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1. Details of Module and its Structure
Module Detail
Subject Name <BOTANY>
Paper Name <Cell Biology>
Module Name/Title < Glyoxysomes and Peroxisomes :structure, enzymes and functions >
Module Id <9>
Pre-requisites Basic knowledge about plant cells, their contents and basic idea about cell organelles
Objectives To make the students aware of the structure and function of glyoxysomes and peroxisomes along with the enzymes present within them.
Keywords Glyoxysomes, peroxisomes, biogenesis, glyoxalate cycle, protein import, catalase. Hydrogen peroxide, photorespiration, β oxidation
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2. 2. Development Team
TABLE OF CONTENTS 1. Introduction
2. Types of peroxisomes 3. Biogenesis of peroxisomes
3.1 Models for peroxisome biogenesis
3.2 Contribution of ER to peroxisome biogenesis 3.3 Import of matrix proteins
3.3.1 Targeting signals
3.3.2 Mechanism of translocation
3.3.3 Peroxisomal membrane protein trafficking 4. Peroxisome growth and division
5. Functions of peroxisomes 5.1 General functions
5.2 The glyoxalate cycle: function of glyoxysomes 5.3 Photorespiration: function of leaf peroxisomes 5.4 Peroxisomal β-oxidation
Role Name Affiliation
National Coordinator <NA>
Subject Coordinator <Dr. Sujata Bhargava>
Paper Coordinator <Dr. Nutan Malpathak>
Content Writer/Author (CW) <Dr. Gauri Abhyankar>
Content Reviewer (CR) <Dr. Nutan Malpathak >
Language Editor (LE) <Dr. Nutan Malpathak >
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1. Introduction:
Peroxisomes are small organelles present in all eukaryotic cells. They are bound by single membrane with diameter ranging between 0.2 to 1.5μm. Inside they contain a coarsely granular matrix. In some cases the matrix contains numerous threads or fibrils.
Peroxisomes can be observed under the electron microscope (Fig 1) and were referred to as microbodies (Donaldson et al, 2001).
Fig 1: An electron micrograph showing peroxisome.
Peroxisomes are variable in size and shape depending on the cell or tissue type.
They can be nearly spherical to ovoid and frequently pleomorphic with large invaginations.
Peroxisomes do not contain their own DNA and ribosomes. So all peroxisomal proteins are encoded by nuclear genes. They are synthesized on free ribosomes in the cytosol and incorporated into pre-existing peroxisomes. They are involved in two fundamental processes: diverse reactions involved in lipid metabolism, and defense systems for in situ scavenging of peroxides and reactive oxygen species.
2. Types of peroxisomes:
There are four classes of peroxisomes commonly known in higher plants. They are – glyoxysomes, leaf peroxisomes, root nodule peroxisomes and unspecialized peroxisomes (Donaldson et al, 2001). All these classes show certain common characteristic features like - a) single membrane, b) high equilibrium density in sucrose gradient centrifugation, and c) finely amorphous matrix. Besides their common features, all of them play distinct metabolic roles depending on the developmental stage of the tissue and the type of cell in
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which they are present. But all of them are meant to carry out activities that produce and destroy hydrogen peroxide.
A) Glyoxysomes are specialized peroxisomes that are observed in germinating seedlings of oil seeds and also in senescent organs (gerontosomes). Glyoxysomes mobilize storage lipids in germinating seedlings through glyoxalate pathway. Succinate that is produced in glyoxysomes by glyoxalate pathway is finally converted to sucrose in the cytoplasm. The presence of glyoxysomes in senescing or old organs is thought to be a response to the mobilization of membrane lipids.
B) Leaf peroxisomes are present in green and photosynthetically active tissues, such as green cotyledons and leaves. They contain enzymes necessary for light dependent processes of photorespiration.
C) Root nodule peroxisomes are present in the root nodules of certain legumes and are involved in nitrogen metabolism. In many tropical legumes like soybean and cowpea, nitrogen is transported in the form of ureides, allantoin and allantoic acid. Reactions of ureide biosynthesis occur in various subcellular compartments. One of the steps in this biosynthesis (conversion of urate to allantoin) is carried out in peroxisomes by urate oxidase.
