Cover Caption: Cytosolic Localization of GLYR1

Glyoxylate reductase isoform 1 (GLYR1) is a key enzyme in the γ‐aminobutryate (GABA) pathway that is linked to a remarkable array of biological processes including stress response, signaling, and redox homeostasis. However, previously the intracellular localization of GLYR1 was unclear. Ching et al. (pp 152–168) show that GLYR1 in plant cells is localized to the cytosol and not peroxisomes, as previously proposed, by using Arabidopsis mesophyll cells co‐expressing GFP‐tagged GLYR1 and a Cherry‐tagged peroxisomal protein.     

Differential subcellular localization of endogenous and transfected soluble epoxide hydrolase in mammalian cells: evidence for isozyme variants

Endogenous, constitutive soluble epoxide hydrolase in mice 3T3 cells was localized via immunofluorescence microscopy exclusively in peroxisomes, whereas transiently expressed mouse soluble epoxide hydrolase (from clofibrate-treated liver) accumulated only in the cytosol of 3T3 and HeLa cells. When the C-terminal lie of mouse soluble epoxide hydrolase was mutated to generate a prototypic putative type 1 PTS (-SKI to -SKL), the enzyme targeted to peroxisomes. The possibility that soluble epoxide hydrolase-SKI was sorted slowly to peroxiosmes from the cytosol was examined by stably expressing rat soluble epoxide hydrolase-SKI appended to the green fluorescent protein. Green fluorescent protein soluble epoxide hydrolase-SKI was strictly cytosolic, indicating that -SKI was not a temporally inefficient putative type 1 PTS. Import of soluble epoxide hydrolase-SKI into peroxisomes in plant cells revealed that the context of -SKI on soluble epoxide hydrolase was targeting permissible. These results show that the C-terminal -SKI is a non-functional putative type 1 PTS on soluble epoxide hydrolase and suggest the existence of distinct cytosolic and peroxisomal targeting variants of soluble epoxide hydrolase in mouse and rat.     

Sorting pathway and molecular targeting signals for the Arabidopsis peroxin 3

Peroxin 3 (Pex3p) has been identified and characterized as a peroxisomal membrane protein in yeasts and mammals. We identified two putative homologs in Arabidopsis (AtPex3p, forms 1 and 2), both with an identical cluster of positively charged amino acid residues (RKHRRK) immediately preceding one of the two predicted transmembrane domains (TMD1). In transiently transformed Arabidopsis and tobacco BY-2 suspension-cultured cells, epitope-tagged AtPex3p (form 2) sorted post-translationally from the cytosol directly to peroxisomes, the first sorting pathway described for any peroxin in plants. TMD1 and RKHRRK were necessary for targeting form 2 to peroxisomes and sufficient for directing chloramphenicol acetyltransferase to peroxisomes in both cell types. The N and C termini of AtPex3p (form 2) extend into the peroxisomal matrix, different from mammal and yeast Pex3 proteins. Thus, two authentic peroxisomal membrane-bound Pex3p homologs possessing a membrane peroxisomal targeting signal, the first one defined for a plant peroxin and for any Pex3p homolog, exist in plant cells.     

Plant peroxisomes respire in the light: some gaps of the photorespiratory C2 cycle have become filled--others remain

The most prominent role of peroxisomes in photosynthetic plant tissues is their participation in photorespiration, a process also known as the oxidative C2 cycle or the oxidative photosynthetic carbon cycle. Photorespiration is an essential process in land plants, as evident from the conditionally lethal phenotype of mutants deficient in enzymes or transport proteins involved in this pathway. The oxidative C2 cycle is a salvage pathway for phosphoglycolate, the product of the oxygenase activity of ribulose 1,5-bisphosphate carboxylase/oxygenase (RubisCO), to the Calvin cycle intermediate phosphoglycerate. The pathway is highly compartmentalized and involves reactions in chloroplasts, peroxisomes, and mitochondria. The H2O2-producing enzyme glycolate oxidase, catalase, and several aminotransferases of the photorespiratory cycle are located in peroxisomes, with catalase representing the major constituent of the peroxisomal matrix in photosynthetic tissues. Although photorespiration is of major importance for photosynthesis, the identification of the enzymes involved in this process has only recently been completed. Only little is known about the metabolite transporters for the exchange of photorespiratory intermediates between peroxisomes and the other organelles involved, and about the regulation of the photorespiratory pathway. This review highlights recent developments in understanding photorespiration and identifies remaining gaps in our knowledge of this important metabolic pathway.     

