Oilseed isocitrate lyases lacking their essential type 1 peroxisomal targeting signal are piggybacked to glyoxysomes.

Isocitrate lyase (IL) is an essential enzyme in the glyoxylate cycle, which is a pathway involved in the mobilization of stored lipids during postgerminative growth of oil-rich seedlings. We determined experimentally the necessary and sufficient peroxisome targeting signals (PTSs) for cottonseed, oilseed rape, and castor bean ILs in a well-characterized in vivo import system, namely, suspension-cultured tobacco (Bright Yellow) BY-2 cells. Results were obtained by comparing immunofluorescence localizations of wild-type and C-terminal-truncated proteins transiently expressed from cDNAs introduced by microprojectile bombardment. The tripeptides ARM-COOH (on cottonseed and castor bean ILs) and SRM-COOH (on oilseed rape IL) were necessary for targeting and actual import of these ILs into glyoxysomes, and ARM-COOH was sufficient for redirecting chloramphenicol acetyltransferase (CAT) from the cytosol into the glyoxysomes. Surprisingly, IL and CAT subunits without these tripeptides were still acquired by glyoxysomes, but only when wild-type IL or CAT-SKL subunits, respectively, were simultaneously expressed in the cells. These results reveal that targeting signal-depleted subunits are being piggybacked as multimers to glyoxysomes by association with subunits possessing a PTS1. Targeted multimers are then translocated through membrane pores or channels to the matrix as oligomers or as subunits before reoligomerization in the matrix.     

Diverse amino acid residues function within the type 1 peroxisomal targeting signal. Implications for the role of accessory residues upstream of the type 1 peroxisomal targeting signal.

The purpose of this study was to determine whether the plant type 1 peroxisomal targeting signal (PTS1) utilizes amino acid residues that do not strictly adhere to the serine-lysine-leucine (SKL) motif (small-basic-hydrophobic residues). Selected residues were appended to the C terminus of chloramphenicol acetyltransferase (CAT) and were tested for their ability to target CAT fusion proteins to glyoxysomes in tobacco (Nicotiana tabacum L.) cv Bright Yellow 2 suspension-cultured cells. CAT was redirected from the cytosol into glyoxysomes by a wide range of residues, i.e. A/C/G/S/T-H/K/ L/N/R-I/L/M/Y. Although L and N at the -2 position (-SLL, -ANL) do not conform to the SKL motif, both functioned, but in a temporally less-efficient manner. Other SKL divergent residues, however, did not target CAT to glyoxysomes, i.e. F or P at the -3 position (-FKL, -PKL), S or T at the -2 position (-SSI, STL), or D at the -1 position (-SKD). The targeting inefficiency of CAT-ANL could be ameliorated when K was included at the -4 position (-KANL). In summary, the plant PTS1 mostly conforms to the SKL motif. For those PTS1s that possess nonconforming residue(s), other residues upstream of the PTS1 appear to function as accessory sequences that enhance the temporal efficiency of peroxisomal targeting.     

Identification of the peroxisomal targeting signal for cottonseed catalase

Catalase is a ubiquitous peroxisomal matrix enzyme, yet the molecular targeting signal(s) for sorting it in plant cells has not been defined. The most common peroxisome targeting signal (PTS) is a C-terminal tripeptide composed of a conserved SKL motif (type 1 PTS). The PTS for cottonseed catalase (Ccat) was elucidated in this study from immunofluorescence microscopic analyses of tobacco BY-2 suspension cells serving as an in vivo import system. To distinguish biolistically introduced Ccat from endogenous tobacco catalase, Ccat was hemagglutinin (HA) epitope-tagged at its N-terminus. Bombardment with HA-Ccat resulted in the import of Ccat into glyoxysomes, the specialized type of peroxisome in BY-2 cells. The C-terminal tripeptide of Ccat, PSI, is necessary for import. Evidence for this were mislocalizations to the cytosol of PSI-truncated Ccat and AGV-substituted (for PSI) Ccat. PSI-COOH, however, was not sufficient to re-route chloramphenicol acetyltransferase (CAT) from the cytosol to glyoxysomes, whereas the Ccat tetrapeptide RPSI-COOH was sufficient. Surprisingly, substitution of K (common at the fourth position in other plant catalases) for the R (CAT-KPSI) decreased import efficiency. However, substitution of K did not affect import, when additional upstream residues in Ccat were included (e.g. CAT-NVKPSI). Other evidence for the importance of upstream residues comprised abolishment of Ccat import due to substitutions with non-conserved residues (e.g. -AGVNVRPSI for -SRLNVRPSI). These data indicate that Ccat is sorted to plant peroxisomes by a degenerate type 1 PTS (PSI-COOH) whose residues are functionally dependent on a strict context of adjacent C-terminal amino acid residues.     