D) Unspecialized peroxisomes are present in plant tissues that are not photosynthetically active and those which lack storage lipids. So these are observed in the roots of most plants. Unspecialized peroxisomes are smaller in size than other types of peroxisomes and are present in low frequency and density compared to glyoxysomes and leaf-type peroxisomes. Their specific role in the cellular metabolism is not known.
3. Biogenesis of peroxisomes:
Peroxisome biogenesis includes various processes like peroxisome membrane formation, import of matrix proteins, and proliferation & inheritance of the organelle.
Proteins responsible for biogenesis of peroxisomes are called as peroxins (PEX proteins) and they are translated in the cytoplasm. It was considered that peroxisomes are formed from endoplasmic reticulum, but it is quite debatable.
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3.1 Models for peroxisome biogenesis:
A) The first model suggested was ER vesicular model (Beevers, 1979). This model suggested that peroxisomes were formed exclusively through vesiculation of specialized ER regions. All soluble and membrane bound protein constituents of the peroxisomes were considered to be synthesized cotranslationally on the ER (Fig 2).
They were then sequestered into a specialized region of the ER. This ER region would form a smooth vesicle that would expand and ultimately bud off to yield (de novo) a nascent, functional peroxisome.
Fig 2: ER vesiculation model (Hu et al, The Plant Cell, 24: 2279–2303, 2012).
B) However, new techniques and reevaluation of older data resulted in the new model called as growth and division model (Lazarow & Fujiki, 1985). This model considered peroxisomes as fully autonomous organelles like chloroplasts and mitochondria. They were postulated to increase in size by post-translational import of protein constituents from the cytosol. All peroxisome membrane proteins (PMPs) and matrix proteins are synthesized on free polyribosomes in the cytoplasm and they are post-translationally sorted to pre-existing and new (daughter) peroxisomes. Peroxisomes were considered to be formed only by the fission of pre-existing organelles (Fig 3). In this model, ER served only as a source of membrane lipids required for the enlargement of preexisting peroxisomes.
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Fig 3: Growth and division model (Hu et al, The Plant Cell, 24: 2279–2303, 2012).
C) The current working model (ER semiautonomous model) for peroxisome biogenesis includes aspects of both earlier models along with the latest data. The model considers that peroxisomes are semi-autonomous and they are formed by two distinct pathways:
1) de novo biogenesis from specific regions of the ER and 2) by growth and fission of pre-existing peroxisomes. This model depicts one important feature, i.e. involvement of ER-derived pre-peroxisomes (Fig 4). These pre-peroxisomes carry phospholipids and some PMPs (peroxisomal membrane proteins) to pre-existing peroxisomes and/or they fuse together in a controlled, step-wise fashion to form a new peroxisome (Trelease &
Lingard, 2006; Titorenko & Rachubinski, 2009). Group I PMPs are post-translationally inserted into the pER either directly or first inserted into general ER and then routed to the pER. These group I PMPs are further transported from the pER to pre-existing and daughter peroxisomes. All matrix proteins and group II PMPs are classified post- translationally from the cytosol and sent to daughter peroxisomes and preexisting peroxisomes, and perhaps pre-peroxisomes.
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Fig 4: ER semiautonomous model (Hu et al, The Plant Cell, 24: 2279–2303, 2012).
There is an increasing understanding about the processes involved in de novo synthesis, growth and fission of peroxisomes. It is now considered that these processes may not be solely controlled independently (Koch & Brocard, 2011) and they may vary considerably depending on the species, cell type, or physiological status of the organism.
So, a cohesive model of peroxisome biogenesis may not be easy to attain.
3.2 Contribution of ER to peroxisome biogenesis:
Earlier studies suggested that peroxisomes originated from ER. This was supported by many facts like – during seed germination some enzymes appeared in the ER first and later they were observed in glyoxysomes. Subsequently, synthesis of peroxisomal enzymes and some PMPs was carried out on free polyribosomes and then the enzymes were imported post-translationally into peroxisomes.
