Known signals for targeting of matrix proteins to peroxisomes

Most peroxisomal matrix proteins are targeted to peroxisomes by a PTS1, a C-terminal tripeptide that interacts directly with a specific cytosolic receptor, Pex5 (Johnson and Olsen, 2001). The first PTS1 to be characterized was that of the firely luciferase, which contains the PTS1 serine-lysine-leucine (SKL>). Site-directed mutagenesis experiments revealed that only a limited number of conservative changes were allowed in the PTS1 SKL>, leading to deduce of the PTS1 consensus sequence [SAC][KRH]L> (Gould et al., 1989). However, this consensus sequence became increasingly degenerated as different organisms were discovered to have related but specialized PTS1 sequences. In Saccharo-myces cerevisiae, a large variety of PTS1 tripeptides were deduced from complementation studies on yeast mutants of peroxisomal malate dehydrogenase (MDH3) using MDH3 constructs, whose native PTS1 tripeptide was replaced by PTS1-related variants. Some of the tripeptides found to work as PTS1 in yeast, including SEL>, SSL>, and EKL>, do not fit the consensus sequence determined for mammals and plants (Elgersma et al., 1996).

Studies comparing the peroxisomal targeting efficiency of β-glucuronidase constructs, to which various C-terminal tripeptides were fused, revealed the first plant-specific PTS1 consensus sequence [SACP][KR][LMV]>, the so-called Hayashi motif (Hayashi et al., 1996).

In a follow-up study, a more permissive consensus motif ([SACGT][KRHLN][LMIY]>) was deduced by using a slightly different experimental system consisting of the reporter protein chloramphenicol acetyltransferase and transient expression in BY-2 suspension-cultured cells of tobacco (Nicotiana tabacum, Mullen et al., 1997). Kragler et al. (1998) deduced the most permissive PTS1 motif based on the interaction of PTS1 tripeptides with tobacco Pex5 in the yeast two-hybrid system, which also allowed a proline residue at position -3 ([SACGTP][KRHLN][LMIY]>).

With the availability of large numbers of DNA sequences in the public databases, bioinformatics approaches can be applied to specify plant PTS1 signals. Reumann (2004) specified the C-terminal tripeptides by retrieving plant cDNAs and expressed sequence tags (ESTs) that are homologous to PTS1-targeted proteins from the public protein sequence and EST databases. According to this study, nine PTS1 tripeptides ([SA][RK][LM] without AKM>

plus SRI> and PRL>) were identified in at least 10 sequences and three different orthologous groups are defined as major PTS1. Furthermore, eleven additional PTS1 tripeptides, inclu-ding some previously unknown plant PTS peptides, were defined as minor PTS1. These analyses strongly suggested that not all 24 tripeptides of the Hayashi motif [SACP][KR][LVM]> and in particular not those 100 to 120 PTS1 peptides of the more permissive PTS consensus motifs are functional PTS1 tripeptides in vivo. Instead, a pronounced preference seems to exist in plants for a small set of specific PTS1 tripeptides.

In addition to the tripeptide at the very C-terminal end, accessory elements upstream of the PTS1 have been hypothesized to be important for peroxisomal targeting, in particular for some weak or non-canonical PTS1 peptides (Reumann, 2004). For instance, a basic residue at position -4 can act as an accessory element in some proteins. As demonstrated for the PTS1s of human catalase (ANL>) and rat and human 2-methylacyl-CoA racemase (ANL> and ASL>, respectively), a lysine residue located at the position immediately upstream of the PTS1 tripeptide was found to be essential for peroxisomal targeting (Purdue et al., 1996; Amery et al., 2000; Kotti et al., 2000). Furthermore, residues about 20 upstream of the PTS1 tripeptide can act as accessory elements. In peptides interacting with human Pex5, hydrophobic residues were found with high frequency especially at positions -2 and -5, whereas peptides interacting with S. cerevisiae Pex5 were more hydrophilic and frequently contained arginine at position -2 (Lametschwandtner, et al., 1998). The PTS1 domain of plant PTS1-targeted proteins is characterized by a high probabilityof proline and a second

basic residue in front of the PTS1, as revealed by bioinformatics analyses (Reumann, 2004).

Finally, some PTS1 tripeptides have been shown to require sequences located even further upstream of the PTS1 for successful targeting to peroxisomes. For instance, the tripeptide KKL> of human alanine-glyoxylate aminotransferase must cooperate with an ancillary targeting element located between amino acid residues Val³²⁴ to Ile³⁴⁵, i.e. about 50 residues upstream of the PTS1 tripeptide (Huber et al., 2005).

1.2.1.2 The peroxisome targeting signal type 2 (PTS2)

Compared to the large number of proteins that carry a PTS1 peptide, only few proteins that contain a PTS are known. The first PTS2 peptide identified was RLx₅HL, which was located in the N-terminal end of rat thiolase (Swinkles et al., 1991). Subsequent analyses revealed that PTS2 nonapeptides are conserved in yeast, plants, and mammals but that species-related differences also exist. Intriguingly, Caenorhabditis elegans lacks any gene encoding the PTS2 receptor Pex7 as well as any PTS2-targeted proteins. In this phylogenetic lineage, the PTS2 targeting pathway seems to have been lost during evolution (Motley et al., 2000). In contrast to yeast, the N-terminal domain including the PTS2 peptide is cleaved off upon arrival of the proteins in the peroxisome matrix in plants and mammals (Johnson and Olsen, 2001).

The PTS2 consensus motif of yeast and mammals has been defined as [RK][LVI]x₅[HQ][LA] by site-directed mutagenesis (Tsukamoto et al., 1994; Glover et al., 1994). The first plant-specific PTS2 consensus motif has been determined by mutational analysis of the PTS2 of citrate synthase and malate dehydrogenase in combination with sequence comparison of known plant PTS2 proteins (R[ILQ]x₅HL, Kato et al., 1996, 1998). A very broad PTS2 motif ([RK]x₆[HQ][ALF]), which is similar to that specified for yeast and mammals, was deduced based on targeting experiments with the mutagenized presequence of rat thiolase (Flynn et al., 1998). In contrast to these experimental studies and similar to the bioinformatics-based definition of the PTS1, a relatively small number of two major (R[LI]x₅HL) and nine minor PTS2 nonapeptides (R[QTMAV]x₅HL and RLx₅H[IF]) have been defined for higher plants (Reumann, 2004).