A new definition for the consensus sequence of the peroxisome targeting signal type 2

All organisms except the nematode Caenorhabditis elegans have been shown to possess an import system for peroxisomal proteins containing a peroxisome targeting signal type 2 (PTS2). The currently accepted consensus sequence for this amino-terminal nonapeptide is -(R/K)(L/V/I)X(5)(H/Q)(L/A)-. Some C.elegans proteins contain putative PTS2 motifs, including the ortholog (CeMeK) of human mevalonate kinase, an enzyme known to be targeted by PTS2 to mammalian peroxisomes. We cloned the gene for CeMeK (open reading frame Y42G9A.4) and examined the subcellular localization of CeMeK and of two other proteins with putative PTS2s at their amino termini encoded by the open reading frames D1053.2 and W10G11.11. All three proteins localized to the cytosol, confirming and extending the finding that C.elegans lacks PTS2-dependent peroxisomal protein import. The putative PTS2s of the proteins encoded by D1053.2 and W10G11.11 did not function in targeting to peroxisomes in yeast or mammalian cells, suggesting that the current PTS2 consensus sequence is too broad. Analysis of available experimental data on both functional and nonfunctional PTS2s led to two re-evaluated PTS2 consensus sequences: -R(L/V/I/Q)XX(L/V/I/H)(L/S/G/A)X(H/Q)(L/A)-, describes the most common variants of PTS2, while -(R/K)(L/V/I/Q)XX(L/V/I/H/Q)(L/S/G/A/K)X(H/Q)(L/A/F)-, describes essentially all variants of PTS2. These redefined PTS2 consensus sequences will facilitate the identification of proteins of unknown cellular localization as possible peroxisomal proteins.     

Structural requirements for interaction of peroxisomal targeting signal 2 and its receptor PEX7

The import of a subset of peroxisomal matrix proteins is mediated by the peroxisomal targeting signal 2 (PTS2). The results of our sequence and physical property analysis of known PTS2 signals and of a mutational study of the least characterized amino acids of a canonical PTS2 motif indicate that PTS2 forms an amphipathic helix accumulating all conserved residues on one side. Three-dimensional structural modeling of the PTS2 receptor PEX7 reveals a groove with an evolutionarily conserved charge distribution complementary to PTS2 signals. Mammalian two-hybrid assays and cross-complementation of a mutation in PTS2 by a compensatory mutation in PEX7 confirm the interaction site. An unstructured linker region separates the PTS2 signal from the core protein. This additional information on PTS2 signals was used to generate a PTS2 prediction algorithm that enabled us to identify novel PTS2 signals within human proteins and to describe KChIP4 as a novel peroxisomal protein.     

Specification of the peroxisome targeting signals type 1 and type 2 of plant peroxisomes by bioinformatics analyses

To specify the C-terminal peroxisome targeting signal type 1 (PTS1) and the N-terminal PTS2 for higher plants, a maximum number of plant cDNAs and expressed sequence tags that are homologous to PTS1- and PTS2-targeted plant proteins was retrieved from the public databases and the primary structure of their targeting domains was analyzed for conserved properties. According to their high overall frequency in the homologs and their widespread occurence in different orthologous groups, nine major PTS1 tripeptides ([SA][RK][LM]> without AKM> plus SRI> and PRL>) and two major PTS2 nonapeptides (R[LI]x5HL) were defined that are considered good indicators for peroxisomal localization if present in unknown proteins. A lower but significant number of homologs contained 1 of 11 minor PTS1 tripeptides or of 9 minor PTS2 nonapeptides, many of which have not been identified before in plant peroxisomal proteins. The region adjacent to the PTS peptides was characterized by specific conserved properties as well, such as a pronounced incidence of basic and Pro residues and a high positive net charge, which probably play an auxiliary role in peroxisomal targeting. By contrast, several peptides with assumed peroxisomal targeting properties were not found in any of the 550 homologs and hence play--if at all--only a minor role in peroxisomal targeting. Based on the definition of these major and minor PTS and on the recognition of additional conserved properties, the accuracy of predicting peroxisomal proteins can be raised and plant genomes can be screened for novel proteins of peroxisomes more successfully.     

