Transcriptome profiling to identify genes involved in peroxisome assembly and function

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Gpd1p is a cytosolic NAD؉ -dependent glycerol 3-phosphate dehydrogenase that also localizes to peroxisomes and plays an essential role in the cellular response to osmotic stress and a role in redox balance. Here, we show that Gpd1p is directed to peroxisomes by virtue of an N-terminal type 2 peroxisomal targeting signal (PTS2) in a Pex7p-dependent manner. Significantly, localization of Gpd1p to peroxisomes is dependent on the metabolic status of cells and the phosphorylation of aminoacyl residues adjacent to the targeting signal. Exposure of cells to osmotic stress induces changes in the subcellular distribution of Gpd1p to the cytosol and nucleus. This behavior is similar to Pnc1p, which is coordinately expressed with Gpd1p, and under conditions of cell stress changes its subcellular distribution from peroxisomes to the nucleus where it mediates chromatin silencing. Although peroxisomes are necessary for the ␤-oxidation of fatty acids in yeast, the localization of Gpd1p to peroxisomes is not. Rather, shifts in the distribution of Gpd1p to different cellular compartments in response to changing cellular status suggests a role for Gpd1p in the spatial regulation of redox potential, a process critical to cell survival, especially under the complex stress conditions expected to occur in the wild.Glycerol 3-phosphate dehydrogenase (Gpd1p) is one of two NAD ϩ -dependent glycerol 3-phosphate dehydrogenases in yeast (1, 2). It is classically defined as a cytosolic enzyme that catalyzes the conversion of dihydroxyacetone phosphate (DHAP) 2 and NADH to glycerol 3-phosphate (Glycerol 3-phosphate) and NAD ϩ (2). In unstressed cells, this reaction prevents the accumulation of DHAP (3), which can otherwise be transformed into methyl glyoxylate (MG) (3, 4), a toxic compound that interacts with proteins (5-7). This reaction also allows for the reoxidation of NADH to NAD ϩ , which can serve as a buffer for cytosolic redox balance, compensating for cellular reactions that produce NADH (2). Moreover, glycerol 3-phosphate is a key metabolite for the synthesis of glyceride lipids and phospholipids, as well as for the formation of glycerol (8 -12).Under hyperosmotic stress, Saccharomyces cerevisiae, as well as other yeasts, accumulates glycerol as a major solute (13,14). This increased production of glycerol is caused mainly by an enhanced activity of Gpd1p, and accordingly, Gpd1p is essential for growth under osmotic stress (2, 15). In addition, GPD1 expression is altered by a wide variety of stresses, including heat, cold, and oxidative stress (16 -18), suggesting its regulation is controlled by stress. Genome-wide monitoring of transcript changes in yeast under various stress conditions also showed that GPD1 belongs to a large group of common stressresponse genes (19, 20); however, its regulation is complex and appears to be controlled by multiple signaling pathways (21). This likely reflects the multiple metabolic changes that yeast cells undergo in response to different stresses (19,22,23) and the function of Gpd1p at the int...

Section: Gpd1pmentioning

confidence: 57%

“…Subcellular Fractionation-Subcellular fractionation was performed as described previously (24,36). Briefly, yeast cells were grown to mid-exponential growth phase (A 600 ϳ1) in SMLeu.…”

Section: Methodsmentioning

confidence: 99%

Dynamic Changes in the Subcellular Distribution of Gpd1p in Response to Cell Stress

Jung

Marelli

Rachubinski

et al. 2010

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“…The microarray data extend to at least 12, the number of genes encoding peroxisomal proteins that are dependent on ADR1 for expression in the absence of oleate (Table III). Many of the ADR1-dependent genes scored high in a genomic profile of genes involved in peroxisome assembly or function (39) and were also identified in a search for oleate-inducible, OAF1-PIP2-dependent genes (40,41). In addition to ␤-oxidation, uptake of fatty acyl-CoA by peroxisomes appears to be ADR1-dependent.…”

Section: Resultsmentioning

confidence: 99%

Multiple Pathways Are Co-regulated by the Protein Kinase Snf1 and the Transcription Factors Adr1 and Cat8