Identification of photorespiratory glutamate:glyoxylate aminotransferase (GGAT) gene in Arabidopsis

In the photorespiratory process, peroxisomal glutamate:glyoxylate aminotransferase (GGAT) catalyzes the reaction of glutamate and glyoxylate to 2-oxoglutarate and glycine. Although GGAT has been assumed to play important roles for the transamination in photorespiratory carbon cycles, the gene encoding GGAT has not been identified. Here, we report that an alanine:2-oxoglutarate aminotransferase (AOAT)-like protein functions as GGAT in peroxisomes. Arabidopsis has four genes encoding AOAT-like proteins and two of them (namely AOAT1 and AOAT2) contain peroxisomal targeting signal 1 (PTS1). The expression analysis of mRNA encoding AOATs and EST information suggested that AOAT1 was the major protein in green leaves. When AOAT1 fused to green fluorescent protein (GFP) was expressed in BY-2 cells, it was found to be localized to peroxisomes depending on PTS1. By screening of Arabidopsis T-DNA insertion lines, an AOAT1 knockout line (aoat1-1) was isolated. The activity of GGAT and alanine:glyoxylate aminotransferase (AGAT) in the above-ground tissues of aoat1-1 was reduced drastically and, AOAT and glutamate:pyruvate aminotransferase (GPAT) activity also decreased. Peroxisomal GGAT was detected in the wild type but not in aoat1-1. The growth rate was repressed in aoat1-1 grown under high irradiation or without sugar, though differences were slight in aoat1-1 grown under low irradiation, high-CO2 (0.3%) or high-sugar (3% sucrose) conditions. These phenotypes resembled those of photorespiration-deficient mutants. Glutamate levels increased and serine levels decreased in aoat1-1 grown in normal air conditions. Based on these results, it was concluded that AOAT1 is targeted to peroxisomes, functions as a photorespiratory GGAT, plays a markedly important role for plant growth and the metabolism of amino acids.     

Integrated temporal regulation of the photorespiratory pathway. Circadian regulation of two Arabidopsis genes encoding serine hydroxymethyltransferase

The photorespiratory pathway is comprised of enzymes localized within three distinct cellular compartments: chloroplasts, peroxisomes, and mitochondria. Photorespiratory enzymes are encoded by nuclear genes, translated in the cytosol, and targeted into these distinct subcellular compartments. One likely means by which to regulate the expression of the genes encoding photorespiratory enzymes is coordinated temporal control. We have previously shown in Arabidopsis that a circadian clock regulates the expression of the nuclear genes encoding both chloroplastic (Rubisco small subunit and Rubisco activase) and peroxisomal (catalase) components of the photorespiratory pathway. To determine whether a circadian clock also regulates the expression of genes encoding mitochondrial components of the photorespiratory pathway, we characterized a family of Arabidopsis serine hydroxymethyltransferase (SHM) genes. We examined mRNA accumulation for two of these family members, including one probable photorespiratory gene (SHM1) and a second gene expressed maximally in roots (SHM4), and show that both exhibit circadian oscillations in mRNA abundance that are in phase with those described for other photorespiratory genes. In addition, we show that SHM1 mRNA accumulates in light-grown seedlings, although this response is probably an indirect consequence of the induction of photosynthesis and photorespiration by illumination.     