The growth and division of peroxisomes was linked with the ER-derived biogenesis model because in yeast cells it was shown that ER derived membrane structures fused with pre-existing peroxisomes (Fig. 5) (Motley and Hettema, 2007). However, ER derived
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de novo peroxisome biogenesis occurred only when pre-existing peroxisomes were absent. Furthermore, peroxisome biogenesis in the cells showing pre-existing peroxisomes was dependent on Vps1 and Dnm1 – dynamin-related proteins (DRPs). If these DRPs were absent, number of peroxisomes was much less or reduced. As against this, the de novo biogenesis of peroxisomes was DRP independent. These observations strongly suggested that fission of preexisting peroxisomes require DRPs but the exit of pre-peroxisomal structures do not require DRPs.
Fig 5: Contribution of the ER to peroxisome biogenesis. Most PMPs are first imported into the ER through the Sec61/SSH1 translocon (left inset), are sorted into a pre-peroxisomal compartment, and bud out in a Pex3/Pex19-dependent manner to form pre-peroxisomal vesicles (right inset). These vesicles form mature peroxisomes after fusion. The fusion process is dependent on Pex1/Pex6 and matrix protein import (de novo pathway). The de novo pathway produces cells having peroxisomes in the biogenesis mutants (e.g., pex3 Δ /pex19 Δ) lacking the organelle when corresponding genes are reintroduced. Alternatively, the pre-peroxisomal vesicles fuse with divided peroxisomes generated from preexisting mature peroxisomes.
Peroxisome division requires Pex11 and a specific set of DRPs. In plants, retrograde trafficking from peroxisomes to the ER is observed (Ma et al, 2011).
3.3 Import of matrix proteins:
3.3.1 Targeting signals:
Peroxisomal matrix proteins mostly contain one out of two peroxisomal targeting signals. PTS1 (peroxisomal targeting signal type 1) is located at the C-terminal of the
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protein and consists of a non-cleavable tripeptide made up of serine–lysine–leucine (SKL), whereas, PTS2 (peroxisomal targeting signal type 2) is a nonapeptide located 20-30 residues from the amino terminus (Ma et al, 2011). PTS1 is conserved between organisms. It belongs to a family of sequences which generally correspond to the pattern of [small side chain amino acid]– [basic amino acid]–[hydrophobic amino acid], although in some cases this sequence pattern can be considerably more diverse (Chowdhary et al, 2012). The diversity in sequence pattern implied that the residues immediately adjacent to the C-terminal tripeptide might have an auxiliary function (Brocard & Hartig, 2006). PTS2 sequences have the consensus R[L/I/Q] X5 HL (Lanyon-Hogg et al, 2010). In most organisms, the PTS1 import pathway functions independently of PTS2 pathway components, but in Arabidopsis thaliana, Pex7 is also required for PTS1 import because it enhances the stability of Pex5.
PEX5 functions as the receptor protein for PTS1. It is a modular protein that conserves its essential features between organisms. The C-terminal domain of PEX5 includes seven tetratricopeptide (TPR) repeats. These are repeats of 34 amino acids which form a pair of helices and thereby bind the PTS1 peptide. TPRs show a series of highly conserved asparagines residues within them (Baker et al, 2016). The N-terminus of PEX5 is a natively unstructured domain (Carvalho et al, 2006) and contains a number of functionally important motifs. PEX5 has binding site for PEX7 in plants. PEX7 is the import receptor for a second class of matrix proteins carrying N-terminal PTS2 signal (Woodward
& Bartel, 2005). PEX5 also includes many lysines and a conserved cysteine residue that are targets for ubiquitination. This modification regulates receptor turnover
and recycling (Francisco et al, 2014).
3.3.2 Mechanism of translocation:
It is well known that peroxisomes have capacity to transport folded and oligomeric proteins. So the translocation machinery can accommodate a wide variety of sizes and shapes. Recent studies show that in many cases monomers are preferred for import (Freitas et al, 2015). Also PEX5 may actively inhibit oligomerization of some proteins (Freitas et al, 2011).
Once translated, PTS1 proteins interact with their receptor PEX5 in the cytosol (Fig.
6). PEX5 is highly conserved and provides a binding pocket for PTS1 (Lanyon-Hogg et al., 2010).
