We have combined classical subcellular fractionation with large-scale quantitative mass spectrometry to identify proteins that enrich specifically with peroxisomes of Saccharomyces cerevisiae. In two complementary experiments, isotope-coded affinity tags and tandem mass spectrometry were used to quantify the relative enrichment of proteins during the purification of peroxisomes. Mathematical modeling of the data from 306 quantified proteins led to a prioritized list of 70 candidates whose enrichment scores indicated a high likelihood of them being peroxisomal. Among these proteins, eight novel peroxisome-associated proteins were identified. The top novel peroxisomal candidate was the small GTPase Rho1p. Although Rho1p has been shown to be tethered to membranes of the secretory pathway, we show that it is specifically recruited to peroxisomes upon their induction in a process dependent on its interaction with the peroxisome membrane protein Pex25p. Rho1p regulates the assembly state of actin on the peroxisome membrane, thereby controlling peroxisome membrane dynamics and biogenesis.

(10,11). Another way to stabilize Fvs would be to connect VH and VL by a disulfide bond. One previous study (9) indicated that this is possible. But in that approach "custom disulfides" were placed in complementaritydetermining regions (CDRs) of an antibody whose structure was known (12)

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...