Peroxisomal hydroxypyruvate reductase is not essential for photorespiration in Arabidopsis but its absence causes an increase in the stoichiometry of photorespiratory CO2 release

Recycling of carbon by the photorespiratory pathway involves enzymatic steps in the chloroplast, mitochondria, and peroxisomes. Most of these reactions are essential for plants growing under ambient CO(2) concentrations. However, some disruptions of photorespiratory metabolism cause subtle phenotypes in plants grown in air. For example, Arabidopsis thaliana lacking both of the peroxisomal malate dehydrogenase genes (pmdh1pmdh2) or hydroxypyruvate reductase (hpr1) are viable in air and have rates of photosynthesis only slightly lower than wild-type plants. To investigate how disruption of the peroxisomal reduction of hydroxypyruvate to glycerate influences photorespiratory carbon metabolism we analyzed leaf gas exchange in A. thaliana plants lacking peroxisomal HPR1 expression. In addition, because the lack of HPR1 could be compensated for by other reactions within the peroxisomes using reductant supplied by PMDH a triple mutant lacking expression of both peroxisomal PMDH genes and HPR1 (pmdh1pmdh2hpr1) was analyzed. Rates of photosynthesis under photorespiratory conditions (ambient CO(2) and O(2) concentrations) were slightly reduced in the hpr1 and pmdh1pmdh2hpr1 plants indicating other reactions can help bypass this disruption in the photorespiratory pathway. However, the CO(2) compensation points (Γ) increased under photorespiratory conditions in both mutants indicating changes in photorespiratory carbon metabolism in these plants. Measurements of Γ*, the CO(2) compensation point in the absence of mitochondrial respiration, and the CO(2) released per Rubisco oxygenation reaction demonstrated that the increase in Γ in the hpr1 and pmdh1pmdh2hpr1 plants is not associated with changes in mitochondrial respiration but with an increase in the non-respiratory CO(2) released per Rubisco oxygenation reaction.     

Two alanine aminotranferases link mitochondrial glycolate oxidation to the major photorespiratory pathway in Arabidopsis and rice

The major photorespiratory pathway in higher plants is distributed over chloroplasts, mitochondria, and peroxisomes. In this pathway, glycolate oxidation takes place in peroxisomes. It was previously suggested that a mitochondrial glycolate dehydrogenase (GlcDH) that was conserved from green algae lacking leaf-type peroxisomes contributes to photorespiration in Arabidopsis thaliana. Here, the identification of two Arabidopsis mitochondrial alanine:glyoxylate aminotransferases (ALAATs) that link glycolate oxidation to glycine formation are described. By this reaction, the mitochondrial side pathway produces glycine from glyoxylate that can be used in the glycine decarboxylase (GCD) reaction of the major pathway. RNA interference (RNAi) suppression of mitochondrial ALAAT did not result in major changes in metabolite pools under standard conditions or enhanced photorespiratroy flux, respectively. However, RNAi lines showed reduced photorespiratory CO(2) release and a lower CO(2) compensation point. Mitochondria isolated from RNAi lines are incapable of converting glycolate to CO(2), whereas simultaneous overexpression of GlcDH and ALAATs in transiently transformed tobacco leaves enhances glycolate conversion. Furthermore, analyses of rice mitochondria suggest that the side pathway for glycolate oxidation and glycine formation is conserved in monocotyledoneous plants. It is concluded that the photorespiratory pathway from green algae has been functionally conserved in higher plants.     