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Fig 6: The import of peroxisomal matrix proteins. The process may be divided into distinct steps (white numbers in closed black circles). Bold numbers indicate corresponding Pex proteins. The steps are: (1) Receptor–cargo interaction in the cytosol (2) Receptor–cargo docking at the peroxisomal membrane with the docking subcomplex, inducing the assembly of the translocon. (3) Translocation of the receptor–cargo complex across the membrane followed by cargo release. (4) Export of cargo-free receptors from the peroxisome matrix to the membrane. (5a) Monoubiquitination of the receptor on a cysteine by Pex4 and Pex2 (for receptor recycling) or (5b) polyubiquitination of the receptor on a lysine by Ubc4/5 and Pex10/12 (for degradation by the RADAR pathway). (6a) Receptor recycling from the peroxisome membrane back to the cytosol by the action of the AAA ATPases (Pex1 and Pex6) and ATP hydrolysis, or (6b) degradation of a receptor that is blocked from recycling via the RADAR pathway involving the proteasome. (7) deubiquitination of the receptor before the next round of import. The squiggly line on Pex5 denotes its disordered N-terminal segment.(Ma et al, 2011).
PTS2 proteins interact with their receptor PEX7 prior to peroxisome entry (Fig 7).
PEX5 is able to mediate interaction with the peroxisome membrane alone, but PEX7 requires accessory proteins. In Arabidopsis, PEX5 acts as the co-receptor for PEX7 (Nito et al., 2002) and its N-terminal domain is necessary for PEX7 interaction.
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Fig 7: Working model for the PTS2-mediated protein import - PTS2-containing proteins are recognized by the cytosolic receptors PEX5 and PEX7 forming a highly stable trimeric complex. The cargo -receptor complex then interacts with the docking/translocation machinery (DTM) at the peroxisome membrane. The strong protein-protein interactions that occur at this stage result in the insertion of the cargo-receptor complex into the DTM. At this stage PEX5 displays a transmembrane topology, whereas PEX7 exposes a part of its polypeptide chain into the peroxisomal matrix. The insertion step also induces conformational alterations in PEX5, disrupting its strong stabilizing effect on the PEX7-PTS2 interaction, and thus triggering the release of the PTS2 protein into the peroxisomal matrix. Here, the PTS2 -containing peptide is cleaved by TYSND1. After cargo release, the receptors are recycled back into the cytosol by an ATP - dependent machinery. (Rodrigues et al, 2015).
PEX14 is an integral PMP essential for PTS1 and PTS2 import (Hayashi et al., 2000). Attachment of PEX5/7 at the peroxisome membrane also involves PEX13, where PEX7 binds to the N-terminus of PEX13 (Mano et al., 2006). There is still uncertainty about the order, stoichiometry, and affinity of binding interactions among PEX5, PEX7, their cargoes, PEX14, and PEX13. Still it is presumed that the import is driven by thermodynamically favorable binding interactions. The mechanism of protein translocation is not very clear but yeast PEX5 and PEX14 believed to form a transient pore opening to a diameter of up to 9 nm (Meinecke et al., 2010).
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Once the protein reaches the matrix, cargo is unloaded and the receptors are recycled back. Cargo unloading again remains as an unclear process. In yeast, Pex5p re- export requires –
a) three RING finger peroxins (Pex2p, Pex10p, and Pex12p),
b) the ubiquitin-conjugating enzyme Pex4p and its membrane anchor Pex22p,
c) and the two AAA ATPases (Pex1p and Pex6p), which are tethered to the membrane by Pex15p.
After cargo release, the cargo-free receptors enter the peroxisome membrane for two reasons –
1) either for shuttling back to the cytoplasm for another round of import, or
2) for degradation by the proteasome (receptor accumulation and degradation in the absence of recycling [RADAR] pathway) when there is some dysfunction in receptor recycling (Léon et al., 2006).
