Identification of peroxisomal targeting signal of pumpkin catalase and the binding analysis with PTS1 receptor

Abstract

SummaryMany peroxisomal proteins are imported into peroxisomes via recognition of the peroxisomal targeting signal (PTS1) present at the C-termini by the PTS1 receptor (Pex5p). Catalase, a peroxisomal protein, has PTS1-like motifs around or at the C-terminus. However, it remains unclear whether catalase is imported into peroxisome via the PTS1 system. In this work, we analyzed the PTS of pumpkin catalase (Cat1). A full or truncated pumpkin Cat1 cDNA fused at the 3 0 end of the green fluorescent protein (GFP) coding sequence was introduced and stably expressed in tobacco BY-2 (Nicotiana tabacum cv. Bright Yellow 2) cells or Arabidopsis thaliana by Agrobacterium-mediated transformation. The cellular localization of GFP was analyzed by fluorescence microscopy. The results showed that the C-terminal 10-amino acid region containing an SKL motif-like tripeptide (SHL) was not required for the import into peroxisomes. Surprisingly, the Cterminal 3-amino acid region was required for the import when the fusion proteins were transiently expressed by using particle gun bombardment, suggesting that the transient expression system is inadequate to analyze the targeting signal. We proposed that the C-terminal amino acid region from 13 to 11 (QKL), which corresponds with the PTS1 consensus sequence, may function as an internal PTS1. Analysis of the binding of Cat1 to PTS1 receptor (Pex5p) by the yeast two-hybrid system revealed that Cat1 can bind with the PTS1 receptor (Pex5p), indicating that Cat1 is imported into peroxisomes by the PTS1 system. Abbreviations PTS1: peroxisomal targeting signal 1; PTS2: peroxisomal targeting signal 2; AtPex5p: Arabidopsis thaliana PTS1 receptor protein; Cat1: pumpkin catalase 1; TPR: tetratricopeptide repeat; CaMV: cauliflower mosaic virus; BD: GAL4 binding domain; AD: GAL4-transactivation domain.

CsCAT3, a catalase gene fromCucumis sativus, confers resistance to a variety of stresses toEscherichia coli

“…The deduced amino-acid sequence of CsCAT3 consists of the conserved catalytic active site and heme-binding site, which play important roles in catalytic function and the heme ligand, suggesting that CsCAT3 is a typical CAT. The subcellular localization prediction of CsCAT3 based on ProtComp, CELLO and Plant-mPLoc indicated that CsCAT3 is targeted to the peroxisome, and the CsCAT3 sequence indicated the presence of a putative peroxisomal targeting signal (PTS1) motif QKIASRMNVRPNI that contains conserved QKL/I/V amino acid residues at the position of 480-482 (Figure 1(B)), which was reported previously [29]. These results clearly demonstrated that CsCAT3 is a peroxisomal catalase, which is consistent with the results of previous studies [18,20,30].…”

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

“…Nonperoxisomal targeting of the deletion construct AtAcd31.2DN:EYFP and the version with mutated PTS2 (RLx 5 HF/RLx 5 DF) demonstrated that RLx 5 HF is a functional PTS2 and argued against the presence of an additional internal PTS similar to those described for CAT and a few other proteins (Kamigaki et al, 2003). Subcellular targeting of AtAcd31.2DN:EYFP to plastids was in line with computer prediction for this fusion protein but is not thought to be of physiological significance, because a second putative alternative translational start codon is lacking in front of the predicted transit peptide and because dual targeting of the full-length fusion protein AtAcd31.2:EYFP to both peroxisomes and plastids was not observed.…”

Protein Quaternary Structure and Expression Levels Contribute to Peroxisomal-Targeting-Sequence-1-Mediated Peroxisomal Import of Human Soluble Epoxide Hydrolase

“…35,36 Furthermore, upstream secondary or tertiary structure is also reported to affect import efficiency in plants. 37 Subsequent steps in the PTS1 pathway in vivo could compensate for the loss of efficiency in the recognition and binding step by Pex5p. For example, substitution of the consensus Leu by Ile in PTS1 (SKL to SKI) disrupts peroxisomal localization in other proteins.…”