Identification and Characterization of a Stress-Inducible and a Constitutive Small Heat-Shock Protein Targeted to the Matrix of Plant Peroxisomes

Abstract

Small heat-shock proteins (sHsps) are widespread molecular chaperones for which a peroxisomal localization has not yet been reported. The Arabidopsis (Arabidopsis thaliana) genome encodes two sHsps with putative peroxisomal targeting signals type 1 or 2 (PTS1 or PTS2). As demonstrated by double-labeling experiments using full-length fusion proteins with enhanced yellow fluorescent protein and deletion constructs lacking the putative targeting domains, AtHsp15.7 (At5g37670) and AtAcd31.2 (At1g06460) are targeted to the peroxisome matrix by a functional PTS1 (SKL.) and a functional PTS2 (RLx 5 HF), respectively. The peroxisomal localization of AtAcd31.2 was further confirmed by isolation of leaf peroxisomes from Arabidopsis by two successive sucrose density gradients, protein separation by one-and two-dimensional gel electrophoresis, and mass spectrometric protein identification. When AtHsp15.7 and AtAcd31.2 were heterologously expressed in yeast (Saccharomyces cerevisiae) and directed to the cytosol by deletion of the PTSs, both sHsps were able to complement the morphological phenotype of yeast mutants deficient in the cytosolic homologs ScHsp42 or ScHsp26. According to expression studies by reverse transcription-PCR, AtAcd31.2 is constitutively expressed, whereas AtHsp15.7 is hardly expressed under normal conditions but strongly induced by heat and oxidative stress, the latter of which was triggered by the catalase inhibitor 3-aminotriazole or the herbicide methyl viologen applied by watering of whole plants or infiltration of rosette leaves. Thus, plants are exceptional among eukaryotes in employing sHsps in the peroxisome matrix to prevent unspecific aggregation of partially denatured proteins under both physiological and stress conditions.

The plant sHSP superfamily: five new members in Arabidopsis thaliana with unexpected properties

“…4b). For comparison, the intracellular distribution pattern of Hsp15.7-Po was similar, but its colocalization with endogenous catalase confirmed the expected targeting of this sHsp to peroxisomes (see also Ma et al 2006). In contrast to Hsp26.5-MII-Myc, the N-terminal-tagged version accumulated in the cytoplasm by forming large protein aggregates, which is probably due to the masking of the Nterminal mitochondrial TP (data not shown).…”

“…Based on this analysis and localization studies (see below), we defined five new subfamilies of sHsps (Fig. 1a, CIV, CV, CVI, CVII, MII) in addition to the previously known seven subfamilies CI, CII, CIII, ER, M, P, and Po (Scharf et al 2001;Ma et al 2006). In contrast to the CI, CII, and MI (former class M) subfamilies, which each consist of more than one member in A. thaliana, each of the newly defined subfamilies is represented by only one gene in A. thaliana, i.e., Hsp18.5-CIV, Hsp15.4-CV, Hsp21.7-CVI, Hsp14.7-CVII, and Hsp26.5-MII.…”

“…Searching for sHsp-homologous genes in the complete genome sequence of A. thaliana revealed a total of 19 genes encoding putative members of the sHsp family. Most of them (14 genes) could be classified on the basis of sequence similarities with homologous plant sHsps described before (Scharf et al 2001 and references therein) or subsequently studied in more detail (Siddique et al 2003;Ma et al 2006). However, five sHsp-encoding genes could not be assigned to any of the known sHsp subfamilies and were defined preliminary based on the phylogentic relationships of the conserved ACDs as related either to the cytoplasmic/ nuclear subfamily CI (CI(r)) or to the plastidial subfamily P (P(r); Scharf et al 2001).…”

“…In addition to the cytoplasmic/nuclear sHsps, there are four subfamilies encoding proteins targeted to the endoplasmic reticulum (ER), plastids (P), mitochondria (M), and peroxisomes (Po), respectively. The latter subfamily was identified initially based on the presence of a putative peroxisomal Type 1 targeting signal (PTS1; Scharf et al 2001) and was subsequently shown to be localized to peroxisomes dependent on the functional C-terminal PTS1 (Ma et al 2006).…”

Characterization of rice small heat shock proteins targeted to different cellular organelles

“…Hsp15.7 in Arabidopsis is the homologue of rice Hsp16. The former was shown to be targeted to the peroxisome and strongly induced by heat and oxidative stress (Ma et al 2006). We demonstrated the in vitro chaperone activity of rice Hsp16.…”