Localization of AtAcd31.2 to the peroxisome matrix

To investigate if the two putative PTSs of AtAcd31.2 are functional in vivo, several constructs were generated as depicted in Fig. 3.6. The fusion protein AtAcd31.2-EYFP with

Figure 3.6: Diagram of constructed fusion proteins between AtAcd31.2 and EYFP.

In order to verify the predicted PTS1 and PTS2 peptides, the cDNA of AtACD31.2 was fused at the 5’- or the 3’-terminal end, respectively, to EYFP and expressed under the control of a double 35S CaMV promoter. Deletion of the putative dimerization loop, which was presumably located between amino acid residues 226 to 252, is indicated by a gap in the protein sequence. In addition, the predicted PTSs (PTS2: RLx₅HF→RLx₅DF; PTS1: PKL→PEL) were mutagenized by site-directed mutagenesis.

accessible putative N-terminal PTS2 nonapeptide RLx5HF was directed to small punctate structures that were identified as peroxisomes in double labelling experiments using gMDH-CFP (Fig. 3.7A-C). To demonstrate that the N-terminal domain was necessary for directing AtAcd31.2 to peroxisomes and that RLx₅HF was the PTS2 nonapeptide of this chaperone, a deletion construct AtAcd31.2∆N-EYFP was generated by removing the N-terminal most 29 residues including the putative PTS2 (position 11 to 19). The fusion protein AtAcd31.2∆N-EYFP was no longer targeted to peroxisomes, but directed to plastids, as indicated by the larger size of the organelles and their characteristic stromuli extensions (Fig. 3.7D). This result was consistent with a moderate prediction of a plastidic transit peptide in the N-terminal end of this deletion construct (e.g. TargetP: score=0.70). Conclusive evidence that AtAcd31.2 possesses a functional PTS2 derived from site-directed mutagenesis of the PTS2, in which the absolutely conserved histidine residue at position 8 of the putative PTS2 nonapeptide was replaced by aspartic acid (RLx₅HF→ RLx5DF). As shown in Fig. 3.7E, the fusion protein with mutagenized PTS2 peptide indeed remained in the cytosol.

Interestingly, the inversely arranged fusion protein EYFP-AtAcd31.2 with accessible C-terminal tripeptide PKL> was also targeted to peroxisomes (Fig. 3.7F, G). In order to investigate whether PKL> is a second PTS of AtAcd31.2 and necessary for peroxisomal targeting, two altered fusion proteins were generated: (i) a deletion construct lacking the tripeptide PKL>, and (ii) a construct with a mutagenized PTS1, in which the conserved lysine residue at position -2 was changed to glutamic acid. However, the deletion construct EYFP-AtAcd31.2∆PTS1 and the construct EYFP-AtAcd31.2(K284E) with mutagenized C-terminal tripeptide (PKL→PEL) still entered peroxisomes (Fig. 3.7 H-K). These data strongly suggested that the putative PTS1 was not necessary for targeting of AtAcd31.2 to peroxi-somes.

In summary, these results demonstrated that AtAcd31.2 is a peroxisomal protein that is targeted to the matrix by a functional PTS2, and that the putative PTS1 of AtAcd31.2 is not required for peroxisome targeting.

Because EYFP-AtAcd31.2 ∆PTS1 was targeted to peroxisomes but apparently did neither follow the PTS1 nor the PTS2 pathway, an alternative targeting pathway had to be postulated. It has been demonstrated that subunits of oligomeric enzymes that lack a PTS still enter peroxisomes when co-expressed in cells together with PTS-containing subunits,

Figure 3.7: Subcellular targeting analysis of AtAcd31.2 in onion epidermal cells (Allium cepa).

For subcellular localization of AtAcd31.2, onion epidermal cells were transformed biolistically with EYFP and/or ECFP constructs, and subcellular protein targeting was analyzed by fluorescence microscopy. The fusion protein AtAcd31.2-EYFP (A to C) with accessible PTS2 (RLx5HF) was targeted to punctate cell structures (A) that were identified as peroxisomes by double labeling (B, C).