Domain mapping of human PEX5 reveals functional and structural similarities to Saccharomyces cerevisiae Pex18p and Pex21p

PEX5 functions as an import receptor for proteins with the type-1 peroxisomal targeting signal (PTS1). Although PEX5 is not involved in the import of PTS2-targeted proteins in yeast, it is essential for PTS2 protein import in mammalian cells. Human cells generate two isoforms of PEX5 through alternative splicing, PEX5S and PEX5L, and PEX5L contains an additional insert 37 amino acids long. Only one isoform, PEX5L, is involved in PTS2 protein import, and PEX5L physically interacts with PEX7, the import receptor for PTS2-containing proteins. In this report we map the regions of human PEX5L involved in PTS2 protein import, PEX7 interaction, and targeting to peroxisomes. These studies revealed that amino acids 1-230 of PEX5L are required for PTS2 protein import, amino acids 191-222 are sufficient for PEX7 interaction, and amino acids 1-214 are sufficient for targeting to peroxisomes. We also identified a 21-amino acid-long peptide motif of PEX5L, amino acids 209-229, that overlaps the regions sufficient for full PTS2 rescue activity and PEX7 interaction and is shared by Saccharomyces cerevisiae Pex18p and Pex21p, two yeast peroxins that act only in PTS2 protein import in yeast. A mutation in PEX5 that changes a conserved serine of this motif abrogates PTS2 protein import in mammalian cells and reduces the interaction of PEX5L and PEX7 in vitro. This peptide motif also lies within regions of Pex18p and Pex21p that interact with yeast PEX7. Based on these and other results, we propose that mammalian PEX5L may have acquired some of the functions that yeast Pex18p and/or Pex21p perform in PTS2 protein import. This hypothesis may explain the essential role of PEX5L in PTS2 protein import in mammalian cells and its lack of importance for PTS2 protein import in yeast.     

Motif refinement of the peroxisomal targeting signal 1 and evaluation of taxon-specific differences

Eukaryote peroxisomes, plant glyoxysomes and trypanosomal glycosomes belong to the microbody family of organelles that compartmentalise a variety of biochemical processes. The interaction between the PTS1 signal and its cognate receptor Pex5 initiates the major import mechanism for proteins into the matrix of these organelles. Relying on the analysis of amino acid sequence variability of known PTS1-targeted proteins and PTS1-containing peptides that interact with Pex5 in the yeast two-hybrid assay, on binding site studies of the Pex5-ligand complex crystal structure, 3D models and sequences of Pex5 proteins from various taxa, we derived the requirements for a C-terminal amino acid sequence to interact productively with Pex5. We found evidence that, at least the 12 C-terminal residues of a given substrate protein are implicated in PTS1 signal recognition. This motif can be structurally and functionally divided into three regions: (i) the C-terminal tripeptide, (ii) a region interacting with the surface of Pex5 (about four residues further upstream), and (iii) a polar, solvent-accessible and unstructured region with linker function (the remaining five residues). Specificity differences are confined to taxonomic subgroups (metazoa and fungi) and are connected with amino acid type preferences in region 1 and deviating hydrophobicity patterns in region 2.     

Antibodies against pex14p block ATP-independent binding of matrix proteins to peroxisomes in vitro.

The membrane protein Pex14p is a key component of the protein import machinery of peroxisomes. Antibodies raised against human Pex14p recognise a 66 kDa protein in sunflower glyoxysomes (HaPex14p) and immunoprecipitate in vitro-translated Arabidopsis Pex14p (AtPex14p). These antibodies inhibit the ATP-independent binding to sunflower peroxisome membranes of peroxisome targeting signal type (PTS) 1- and PTS2-targeted matrix proteins, but not an integral membrane protein. These results suggest that Pex14p functions before the ATP-dependent step of peroxisome assembly.     

Pex20p functions as the receptor for non‐PTS1/non‐PTS2 acyl‐CoA oxidase import into peroxisomes of the yeast Yarrowia lipolytica

