown component required for maturation of cytosolic and nuclear proteins, a step carried out by members of the cytosolic Fe/S protein assembly (CIA) system (37, 38). The ISC and CIA proteins involved in Fe/S maturation are highly conserved in eukaryotes and several are essential for viability, underscoring the importance of Fe/S proteins for the eukaryotic cell.Fe/S cluster assembly in mitochondria is initiated by the cysteine desulfurase Nfs1p which serves as the sulfur donor (32). The sulfur is transferred to the essential protein pair Isu1p/Isu2p, which serves as a scaffold for the de novo synthesis of the Fe/S clusters (24, 53). This biosynthetic reaction involves an electron tran...
Glycine specifically induces genes encoding subunits of the glycine decarboxylase complex (GCV1, GCV2, and GCV3), and this is mediated by a fall in cytoplasmic levels of 5,10-methylenetetrahydrofolate caused by inhibition of cytoplasmic serine hydroxymethyltransferase. Here it is shown that this control system extends to genes for other enzymes of one-carbon metabolism and de novo purine biosynthesis. Northern analysis of the response to glycine demonstrated that the induction of the GCV genes and the induction of other amino acid metabolism genes are temporally distinct. The genomewide response to glycine revealed that several other genes are rapidly co-induced with the GCV genes, including SHM2, which encodes cytoplasmic serine hydroxymethyltransferase. These results were refined by examining transcript levels in an shm2⌬ strain (in which cytoplasmic 5,10-methylenetetrahydrofolate levels are reduced) and a met13⌬ strain, which lacks the main methylenetetrahydrofolate reductase activity of yeast and is effectively blocked at consumption of 5,10-methylene tetrahydrofolate for methionine synthesis. Glycine addition also caused a substantial transient disturbance to metabolism, including a sequence of changes in induction of amino acid biosynthesis and respiratory chain genes. Analysis of the glycine response in the shm2⌬ strain demonstrated that apart from the one-carbon regulon, most of these transient responses were not contingent on a disturbance to onecarbon metabolism. The one-carbon response is distinct from the Bas1p purine biosynthesis regulon and thus represents the first example of transcriptional regulation in response to activated one-carbon status.
Up to 1 in 3000 individuals in the United States have a-1 antitrypsin deficiency, and the most common cause of this disease is homozygosity for the antitrypsin-Z variant (ATZ). ATZ is inefficiently secreted, resulting in protein deficiency in the lungs and toxic polymer accumulation in the liver. However, only a subset of patients suffer from liver disease, suggesting that genetic factors predispose individuals to liver disease. To identify candidate factors, we developed a yeast ATZ expression system that recapitulates key features of the disease-causing protein. We then adapted this system to screen the yeast deletion mutant collection to identify conserved genes that affect ATZ secretion and thus may modify the risk for developing liver disease. The results of the screen and associated assays indicate that ATZ is degraded in the vacuole after being routed from the Golgi. In fact, one of the strongest hits from our screen was Vps10, which can serve as a receptor for the delivery of aberrant proteins to the vacuole. Because genome-wide association studies implicate the human Vps10 homolog, sortilin, in cardiovascular disease, and because hepatic cell lines that stably express wild-type or mutant sortilin were recently established, we examined whether ATZ levels and secretion are affected by sortilin. As hypothesized, sortilin function impacts the levels of secreted ATZ in mammalian cells. This study represents the first genome-wide screen for factors that modulate ATZ secretion and has led to the identification of a gene that may modify disease severity or presentation in individuals with ATZ-associated liver disease.A N inherited disorder, a-1 AT deficiency (ATD) is linked to decreased levels and activity of the proteinase inhibitor a-1 antitrypsin (AT). It is one of the most common genetic disorders in the United States, with the most severe form affecting between 1 in 3000 and 1 in 6000 individuals (Kimpen et al. 1988;Silverman et al. 1989;Spence et al. 1993;de Serres et al. 2007de Serres et al. , 2010.Wild-type AT (referred to here as the M variant, or ATM) is an abundant plasma protein secreted by hepatocytes that protects lung tissue from the action of neutrophil elastase. The most common cause of ATD is homozygosity for the mutation that gives rise to the Z variant of AT (ATZ), which exhibits folding and thus secretion defects. Retention of ATZ within hepatocytes results in AT deficiency in the lungsconsidered a loss-of-function phenotype-but can also result in an accumulation of polymeric and aggregated ATZ within the liver, which manifests as a gain-of-function phenotype (Bathurst et al. 1984Foreman et al. 1984;Errington et al. 1985;Janus et al. 1985; Perlmutter et al. 1985a,b;Dycaico et al. 1988;Carlson et al. 1989;Lomas et al. 1992). These organ-specific effects of ATZ are consequently responsible for the two most common clinical manifestations of ATD, lung disease and liver disease.Interestingly, there is considerable variability in the ageof-onset and severity of these diseases, particular...
Genome-wide transcriptional analysis of a Saccharomyces cerevisiae batch culture revealed that more than 829 genes were regulated in response to an environmental shift from pH 6 to pH 3 by added sulfuric acid. This shift in pH was not detrimental to the rate of growth compared to a control culture that was maintained at pH 6 and the transcriptional changes most strikingly implicated not up- but down-regulation of stress responses. In addition, the transcriptional changes upon acid addition indicated remodeling of the cell wall and central carbon metabolism. The overall trend of changes was similar for the pH-shift experiment and the pH 6 control. However, the changes in the pH 6 control were much weaker and occurred 2.5 h later than in the pH-shift experiment. Thus, the reaction to the steep pH decrease was an immediate response within the normal repertoire of adaptation shown in later stages of fermentation at pH 6. Artificially preventing the yeast from acidifying the medium may be considered physiologically stressful under the tested conditions.
The most frequent cause of a 1 -antitrypsin (here referred to as AT) deficiency is homozygosity for the AT-Z allele, which encodes AT-Z. Such individuals are at increased risk for liver disease due to the accumulation of aggregation-prone AT-Z in the endoplasmic reticulum of hepatocytes. However, the penetrance and severity of liver dysfunction in AT deficiency is variable, indicating that unknown genetic and environmental factors contribute to its occurrence. There is evidence that the rate of AT-Z degradation may be one such contributing factor. Through the use of several AT-Z model systems, it is now becoming appreciated that AT-Z can be degraded through at least two independent pathways. One model system that has contributed significantly to our understanding of the AT-Z disposal pathway is the yeast, Saccharomyces cerevisiae.
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