Characterization of the targeting signal of the Arabidopsis 22-kD integral peroxisomal membrane protein

Using a combination of in vivo and in vitro assays, we characterized the sorting pathway and molecular targeting signal for the Arabidopsis 22-kD peroxisome membrane protein (PMP22), an integral component of the membrane of all peroxisomes in the mature plant. We show that nascent PMP22 is sorted directly from the cytosol to peroxisomes and that it is inserted into the peroxisomal boundary membrane with its N- and C-termini facing the cytosol. This direct sorting of PMP22 to peroxisomes contrasts with the indirect sorting reported previously for cottonseed (Gossypium hirsutum) ascorbate peroxidase, an integral PMP that sorts to peroxisomes via a subdomain of the endoplasmic reticulum. Thus, at least two different sorting pathways for PMPs exist in plant cells. At least four distinct regions within the N-terminal one-half of PMP22, including a positively charged domain present in most peroxisomal integral membrane-destined proteins, functions in a cooperative manner in efficient peroxisomal targeting and insertion. In addition, targeting with high fidelity to peroxisomes requires all four membrane-spanning domains in PMP22. Together, these results illustrate that the PMP22 membrane peroxisomal targeting signal is complex and that different elements within the signal may be responsible for mediating unique aspects of PMP22 biogenesis, including maintaining the solubility before membrane insertion, targeting to peroxisomes, and ensuring proper assembly in the peroxisomal boundary membrane.     

Light regulates alternative splicing of hydroxypyruvate reductase in pumpkin

Hydroxypyruvate reductase (HPR) is a leaf peroxisomal enzyme that functions in the glycolate pathway of photorespiration in plants. We have obtained two highly similar cDNAs for pumpkin HPR (HPR1 and HPR2). It has been revealed that two HPR mRNAs might be produced by alternative splicing from a single type of pre-mRNA. The HPR1 protein, but not the HPR2 protein, was found to have a targeting sequence into leaf peroxisomes at the C-terminus, suggesting that alternative splicing controls the subcellular localization of the two HPR proteins. Immunoblot analysis and subcellular fractionation experiments showed that HPR1 and HPR2 proteins are localized in leaf peroxisomes and the cytosol, respectively. Moreover, indirect fluorescence microscopy and analyses of transgenic tobacco cultured cells and Arabidopsis thaliana expressing fusion proteins with green fluorescent protein (GFP) revealed the different subcellular localizations of the two HPR proteins. Both mRNAs were induced developmentally and by light, but with quantitative differences. Almost equal amounts of the mRNAs were detected in pumpkin cotyledons grown in darkness, but treatment with light greatly enhanced the production of HPR2 mRNA. These findings indicate that light regulates alternative splicing of HPR mRNA, suggesting the presence of a novel mechanism of mRNA maturation, namely light-regulated alternative splicing, in higher plants.     

Localization of the tomato bushy stunt virus replication protein p33 reveals a peroxisome-to-endoplasmic reticulum sorting pathway

Tomato bushy stunt virus (TBSV), a positive-strand RNA virus, causes extensive inward vesiculations of the peroxisomal boundary membrane and formation of peroxisomal multivesicular bodies (pMVBs). Although pMVBs are known to contain protein components of the viral membrane-bound RNA replication complex, the mechanisms of protein targeting to peroxisomal membranes and participation in pMVB biogenesis are not well understood. We show that the TBSV 33-kD replication protein (p33), expressed on its own, targets initially from the cytosol to peroxisomes, causing their progressive aggregation and eventually the formation of peroxisomal ghosts. These altered peroxisomes are distinct from pMVBs; they lack internal vesicles and are surrounded by novel cytosolic vesicles that contain p33 and appear to be derived from evaginations of the peroxisomal boundary membrane. Concomitant with these changes in peroxisomes, p33 and resident peroxisomal membrane proteins are relocalized to the peroxisomal endoplasmic reticulum (pER) subdomain. This sorting of p33 is disrupted by the coexpression of a dominant-negative mutant of ADP-ribosylation factor1, implicating coatomer in vesicle formation at peroxisomes. Mutational analysis of p33 revealed that its intracellular sorting is also mediated by several targeting signals, including three peroxisomal targeting elements that function cooperatively, plus a pER targeting signal resembling an Arg-based motif responsible for vesicle-mediated retrieval of escaped ER membrane proteins from the Golgi. These results provide insight into virus-induced intracellular rearrangements and reveal a peroxisome-to-pER sorting pathway, raising new mechanistic questions regarding the biogenesis of peroxisomes in plants.     