Most soluble proteins targeted to the peroxisomal matrix contain a C‐terminal peroxisome targeting signal type 1 (PTS1) or an N‐terminal PTS2 that is recognized by the receptors Pex5p and Pex7p, respectively. These receptors cycle between the cytosol and peroxisome and back again for multiple rounds of cargo delivery to the peroxisome. A small number of peroxisomal matrix proteins, including all six isozymes of peroxisomal fatty acyl‐CoA oxidase (Aox) of the yeast Yarrowia lipolytica, contain neither a PTS1 nor a PTS2. Pex20p has been shown to function as a co‐receptor for Pex7p in the import of PTS2 cargo into peroxisomes. Here we show that cells of Y. lipolytica deleted for the PEX20 gene fail to import not only the PTS2‐containing protein 3‐ketoacyl‐CoA thiolase (Pot1p) but also the non‐PTS1/non‐PTS2 Aox isozymes. Pex20p binds directly to Aox isozymes Aox3p and Aox5p, which requires the C‐terminal Wxxx(F/Y) motif of Pex20p. A W411G mutation in the C‐terminal Wxxx(F/Y) motif causes Aox isozymes to be mislocalized to the cytosol. Pex20p interacts physically with members of the peroxisomal import docking complex, Pex13p and Pex14p. Our results are consistent with a role for Pex20p as the receptor for import of the non‐PTS1/non‐PTS2 Aox isozymes into peroxisomes.     

Calmodulin-like protein AtCML3 mediates dimerization of peroxisomal processing protease AtDEG15 and contributes to normal peroxisome metabolism

Matrix enzymes are imported into peroxisomes and glyoxysomes, a subclass of peroxisomes involved in lipid mobilization. Two peroxisomal targeting signals (PTS), the C-terminal PTS1 and the N-terminal PTS2, mediate the translocation of proteins into the organelle. PTS2 processing upon import is conserved in higher eukaryotes, and in watermelon the glyoxysomal processing protease (GPP) was shown to catalyse PTS2 processing. GPP and its ortholog, the peroxisomal DEG protease from Arabidopsis thaliana (AtDEG15), belong to the Deg/HtrA family of ATP-independent serine proteases with Escherichia coli DegP as their prototype. GPP existes in monomeric and dimeric forms. Their equilibrium is shifted towards the monomer upon Ca²⁺-removal and towards the dimer upon Ca²⁺-addition, which is accompanied by a change in substrate specificity from a general protease (monomer) to the specific cleavage of the PTS2 (dimer). We describe the Ca²⁺/calmodulin (CaM) mediated dimerization of AtDEG15. Dimerization is mediated by the CaM-like protein AtCML3 as shown by yeast two and three hybrid analyses. The binding of AtCML3 occurs within the first 25 N-terminal amino acids of AtDEG15, a domain containing a predicted CaM-binding motif. Biochemical analysis of AtDEG15 deletion constructs in planta support the requirement of the CaM-binding domain for PTS2 processing. Phylogenetic analyses indicate that the CaM-binding site is conserved in peroxisomal processing proteases of higher plants (dicots, monocots) but not present in orthologs of animals or cellular slime molds. Despite normal PTS2 processing activity, an atcml3 mutant exhibited reduced 2,4-DB sensitivity, a phenotype previously reported for the atdeg15 mutant, indicating similarly impaired peroxisome metabolism.     

Differential protein import deficiencies in human peroxisome assembly disorders

Two peroxisome targeting signals (PTSs) for matrix proteins have been well defined to date. PTS1 comprises a COOH-terminal tripeptide, SKL, and has been found in several matrix proteins, whereas PTS2 has been found only in peroxisomal thiolase and is contained within an NH2- terminal cleavable presequence. We have investigated the functional integrity of the import routes for PTS1 and PTS2 in fibroblasts from patients suffering from peroxisome assembly disorders. Three of the five complementation groups tested showed a general loss of PTS1 and PTS2 import. Two complementation groups showed a differential loss of peroxisomal protein import: group I cells were able to import a PTS1- but not a PTS2- containing reporter protein into their peroxisomes, and group IV cells were able to import the PTS2 but not the PTS1 reporter into aberrant, peroxisomal ghostlike structures. The observation that the PTS2 import pathway is intact only in group IV cells is supported by the protection of endogenous thiolase from protease degradation in group IV cells and its sensitivity in the remaining complementation groups, including the partialized disorder of group I. The functionality of the PTS2 import pathway and colocalization of endogenous thiolase with the peroxisomal membranes in group IV cells was substantiated further using immunofluorescence, subcellular fractionation, and immunoelectron microscopy. The phenotypes of group I and IV cells provide the first evidence for differential import deficiencies in higher eukaryotes. These phenotypes are analogous to those found in Saccharomyces cerevisiae peroxisome assembly mutants.     