Pex20 is a PTS2 receptor and its export to the peroxisome membrane requires the RING subcomplex. PTS receptor/co-receptor recycling from peroxisomes requires a ubiquitination step. This step is followed by a ATP-driven dislocation step catalyzed by Pex1 and Pex6 (Miyata and Fujiki, 2005). Both Pex5 and Pex18/Pex20 can be modified by
a) monoubiquitination (linkage of a single ubiquitin molecule) that serves as mandatory signal for receptor recycling, or
b) polyubiquitination (conjugation of at least four ubiquitin molecules), which serve as obligatory signal for proteasomal degradation. (Purdue and Lazarow, 2001).
The ubiquitination pathway requires a ubiquitin-activating enzyme (E1), ubiquitin- conjugating enzyme (E2), and ubiquitin ligase (E3) to conjugate ubiquitin to its target protein (Kerscher et al., 2006). The monoubiquitination of Pex5 depends on Pex4 in yeast and in plants.
The polyubiquitination of Pex5 and Pex20 occurs in cells lacking any component of the receptor recycling machinery (Platta et al., 2004). It is presumed that Pex5 and Pex20 may be polyubiquitinated under certain physiological conditions, like either when they are dysfunctional after multiple rounds of recycling or when Pex1 and Pex6 are transiently nonfunctional due to low ATP levels.
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3.3.3 Peroxisomal membrane protein trafficking:
Peroxisomal membrane proteins are present in the single membrane surrounding the peroxisomes. All these membrane proteins are synthesized in the cytoplasm and then transported to peroxisomes. So other organelles are not involved in biogenesis of peroxisomes. But we need to know how these proteins are transported and anchored in the peroxisomal membrane.
Pex3p and Pex19p are essential for targeting membrane proteins to peroxisomes.
PMPs contain a divergent hydrophobic sequence known as the membrane peroxisomal targeting sequence (mPTS) and this is bound by Pex19p in the cytosol. Pex19p binds to cargo PMP and then the complex (mPTS + Pex19p + cargo PMP) binds to Pex3p on the peroxisomal membrane. The binding to Pex3p facilitates insertion and orientation of the PMP into the peroxisomal membrane (Heiland & Erdmann, 2005).The entire process of protein integration into the peroxisomal membrane is energy independent which does not require ATP or GTP hydrolysis (Pinto et al, 2006).
Pex19p is farnesylated at its COOH terminus which might help in interrupting membrane lipid bilayer and/or stabilizing transmembrane domain of a nascent PMP. But farnesylation is not required for Pex19p function. Alternatively, PMPs can be incorporated through ER translocation apparatus and/or the mitochondrial outer membrane translocase into the membranes of these organelles and then to the membranes of peroxisomes. To exactly know the transport mechanism, a complex model of PMP biogenesis has been putforth (Fig 8). The model requires extensive and careful analysis to define the actual sequence of events involved in the targeting and insertion of the different and varied PMPs.
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Fig 8: Protein insertion into the peroxisomal membrane: Translation of all peroxisomal membrane proteins begins in the cytosol. Cotranslational insertion into the ER is mediated by the signal recognition particle (SRP) pathway. Posttranslational insertion of proteins into the peroxisomal membrane can be achieved through four routes: HSP70/HSP40 chaperones can maintain membrane proteins in an insertion competent state and direct proteins to either the ER translocon or the translocase of the outer mitochondrial membrane (TOM) complex; COOH-terminally anchored proteins rely on the guided entry of tail-anchored proteins (Get) pathway for entry into the ER. Import into both the ER and mitochondria necessitates the existence of a trafficking mechanism to bring these PMPs to peroxisomes, which is not depicted here.PMPs can also target to the peroxisome using the PMP-specific cytosolic chaperone, Pex19, and its two docking partners, Pex3 and Pex16 (Mast et al, 2010).
4. Peroxisome growth and division:
Peroxisomes grow in number by monitoring the levels of their matrix proteins and divide only after a particular threshold has been reached. The threshold is relative, because the diameter of individual peroxisomes ranges from 0.1 to 1 µm. Many factors like dynamin-like proteins and their associated recruitment factors function to coordinate the division of peroxisomes with other cellular processes (Mast et al, 2010).
Currently, two modes of peroxisome division are predicted – a) requirement of peroxisome proliferation in response to environmental stimuli, i.e., a diet rich in
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compounds requiring metabolism by peroxisomes, and b) organelle division or increase in number with respect to a cell’s need in response to cell division.