Young¹,

Dombek²,

Tachibana

et al. 2003

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255

310

ADR1 and CAT8 encode carbon source-responsive transcriptional regulators that cooperatively control expression of genes involved in ethanol utilization. These transcription factors are active only after the diauxic transition, when glucose is depleted and energy-generating metabolism has shifted to the aerobic oxidation of non-fermentable carbon sources. The Snf1 protein kinase complex is required for activation of their downstream target genes described previously. Using DNA microarrays, we determined the extent to which these three factors collaborate in regulating the expression of the yeast genome after glucose depletion. The expression of 108 genes is significantly decreased in the absence of ADR1. The importance of ADR1 during the diauxic transition is illustrated by the observation that expression of almost one-half of the 40 most highly glucose-repressed genes is ADR1-dependent. ADR1-dependent genes fall into a variety of functional classes with carbon metabolism containing the largest number of members. Most of the genes in this class are involved in the oxidation of different non-fermentable carbon sources. These microarray data show that ADR1 coordinates the biochemical pathways that generate acetylCoA and NADH from non-fermentable substrates. Only a small number of ADR1-dependent genes are also CAT8-dependent. However, nearly one-half of the ADR1-dependent genes are also dependent on the Snf1 protein kinase for derepression. Many more genes are SNF1-dependent than are either ADR1-or CAT8-dependent suggesting that SNF1 plays a broader role in gene expression than either ADR1 or CAT8. The largest class of SNF1-dependent genes encodes regulatory proteins that could extend SNF1 dependence to additional pathways.During the diauxic shift, when yeast cells deplete the glucose in the medium, the flow of metabolites changes dramatically to adapt to the use of alternative energy and carbon sources, primarily ethanol produced during fermentation. The changes in gene expression that are required for this metabolic reorganization were dramatically revealed by DNA microarray analysis (1). The expression of more than one-quarter of the yeast genome is altered when glucose is exhausted. These alterations allow the cell to utilize carbon sources other than glucose to channel metabolites into the tricarboxylic acid and glyoxylate cycles to generate energy and synthesize intermediates for gluconeogenesis. In addition, these changes allow the cell to alter its growth rate and prepare for growth arrest and stationary phase.Numerous transcription factors and regulatory proteins are responsible for the altered transcriptional program that accompanies the depletion of glucose. Two transcription factors that play important roles during the diauxic transition are Adr1 1 and Cat8.

“…(20), and pRS406 (21) have been described previously. pmRFP-SKL was constructed by replacing the gene for red fluorescent protein in the plasmid pDsRed-PTS1 (22) with the gene encoding monomeric red fluorescent protein (mRFP) (23). pGFPϩ/HIS5 and pmRFP/HIS5 were constructed by replacing the gene for GFP in pGFP/HIS5 (24) by the genes encoding GFPϩ (25) and mRFP, respectively.…”

Section: Methodsmentioning

confidence: 99%

“…Analytical Procedures-Subcellular fractionation was performed as described previously (22). Whole cell lysates were prepared as described previously (27).…”

Section: Aa-gfp (His5)/pex3::gal1pex3-mrfp (His5)mentioning

confidence: 99%

Pex3p Initiates the Formation of a Preperoxisomal Compartment from a Subdomain of the Endoplasmic Reticulum in Saccharomyces cerevisiae

Tam¹,

Fagarasanu

et al. 2005

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151

172

Peroxisomes are dynamic organelles that often proliferate in response to compounds that they metabolize. Peroxisomes can proliferate by two apparent mechanisms, division of preexisting peroxisomes and de novo synthesis of peroxisomes. Evidence for de novo peroxisome synthesis comes from studies of cells lacking the peroxisomal integral membrane peroxin Pex3p. These cells lack peroxisomes, but peroxisomes can assemble upon reintroduction of Pex3p. The source of these peroxisomes has been the subject of debate. Here, we show that the amino-terminal 46 amino acids of Pex3p of Saccharomyces cerevisiae target to a subdomain of the endoplasmic reticulum and initiate the formation of a preperoxisomal compartment for de novo peroxisome synthesis. In vivo video microscopy showed that this preperoxisomal compartment can import both peroxisomal matrix and membrane proteins leading to the formation of bona fide peroxisomes through the continued activity of full-length Pex3p. Peroxisome formation from the preperoxisomal compartment depends on the activity of the genes PEX14 and PEX19, which are required for the targeting of peroxisomal matrix and membrane proteins, respectively. Our findings support a direct role for the endoplasmic reticulum in de novo peroxisome formation.A characteristic of eukaryotic cells is the presence of seemingly distinct subcellular compartments or organelles possessing specific sets of proteins required for specialized cellular functions. However, organelles do not exist in isolation, and interorganellar communication through the movement and exchange of different molecules is required for normal cell function. This interdependence of organelles extends beyond their biochemical and metabolic roles and necessitates that their biogenesis and turnover also be coordinated.The peroxisome has long been considered an autonomous organelle that proliferates by the growth and division of preexisting peroxisomes (1) and is inherited as a functional organelle at cell division. But what of the concept of de novo peroxisome biogenesis? From an evolutionary point of view, peroxisome proliferation and inheritance could have evolved as a response to a slow and perhaps unreliable mechanism of de novo peroxisome biogenesis. However, de novo peroxisome biogenesis, when combined with peroxisome growth, division, and inheritance, would provide the cell with a fail-safe system for peroxisome maintenance and ultimately for its survival.Evidence implicating the endoplasmic reticulum (ER) 3 in peroxisome biogenesis has accumulated in recent years (reviewed in Refs. 2-4). The amino-terminal 16 amino acids of the peroxisomal integral membrane protein Pex3p of Hansenula polymorpha were shown to be sufficient to target a reporter protein to the ER (5), whereas treatment of cells of this yeast with brefeldin A led to the accumulation of newly synthesized peroxisomal membrane and matrix proteins at the ER (6). In the yeast Yarrowia lipolytica, the peroxisomal membrane proteins Pex2p and Pex16p were shown to traffic through the E...