Import of the peroxisomal targeting signal type 2 protein 3-ketoacyl-coenzyme a thiolase into glyoxysomes

Most peroxisomal matrix proteins possess a carboxy-terminal tripeptide targeting signal, termed peroxisomal targeting signal type 1 (PTS1), and follow a relatively well-characterized pathway of import into the organelle. The peroxisomal targeting signal type 2 (PTS2) pathway of peroxisomal matrix protein import is less well understood. In this study, we investigated the mechanisms of PTS2 protein binding and import using an optimized in vitro assay to reconstitute the transport events. The import of the PTS2 protein thiolase differed from PTS1 protein import in several ways. Thiolase import was slower than typical PTS1 protein import. Competition experiments with both PTS1 and PTS2 proteins revealed that PTS2 protein import was inhibited by addition of excess PTS2 protein, but it was enhanced by the addition of PTS1 proteins. Mature thiolase alone, lacking the PTS2 signal, was not imported into peroxisomes, confirming that the PTS2 signal is necessary for thiolase import. In competition experiments, mature thiolase did not affect the import of a PTS1 protein, but it did decrease the amount of radiolabeled full-length thiolase that was imported. This is consistent with a mechanism by which the mature protein competes with the full-length thiolase during assembly of an import complex at the surface of the membrane. Finally, the addition of zinc to PTS2 protein imports increased the level of thiolase bound and imported into the organelles.     

Pex5p imports folded tetrameric catalase by interaction with pex13p

Human catalase forms a 240-kDa tetrameric complex and degrades H(2) O(2) in peroxisomes. Human catalase is targeted to peroxisomes by the interaction of its peroxisomal targeting signal type 1 (PTS1)-like KANL sequence with the cytosolic PTS1 receptor Pex5p. We show herein that human catalase tetramers are formed in the cytoplasm and that the expression of a PTS signal on each of the four subunits is not necessary for peroxisomal transport. We previously demonstrated that a Pex5p mutant defective in binding to Pex13p, designated Pex5p(Mut234), imports typical PTS1-type proteins but not catalase. This impaired catalase import is not rescued by replacing its C-terminal KANL sequence with a typical PTS1 sequence, SKL, indicating that the failure of catalase import in Mut234-expressing cells is not due to its weak PTS1. In contrast, several enzymatically inactive and monomeric mutants of catalase are efficiently imported in Mut234-expressing cells. Moreover, trimeric chloramphenicol acetyltransferase (CAT) harboring SKL is not imported in Pex5p(Mut234)-expressing cells, but CAT-SKL trimers are transported to peroxisomes in the wild-type cells. These findings suggest that the Pex5p-Pex13p interaction likely plays a pivotal role in the peroxisomal import of folded and oligomeric proteins.     

Identification of novel antifungal nonapeptides through the screening of combinatorial peptide libraries based on a hexapeptide motif

Four sets of mixture based nonapeptide libraries derived from an antifungal hexapeptide pharmacophore Arg-D-Trp-D-Phe-Ile-D-Phe-His-NH(2) (II) have been synthesized. The three C-terminal positions 7, 8 and 9 were subject to randomization using 19 genetically coded amino acids. They were then screened for their antifungal activity against Candida albicans and Cryptococcus neoformans in order to quantify inhibition at each step of the nonapeptide sublibrary deconvolution. The studies led to the identification of several novel nonapeptides with potent antifungal activity. Two of the nonapeptides exhibited approximately 17-fold increase in the activity in comparison to the lead hexapeptide motif His-D-Trp-D-Phe-Phe-D-Phe-Lys-NH(2) (I) against C. albicans.     

A novel pex2 mutant: catalase-deficient but temperature-sensitive PTS1 and PTS2 import

We searched for Chinese hamster ovary (CHO) cell mutants defective in peroxisome biogenesis by using peroxisome targeting sequence (PTS) of Pex3p (amino acid residues 1-40)-fused enhanced green fluorescent protein (EGFP). From mutagenized wild-type CHO-K1 cells stably expressing rat Pex2p and Pex3p(1-40)-EGFP, cell colonies resistant to the 9-(1(')-pyrene)nonanol/ultraviolet treatment were examined for intracellular location of peroxisomal proteins, including EGFP chimera, catalase, and matrix proteins with PTS types 1 and 2. One clone, ZPEG309, showed a distinct phenotype: import defect of catalase, but normal transport of PTS1 and PTS2 proteins at 37 degrees C. PTS1 and PTS2 import was abrogated when ZPEG309 was cultured at 39 degrees C. Genetic defect of ZPEG309 was a nonsense point mutation in a codon for Arg50 in CHO PEX2 and a mutation resulting in a C-terminal truncation of the introduced rat Pex2p. Therefore, ZPEG309 is a novel pex2, catalase-deficient mutant with temperature-sensitive PTS1 and PTS2 import.