One undetermined aspect of peroxisome division is whether it is symmetrical, asymmetrical, or both (Fig 9). Electron micrographs show dimples or tubules formed from the body of the peroxisomes which point towards asymmetrical division of peroxisomes.
Hansenuela polymorpha, a yeast showed formation of prominent tubule which confirms asymmetrical division in peroxisomes. Asymmetry is not restricted to the peroxisome body but is also seen in constituents of the peroxisomal membrane. The membrane protein Inp2p is polarized toward the leading edge of peroxisomes in yeast cells lacking the dynamin-related protein Vps1p (Fagarasanu et al, 2009). Matrix proteins also show asymmetrical distribution between the two peroxisomes arising from peroxisome division.
The distribution of lipids within the peroxisomal membrane is an important aspect of the current model of peroxisome division. Mature peroxisomes do not fuse with each other, although the fusion of immature, precursor peroxisomes may play a role in their development.
Fig 9. Peroxisome division can be symmetrical or asymmetrical. After receiving a signal to divide, peroxisomes elongate and are constricted into divisible units, making them competent for the final scission event. Whether symmetric or asymmetric division represents the primary mode of peroxisome division and whether peroxisome division, irrespective of the mode of cleavage, leads to matrix protein asymmetry remain unresolved (Mast et al, 2010).
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5. Functions of peroxisomes:
5.1 General functions:
Peroxisomal functions are very important for cell survival. Peroxisomes contain at least 50 different enzymes, which function in a variety of biochemical pathways in various cell types. Originally, peroxisomes were defined as organelles that carry out oxidation reactions leading to the production of hydrogen peroxide which is toxic to the cell.
Peroxisomes contain the enzyme catalase, which degrades hydrogen peroxide either by converting it to water or by using it to oxidize another compound. Peroxisomes bring about oxidative breakdown of many substrates like uric acid, amino acids, and fatty acids.
Peroxisomes carry out β-oxidation of very long-chain fatty acids. The process provides a major source of metabolic energy. Peroxisomes are also involved in lipid biosynthesis.
Leaf peroxisomes are involved in photorespiration, wherein glycolate is metabolized (Hu et al, 2012). Photorespiration allows most of the carbon in glycolate to be recovered and utilized. Glyoxysomes are responsible for the conversion of stored fatty acids to carbohydrates. This conversion provides energy and raw materials for growth of the germinating plant.
5.2 The glyoxalate cycle: function of glyoxysomes
Plant seedlings as well as some algae can use acetate as source of carbon to produce carbohydrates. In tricarboxylic acid (TCA) cycle (which is primarily meant for energy production), for every 2C acetate group entering the cycle, 2CO2 are wasted. So TCA cycle cannot produce biosynthetic intermediates on a large scale if large amount of acetate (acetyl CoA) groups enter in the pathway. So plants employ a modification of TCA cycle called glyoxalate cycle. This cycle eliminates release of CO2 and enhances net production of 4C compounds (oxaloacetate). The cycle bypasses two oxidative decarboxylation steps from TCA cycle.
Glyoxylate serves as intermediate in many metabolic processes. But high concentrations of glyoxalate are toxic to the cell. Enzymes involved in glyoxalate cycle are located on both sides of the peroxisomal membrane. Glyoxylate can be produced from different precursor molecules (Fig. 10) and converted into stable metabolites for further utilization. The proteins involved are ureidoglycolate hydrolase (UGH), isocitrate lyase (ICL), glycolate oxidase (GO), malate synthase (MLS), glutamine:glyoxylate aminotransferase (GGAT).
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Fig 10: Generation of glyoxalate (Kunze & Hartig, 2013).
