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

Abstract

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...

Novel insights into renal d-amino acid oxidase accumulation: propiverine changes DAAO localization and peroxisomal size in vivo

“…Interestingly, it is reported that peroxisomal proteins can be differentially localized within the cell depending on differential splicing, multiple targeting signals or phosphorylation (Fordor et al 2012;Ast et al 2013). However, dual localization of peroxisomal proteins was predominantly reported to occur into the cytosol and into mitochondria (Ast et al 2013), merely one study in yeast described nuclear translocation of a peroxisomal NAD + -dependent glycerol 3-phosphate dehydrogenase (Jung et al 2010). The concurrent analysis of the intracellular distribution of catalase, another peroxisomal protein, demonstrated a comparable propiverine-mediated mislocalization from the peroxisomes to the cytosol and nucleus, thereby suggesting the presence of more generalized compound-mediated reduced or abrogated translocation to or import of peroxisomal proteins into peroxisomes.…”

Role of Pex21p for Piggyback Import of Gpd1p and Pnc1p into Peroxisomes of Saccharomyces cerevisiae

“…Post-translational modifications could also contribute to specificity of co-receptor binding of cargo proteins. To this end, it has been shown that phosphorylation of two serine residues close to the PTS2 signal of Gpd1p is important for efficient peroxisomal import (27).…”

“…4, left panel). Most of Pnc1p-GFP was found in cytosolic fractions (fractions [25][26][27]. However, a significant portion was present in fractions 2 and 3, co-fractionating with the peroxisomal matrix proteins Fox3p and Pcs60p.…”

“…To characterize alternative peroxisomal import pathways, we analyzed the PTS2-containing Gpd1p, a key enzyme for glycerol production that is essential for growth under hyperosmotic stress conditions (27). To compare the expression of Pex18p, Pex21p, Gpd1p, and Fox3p under different conditions, all four proteins were genomically tagged with protein A.…”

“…These are the ␤-oxidation enzyme 3-ketoacyl-CoA thiolase (Fox3p) and the glycerol-3-phosphate dehydrogenase (Gpd1p). For both, the PTS2 sequence is essential and sufficient to target these proteins in a Pex7p-dependent way into peroxisomes (19,27). Another enzyme, the nicotinamidase Pnc1p, also enters peroxisomes in a Pex7p-dependent manner despite the lack of a PTS (28).…”