The shortened construct lacking the most 29 amino acid residues of the N-terminus was targeted to chloroplasts in line with a moderate prediction of a plastidic transit peptide in the N-terminus (D), (cont. next page)

A B C

F G

J K

H I D E

L M

N O

either by oligomerization with native enzyme subunits or subunits of closely related homologs. The protein import mechanism is referred to as peroxisome piggyback import (Purdue and Lazarow, 2001).

The import of specific peroxisomal matrix proteins into peroxisomes in a piggyback fashion has profound consequences of physiological relevance for (i) subunit hetero-oligomerization and (ii) the import of homologous subunits, belonging to the same protein superfamily but lacking putative PTS peptides, into peroxisomes. Therefore, as a side aspect of this thesis, we intended to clarify the targeting mechanism of EYFP-AtAcd31.2 into peroxisomes.

To import EYFP-AtAcd31.2 into peroxisomes of onion epidermal cells in a piggybacked fashion, a gene orthologous to AtAcd31.2 had to be encoded and expressed in onion epidermal cells, and the protein had to form dimers or oligomers with EYFP-AtAcd31.2 (see also chapter 4.2). In fact, homologs of AtAcd31.2 from diverse plant species, which were retrieved by translating EST sequences from publicly available databases, also contained a conserved PTS2 nonapeptide (chapter 3.1.1), suggesting that these proteins were probably orthologous to AtAcd31.2 and targeted to peroxisomes as well. Thus, Allium cepa was likely to possess a gene for a peroxisome-targeted ortholog of AtAcd31.2 as well.

In addition and unlike most sHsps, AtACD31.2 was constitutively expressed in leaves under physiological conditions (chapter 3.1.5). This atypical expression pattern of AtACD31.2 led to the reasonable prediction that the gene was probably also expressed under standard conditions in non-green tissue such as storage organs like onions. Thus, it was hypothesized that the fusion proteins EYFP-AtAcd31.2(K284E) and EYFP-AtAcd31.2∆PTS1 may be targeted to peroxisomes via piggybacking by the formation of mixed oligomers with the onion ortholog of AtAcd31.2 or another peroxisome-targeted sHsp homolog.

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(Figure 3.7 length continued),

whereas site-directed mutagenesis of the PTS2 from RLx5HF to RLx5DF abolished peroxisome targeting (E). The inverse fusion protein EYFP-AtAcd31.2 was targeted to peroxisomes as well (F, G).

In this case, however, deletion (EYFP-AtAcd31.2∆PKL, H, I) or mutagenesis of the C-terminal tripeptide (PKL→PEL, J, K) did not abolish peroxisome targeting. Deletion of the dimerization loop of AtAcd31.2 also failed to abolish peroxisome targeting (AtAcd31.2∆226-252, L, M; EYFP-AtAcd31.2∆226-252∆PKL, N, O). For imaging either EYFP- (A, B, D, E, F, H, J, L, N) or ECFP-specific filters were used (C, G, I, K, M, O). The bar represents 20 µm.

According to two reported crystal structures of sHsps, namely that of Hsp16.5 from Methanococcus jannaschii and that of Hsp16.9 from Triticum aestivum, the β-strand β6 of the Ac domain is exchanged between two subunits and thus stabilizes the formation of a dimer, which is the building block of larger sHsp oligomers (Kim et al., 1998; van Montfort et al., 2001). Based on a multiple sequence alignment of Hsp16.5 of M. jannaschii, the homolog of Triticum aestivum, and AtAcd31.2, the putative dimerization loop of AtAcd31.2 was deduced to be located between amino acid residue 226 and 252 (data not shown). To dissect whether the fusion proteins EYFP-AtAcd31.2(K284E) and EYFP-AtAcd31.2∆PTS1 were targeted to peroxisomes by piggybacking, the dimerization loop was deleted in both cDNAs (yielding EYFP-AtAcd31.2∆226-252 and EYFP-AtAcd31.2∆226-252∆PTS1) by two successive PCR reactions. The fusion protein EYFP-AtAcd31.2∆226-252, however, still entered peroxisomes (Fig. 3.7L, M). The peroxisome targeting of this fusion protein was clearly not mediated by the retained C-terminal PKL> since the fusion protein lacking both the dimerization loop and the putative PTS1 PKL> was likewise targeted to peroxisomes (Fig. 3.7N, O). Deletion of the putative dimerization loop of AtAcd31.2 thus did not affect peroxisome targeting.