The glyoxylate cycle makes use of two carbon compounds to form 4-carbon compounds (Fig. 11). The resulting succinate refills the TCA cycle and thus functions as the major collector and distributor of small carbon units. Also succinate and oxaloacetate serve as precursors for various biosynthetic processes. Glyoxalate cycle is absent in all animals except nematodes. Acetyl-CoA used in the glyoxylate cycle is derived from different sources, such as β-oxidation of fatty acids, degradation of amino acids or in case of microbial organisms from external carbon sources such as ethanol or acetate. The glyoxylate cycle shares a series of three enzymatic activities with the TCA cycle, namely malate dehydrogenase (MDH), citrate synthase (CIT), and aconitase (ACO) activity. The two unique activities, isocitrate lyase (ICL) and malate synthase (MLS) generate and consume glyoxylate. The cleavage of isocitrate bypasses the decarboxylation reactions and the synthase reaction leads to the net condensation of acetyl-CoA units. In many organisms these activities can be carried out by two or more isoenzymes with different localization signals and different expression patterns catalyzing the respective reactions.
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Fig 11: The glyoxalate cycle (Kunze & Hartig, 2013).
The enzymes of the glyoxylate cycle are present on different sides of the peroxisomal membrane. So for effective functioning of the pathway, efficient transport of intermediates across the peroxisomal membrane is very crucial (Fig 12). When acetyl-CoA is generated inside the peroxisomal matrix it remains confined to peroxisomes. Citrate synthase catalyzes the condensation of acetyl-CoA with oxaloacetate to form citrate.
Citrate produced is exported and serves as substrate for extra-peroxisomal aconitase activity. The resulting isocitrate is imported into peroxisomes in those organisms in which the corresponding isocitrate lyase activity is carried out inside peroxisomes (A. thaliana). In this way, the net product of the cycle (succinate) is released within peroxisomes and requires an additional export mechanism. On the contrary, glyoxylate is directly handed over to the second acetyl-CoA.
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Fig 12: Metabolites of the glyoxylate cycle crossing the peroxisomal membrane. The peroxisomal membrane facilitates the transfer of small metabolites. The transport of glyoxylate cycle intermediates and of C2 units is shown for Arabidopsis thaliana. Hypothetical pore forming proteins permitting the export of intermediates are colored orange, hypothetical pore forming proteins permitting the import of intermediates are colored green. Broken lines are drawn to close the glyoxylate cycle. Intermediates that cross the peroxisomal membrane are indicated bold. Px, peroxisomal side of the membrane, Cyt, cytosolic side of the membrane (Kunze & Hartig, 2013).
5.3 Photorespiration: function of leaf peroxisomes
Leaf peroxisomes play an essential role in photorespiration – a photosynthesis- related pathway. Photorespiration is a light-dependent process reminiscent of mitochondrial respiration because O2 is taken up and CO2 is released. Because of the release of CO2, photorespiration appears as a wasteful pathway that results in a decline in plant productivity. However, photorespiration also provides protection against abiotic stress conditions caused by high light intensity, drought, and salinity (Wingler et al, 2000).
RubisCO (ribulose 1,5-bisphosphate carboxylase/oxygenase) can catalyze both carboxylation and oxygenation of RuBP (ribulose 1,5-bisphosphate). The pathway is highly compartmentalized and involves reactions in chloroplasts, peroxisomes, and mitochondria (Fig 13). Enzymes like glycolate oxidase, catalase, and many aminotransferases of the photorespiratory cycle are located in peroxisomes. Catalase is the major constituent of the peroxisomal matrix in photosynthetic tissues.
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Fig. 13: The photorespiratory reactions comprise eleven directly involved enzymes (RubisCO, ribulose-1.5- bisphosphate carboxylase/oxygenase; PGP, phosphoglycolate phosphatase; GOX, glycolate oxidase; CAT, catalase; GGT, Glu:glyoxylate aminotransferase; SGT, Ser:glyoxylate aminotransferase; GDC, Gly decarboxylase; SHMT, Ser hydroxymethyl transferase; HPR, hydroxypyruvate reductase; pMDH, peroxisomal malate dehydrogenase; GLYK, glycerate kinase) and four indirectly participating enzymes (GS, Gln synthase; GOGAT, Glu synthase, Glu:oxoglutarate aminotransferase; mMDH/cMDH, mitochondrial /chloroplastic malate dehydrogenase), all of which are compartmentalized between chloroplasts, leaf peroxisomes, and mitochondria. For the transport of photorespiratory intermediates different translocators and a porin-like channel have been characterized biochemically (white) or the corresponding genes been cloned (black). Photorespiratory metabolites are abbreviated as follows: RuBP, ribulose-bisphosphate; 3- PGA, 3-phosphoglycerate; THF, tetrahydrofolate.(Reumann & Weber, 2006).
