ABSTRACT
Intracellular levels of iron are tightly regulated. Saccharomyces cerevisiae uses well-defined pathways to extract iron molecules from the environment. Once inside the cell, the iron molecules must be transferred to target sites via an intracellular iron transporter. Although analogous carriers have been described for other metals, such as copper, an iron transporter has yet to be identified. We used two-dimensional gel electrophoresis and mass spectrometry techniques to attempt to identify the iron transporter from cytosolic fraction of S. cerevisiae. In this study, we identified the iron-binding activity of thioredoxin reductase, and our data suggest a potential role for this enzyme in intracellular iron transport.
Subject(s)
Iron-Binding Proteins/physiology , Iron/metabolism , Saccharomyces cerevisiae/enzymology , Thioredoxin Reductase 1/physiology , Chromatography, Affinity/methods , Circular Dichroism , Culture Media , Iron-Binding Proteins/genetics , Iron-Binding Proteins/isolation & purification , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae/growth & development , Spectrophotometry, Ultraviolet , Thioredoxin Reductase 1/genetics , Thioredoxin Reductase 1/isolation & purificationABSTRACT
Psychrophiles have been known as efficient organism to degrade organic solvent. To investigate the mechanism of solvent stress and identify the factors that affect the solvent stress in psychrophiles, we selected Bacillus psychrosaccharolyticus one of the psychrophiles and two-dimensional gel electrophoresis was performed. Among the protein spots analyzed by 2-DE, five spots induced in 3% IPA stress conditions were identified by MS/MS, and one of these spots was identified as a Hsp33 family. The Hsp33 protein sequence of B. psychrosaccharolyticus exhibited a high similarity with the corresponding proteins of other bacteria. The Hsp33 protein of B. psychrosaccharolyticus has a highly conserved zinc-binding domain (CXCX, CXXC) that includes four cysteine residues in the C-terminus. In addition, the transcriptional induction of the HSP33 of B. psychrosaccharolyticus was confirmed by Northern blot analysis, and formation of free thiol linkage was induced under stress conditions such as exposure to solvents, heat-shock, and oxidative stress. Furthermore, over-expressed strains of HSP33 of B. psychrosaccharolyticus in Escherichia coli improved stress tolerance to the organic solvent when compared with the wild-type. These data suggest that the solvent stress condition was similar to heat-shock or oxidative stress, especially through the triggering of induction and activation of a redox-regulatory chaperone, Hsp33, and Hsp33 plays a critical role in the tolerance to stress.