Cycloheximide as a tool to investigate protein import in peroxisomes: a case study of the subcellular localization of isoprenoid biosynthetic enzymes

Cytosolic background fluorescence is often observed when native low-abundance peroxisomal proteins carrying a weak peroxisomal targeting sequence are expressed as fluorescent fusion protein using a strong constitutive promoter in transiently transformed plant cells. This cytosolic fluorescence usually comes from the strong expression of the low-abundance proteins exceeding the peroxisome import efficiency. This often results in a misinterpretation of the protein subcellular localization, as there is doubt as to whether proteins are dually targeted to the cytosol and peroxisome or are exclusively localized to peroxisomes. To circumvent this experimental difficulty, the protein peroxisome import study can be optimized by de novo protein synthesis inhibition in transiently transformed cells using the translation inhibitor cycloheximide. This approach was used here successfully for the study of the subcellular localization of distinct plant isoprenoid biosynthetic enzymes, allowing us to clearly demonstrate that 5-phosphomevalonate kinase, mevalonate 5-diphosphate decarboxylase and a short isoform of farnesyl diphosphate synthase from Catharanthus roseus are exclusively localized to peroxisomes.     

Peroxisome function regulates growth on glucose in the basidiomycete fungus Cryptococcus neoformans

The function of the peroxisomes was examined in the pathogenic basidiomycete Cryptococcus neoformans. Recent studies reveal the glyoxylate pathway is required for virulence of diverse microbial pathogens of plants and animals. One exception is C. neoformans, in which isocitrate lyase (encoded by ICL1) was previously shown not to be required for virulence, and here this was extended to exclude also a role for malate synthase (encoded by MLS1). The role of peroxisomes, in which the glyoxylate pathway enzymes are localized in many organisms, was examined by mutation of two genes (PEX1 and PEX6) encoding AAA (ATPases associated with various cellular activities)-type proteins required for peroxisome formation. The pex1 and pex6 deletion mutants were unable to localize the fluorescent DsRED-SKL protein to peroxisomal punctate structures, in contrast to wild-type cells. pex1 and pex6 single mutants and a pex1 pex6 double mutant exhibit identical phenotypes, including abolished growth on fatty acids but no growth difference on acetate. Because both icl1 and mls1 mutants are unable to grow on acetate as the sole carbon source, these findings demonstrate that the glyoxylate pathway can function efficiently outside the peroxisome in C. neoformans. The pex1 mutant exhibits wild-type virulence in a murine inhalation model and in an insect host, demonstrating that peroxisomes are not required for virulence under these conditions. An unusual phenotype of the pex1 and pex6 mutants was that they grew poorly with glucose as the carbon source, but nearly wild type with galactose, which suggested impaired hexokinase function and that C. neoformans peroxisomes might function analogously to the glycosomes of the trypanosomid parasites. Deletion of the hexokinase HXK2 gene reduced growth in the presence of glucose and suppressed the growth defect of the pex1 mutant on glucose. The hexokinase 2 protein of C. neoformans contains a predicted peroxisome targeting signal (type 2) motif; however, Hxk2 fused to fluorescent proteins was not localized to peroxisomes. Thus, we hypothesize that glucose or glycolytic metabolites are utilized in the peroxisome by an as yet unidentified enzyme or regulate a pathway required by the fungus in the absence of peroxisomes.     

