Distinct Physiological Functions of Thiol Peroxidase Isoenzymes in Saccharomyces cerevisiae

Sung Goo Park,Mee-Kyung Cha,Woojin Jeong,Il-Han Kim

doi:10.1074/jbc.275.8.5723

Abstract

A new type of peroxidase ("thiol peroxidase"; TPx) having cysteine as the primary site of catalysis has been discovered from prokaryotes to eukaryotes. In addition to two yeast TPx isoforms (TSA I and TSA II/AHPC1) previously described, three additional TPx homologues were identified by analysis of the open reading frame data base for Saccharomyces cerevisiae. Three novel isoforms showed a distinct thiol peroxidase activity supported by thioredoxin, and appeared to be distinctively localized in cytoplasm, mitochondria, and nucleus. Each isoform was named after its subcellular localization such as cytoplasmic TPx I (cTPx I or TSA I), cTPx II, cTPx III (TSA II/AHPC1), mitochondrial TPx (mTPx), and nuclear TPx (nTPx). Their transcriptional activities suggest that cTPx I and cTPx III are the most predominant isoforms among the five type isoforms. Transcriptional activities of TPx isoenzymes during yeast life span were quite different from each other. Unlike other TPx null mutants, cTPx I null mutant was hypersensitive to various oxidants except for 4-nitroquinoline N-oxide. The null mutant was more resistant toward 4-nitroquinoline N-oxide and acidic culture than its wild type. The severe growth retardation of cTPx II mutant resulted in accumulation of G(1)-phased cells. Based on kinetic properties of five isoforms, their subcellular localizations, and distinct physiology of each null mutant, we discussed the physiological functions of five types of TPx isoenzymes in yeast throughout the full growth cycle.

Highlights

The incomplete reduction of molecular oxygen during respiration, lipid metabolism in peroxisomes, and aerobic metabolism cause to form reactive oxygen species (ROS)1 such as H2O2, alkyl hydroperoxides, and superoxide anion
In addition to targeting signal was identified in both YML028W (TSA I) and TSA II/AHP1, we reported for the first time the distinct subcellular localization and physiological function of three new types of thiol peroxidase (TPx) isoenzymes in yeast cells
Cytoplasmic TPx I Acts as a Principal Antioxidant—Based on the subcellular localization of five types of TPx isoenzymes in yeast cell, we discussed the physiological functions of TPx isoenzymes during the full life span

Summary

EXPERIMENTAL PROCEDURES

Yeast Culture and Media—Standard methods were used for growth, transformation, and genetic manipulation of S. cerevisiae [14]. Total RNA (25 ␮g) was fractionated in a 1% denaturing agarose gel, blotted, and probed with PCR-amplified labeled ORF by using standard method [15]. ␤-Galactosidase assay was performed as described [18]. Plasmid Construction for Expression of Five Yeast TPx—Five yeast TPx genes were amplified from yeast genomic DNA by the polymerase chain reaction (PCR) with two pairs of primers covering whole coding sequence. The following primers were used for the PCR of TPx genes and enzyme sites are underlined: cTPx I (5Ј-CGATCCATATGGTCGCTCAAGTTCAAAAG (forward) and 5Ј-CGCGGATCCTTATTTGTTGGCAGCTTCGA (reverse)), cTPx II

Physiological Functions of Five Thiol Peroxidases in Yeast

RESULTS

DISCUSSION

Cytoplasmic TPx III Acts as an Important Cytoplasmic Alkyl