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Glycolate is produced in high amounts in chloroplasts during photorespiration. This glycolate leaves chloroplast through a specific transporter protein in the inner chloroplast membrane and diffuses to peroxisomes. So this transport across the membrane of leaf peroxisomes requires efficient transport systems. We know that the peroxisomal membrane is permeable for small metabolites but not for larger molecules like NADH and CoA (van Roemund et al, 1995). Transport usually occurs through porin-like channels in the peroxisome membrane. These porin-like channels have been characterized in many organisms (Reumann, 2002). In spinach (Spinacia oleracea L.), large number of porin-like channels have been characterized (Reumann et al, 1995). The channel is strongly anion selective and shows a relatively broad permeability for diverse inorganic and organic anions. The channel diameter is about 0.6 nm and so it restricts the permeability of the peroxisomal membrane to low molecular weight metabolites up to C6. The leaf peroxisomal channel is permeable to the photorespiratory intermediates (glycolate, glycerate, and Glutamate) and possesses an internal binding site for dicarboxylates, i.e.
the photorespiratory intermediates malate, OAA, and α-ketoglutarate, and tricarboxylates.
5.4 Peroxisomal β-oxidation
Fatty acid degradation in most organisms occurs primarily via the β-oxidation cycle.
In mammals, β-oxidation occurs in both mitochondria and peroxisomes, whereas plants and most fungi (Fig. 14) show the β-oxidation cycle only in the peroxisomes (Poirier et al, 2006). Here we will focus only on peroxisomal β-oxidation.
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Fig. 14: Schematic representation of fatty acid β-oxidation in yeast peroxisomes. Protein Pxa1p-Pxa2p embedded in peroxisomal membrane translocate activated fatty acids into peroxisomes for degradations.
The β-oxidation reactions are catalyzed by enzymes Pox1p/Fox1p, Mfe2p/Fox2p and Pot1p/Fox3p (Chen et al, 2016).
This pathway catalyzes the chain shortening of acyl-CoA esters between carbons 2 and 3, yielding chain-shortened acyl-CoA and acetyl-CoA or propionyl-CoA as products depending on substrates.
To date, two transporter families are known that carry out transport through peroxisomal membrane. A) The peroxisomal ATP-binding cassette (ABC) transporter involved in the import of substrates for β-oxidation and B) three members of the mitochondrial carrier (MC) family required for the influx of the cofactors ATP or NAD.
The peroxisomal ABC transporter in Arabidopsis is called as COMATOSE(CTS) (Fig 15) and it is encoded by a single gene. Arabidopsis AtABCD1 is the most studied and best understood plant peroxisomal ABC transporter. The CTS gene is expressed
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throughout the plant and encodes a full-sized transporter with two homologous but distinct halves fused in a heterodimer in the arrangement [TMD1] (the transmembrane domain) – [NBD1] (nucleotide-binding domain)–[TMD2]– [NBD2].
Fig 15. Molecular models showing of putative inward and outward conformations Arabidopsis CTS - Models of CTS are based on the structures of (A) ABCB10 with bound AMP–PCP (β,γ -methylene adenosine 5_-triphosphate) (green spheres) in an inward-facing conformation (B) Sav1866 open-outward ADP-bound complex in which the two NBDs (blue and purple) are closely packed with two nucleotides (green spheres), sandwiched between them. The NBDs face the cytosol, views are from side-on (top image) and bottom-up (bottom image) (Baker et al, 2015).
Eukaryotic ABC transporter moves various molecules across the membrane, often against concentration gradients. CTS mediates uptake of several biologically important molecules like fatty acids or fatty acid derived signaling molecules (precursors of auxin and jasmonic acid) into peroxisomes (Theodoulou etal., 2006).
In addition to mobilization of storage oil during early seedling growth, CTS also provides fatty acids hydrolyzed from membrane lipids. This process has dual role – a) it shows housekeeping function and b) it provides substrates when carbon and energy status are low (Slocombe et al, 2009).
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