A cytosolic pathway for the conversion of hydroxypyruvate to glycerate during photorespiration in Arabidopsis

Deletion of any of the core enzymes of the photorespiratory cycle, one of the major pathways of plant primary metabolism, results in severe air-sensitivity of the respective mutants. The peroxisomal enzyme hydroxypyruvate reductase (HPR1) represents the only exception to this rule. This indicates the presence of extraperoxisomal reactions of photorespiratory hydroxypyruvate metabolism. We have identified a second hydroxypyruvate reductase, HPR2, and present genetic and biochemical evidence that the enzyme provides a cytosolic bypass to the photorespiratory core cycle in Arabidopsis thaliana. Deletion of HPR2 results in elevated levels of hydroxypyruvate and other metabolites in leaves. Photosynthetic gas exchange is slightly altered, especially under long-day conditions. Otherwise, the mutant closely resembles wild-type plants. The combined deletion of both HPR1 and HPR2, however, results in distinct air-sensitivity and a dramatic reduction in photosynthetic performance. These results suggest that photorespiratory metabolism is not confined to chloroplasts, peroxisomes, and mitochondria but also extends to the cytosol. The extent to which cytosolic reactions contribute to the operation of the photorespiratory cycle in varying natural environments is not yet known, but it might be dynamically regulated by the availability of NADH in the context of peroxisomal redox homeostasis.     

Peroxisomal Delta(3),Delta(2)-enoyl CoA isomerases and evolution of cytosolic paralogues in embryophytes

Delta(3),Delta(2)-enoyl CoA isomerase (ECI) is an enzyme that participates in the degradation of unsaturated fatty acids through the beta-oxidation cycle. Three genes encoding Delta(3),Delta(2)-enoyl CoA isomerases and named AtECI1, AtECI2 and AtECI3 have been identified in Arabidopsis thaliana. When expressed heterologously in Saccharomyces cerevisiae, all three ECI proteins were targeted to the peroxisomes and enabled the yeast Deltaeci1 mutant to degrade 10Z-heptadecenoic acid, demonstrating Delta(3),Delta(2)-enoyl CoA isomerase activity in vivo. Fusion proteins between yellow fluorescent protein and AtECI1 or AtECI2 were targeted to the peroxisomes in onion epidermal cells and Arabidopsis root cells, but a similar fusion protein with AtECI3 remained in the cytosol for both tissues. AtECI3 targeting to peroxisomes in S. cerevisiae was dependent on yeast PEX5, while expression of Arabidopsis PEX5 in yeast failed to target AtECI3 to peroxisomes. AtECI2 and AtECI3 are tandem duplicated genes and show a high level of amino acid conservation, except at the C-terminus; AtECI2 ends with the well conserved peroxisome targeting signal 1 (PTS1) terminal tripeptide PKL, while AtECI3 possesses a divergent HNL terminal tripeptide. Evolutionary analysis of ECI genes in plants revealed several independent duplication events, with duplications occurring in rice and Medicago truncatula, generating homologues with divergent C-termini and no recognizable PTS1. All plant ECI genes analyzed, including AtECI3, are under negative purifying selection, implying functionality of the cytosolic AtECI3. Analysis of the mammalian and fungal genomes failed to identify cytosolic variants of the Delta(3),Delta(2)-enoyl CoA isomerase, indicating that evolution of cytosolic Delta(3),Delta(2)-enoyl CoA isomerases is restricted to the plant kingdom.     

Malate dehydrogenase isoenzymes: cellular locations and role in the flow of metabolites between the cytoplasm and cell organelles.

Malate dehydrogenases belong to the most active enzymes in glyoxysomes, mitochondria, peroxisomes, chloroplasts and the cytosol. In this review, the properties and the role of the isoenzymes in different compartments of the cell are compared, with emphasis on molecular biological aspects. Structure and function of malate dehydrogenase isoenzymes from plants, mammalian cells and ascomycetes (yeast, Neurospora) are considered. Significant information on evolutionary aspects and characterisation of functional domains of the enzymes emanates from bacterial malate and lactate dehydrogenases modified by protein engineering. The review endeavours to give up-to-date information on the biogenesis and intracellular targeting of malate dehydrogenase isoenzymes as well as enzymes cooperating with them in the flow of metabolites of a given pathway and organelle.     

