Defining the plant disulfide proteome

Abstract

There is considerable interest in redox regulation and new targets for thioredoxin and glutaredoxin are now being identified. It would be of great benefit to the field to have a list of all possible candidates for redox regulation--that is all disulfide proteins in plant. We developed a simple and very powerful method for identifying proteins with disulfide bonds in vivo. In this method, free thiols in proteins are fully blocked by alkylation, following which disulfide cysteines are converted to sulfhydryl groups by reduction. Finally, proteins with sulfhydryls are isolated by thiol affinity chromatography. Our method is unique in that membrane proteins as well as water-soluble proteins are examined for their disulfide nature. By applying this method to Arabidopsis thaliana we identified 65 putative disulfide proteins, including 20 that had not previously been demonstrated to be regulated by redox state. The newly identified, possibly redox-regulated proteins include: violaxanthin de-epoxidase, two oxygen-evolving enhancer proteins, carbonic anhydrase, photosystem I reaction center subunit N, photosystem I subunit III, S-adenosyl-L-methionine carboxyl methyltransferase, guanylate kinase, and bacterial mutT homolog. Possible functions of disulfide bonding in these proteins are discussed.

Regulation of the protein disulfide proteome by mitochondria in mammalian cells

“…4]. Affinity purification and identification of cellular disulfide-containing proteins by mass spectrometry (SI Table 1) reveal nine proteins, seven of which are cell-surface membrane proteins known to contain disulfides, which is consistent with studies in plants (11) and E. coli (12) reporting that, under normal growth conditions, most proteins containing disulfides are secreted or membrane-bound. Upon treatment with the mitochondrial uncoupler carbonylcyanide m-chlorophenylhydrazone (CCCP), the overall signal decreased (Fig.…”

Identification of Novel Nuclear Targets of Human Thioredoxin 1

“…Yano et al used monobromobimane to label free thiols in proteins reduced by Trx that were resolved via two-dimensional gel electrophoresis and identified five Trx targets in peanut seeds (26). The Trx affinity technique was subsequently developed to identify Trx targets in diverse cell types, including spinach (27)(28)(29)(30)(31), Arabidopsis (32)(33)(34)(35), Escherichia coli (36,37), wheat seeds (38,39), Synechocystis (40 -42), and HeLa cells (16,43). The most successful of these studies identified up to 80 putative Trx targets (37).…”

“…More recently, we used the isotope-coded affinity tag approach to identify over 70 targets of Trx1 transnitrosylation and 50 targets of Trx1-mediated denitrosylation (43). Although a Trx mutant has been used to identify Trx targets in non-mammalian cells (25)(26)(27)(28)(29)(30)(31)(32)(33)(34)(35)(36)(37)(38)(39)(40)(41)(42)44), its application in the identification of mammalian targets has been rare, presumably because of the presence of highly abundant cytosolic targets in the total cell lysates, such as Prx1, that compete for a limited number of binding sites on the Trx1 C35S mutant. This is the first report demonstrating that an nTrx1 C35S mutant can be used to identify novel Trx1 targets in the nucleus of mammalian cells.…”

Structural analysis uncovers a role for redox in regulating FKBP13, an immunophilin of the chloroplast thylakoid lumen

“…Such a mode of activation, in which an enzyme of the lumen is rendered active when oxidized in the light, contrasts with the mechanism functional in the stroma. The relation of electron transfer proteins of the lumen (36) [HCF164 (37), thylakoid luminal protein (38), oxygen-evolving enhancer protein (39), and CcdA (40)] to the scheme depicted in Fig. 5 …”