The significance of peroxisome function in chronological aging of Saccharomyces cerevisiae

We studied the chronological lifespan of glucose‐grown Saccharomyces cerevisiae in relation to the function of intact peroxisomes. We analyzed four different peroxisome‐deficient (pex) phenotypes. These included Δpex3 cells that lack peroxisomal membranes and in which all peroxisomal proteins are mislocalized together with Δpex6 in which all matrix proteins are mislocalized to the cytosol, whereas membrane proteins are still correctly sorted to peroxisomal ghosts. In addition, we analyzed two mutants in which the peroxisomal location of the β‐oxidation machinery is in part disturbed. We analyzed Δpex7 cells that contain virtually normal peroxisomes, except that all matrix proteins that contain a peroxisomal targeting signal type 2 (PTS2, also including thiolase), are mislocalized to the cytosol. In Δpex5 cells, peroxisomes only contain matrix proteins with a PTS2 in conjunction with all proteins containing a peroxisomal targeting signal type 1 (PTS1, including all β‐oxidation enzymes except thiolase) are mislocalized to the cytosol. We show that intact peroxisomes are an important factor in yeast chronological aging because all pex mutants showed a reduced chronological lifespan. The strongest reduction was observed in Δpex5 cells. Our data indicate that this is related to the complete inactivation of the peroxisomal β‐oxidation pathway in these cells due to the mislocalization of thiolase. Our studies suggest that during chronological aging, peroxisomal β‐oxidation contributes to energy generation by the oxidation of fatty acids that are released by degradation of storage materials and recycled cellular components during carbon starvation conditions.     

Localization of peroxisomal matrix proteins by photobleaching

The distribution of some enzymes between peroxisomes and cytosol, or a dual localization in both these compartments, can be difficult to reconcile. We have used photobleaching in live cells expressing green fluorescent protein (GFP)-fusion proteins to show that imported bona fide peroxisomal matrix proteins are retained in the peroxisome. The high mobility of the GFP-fusion proteins in the cytosol and absence of peroxisomal escape makes it possible to eliminate the cytosolic fluorescence by photobleaching, to distinguish between exclusively cytosolic proteins and proteins that are also present at low levels in peroxisomes. Using this technique we found that GFP tagged bile acid-CoA:amino acid N-acyltransferase (BAAT) was exclusively localized in the cytosol in HeLa cells. We conclude that the cytosolic localization was due to its carboxyterminal non-consensus peroxisomal targeting signal (-SQL) since mutation of the -SQL to -SKL resulted in BAAT being efficiently imported into peroxisomes.     

A newly discovered function of peroxisomes: Involvement in biotin biosynthesis

In plants, peroxisomes are the organelles involved in various metabolic processes and physiological functions including β-oxidation, mobilization of seed storage lipids, photorespiration, and hormone biosynthesis. We have recently shown that, in fungi and plants, peroxisomes play a vital role in biosynthesis of biotin, an essential cofactor required for various carboxylation and decarboxylation reactions. In fungi, the mutants defective in peroxisomal protein import exhibit biotin auxotrophy. The fungal BioF protein, a 7-keto-8-aminopelargonic acid (KAPA) synthase catalyzing the conversion of pimeloyl-CoA to KAPA in biotin biosynthesis, contains the peroxisomal targeting sequence 1 (PTS1), and its peroxisomal targeting is required for biotin biosynthesis. In plants, biotin biosynthesis is essential for embryo development. We have shown that the peroxisomal targeting sequences of the BioF proteins are conserved throughout the plant kingdom, and the Arabidopsis thaliana BioF protein is indeed localized in peroxisomes. Our findings suggest that peroxisomal localization of the BioF protein is evolutionarily conserved among eukaryotes, and required for biotin biosynthesis and plant growth and development.