Post-translational
Transport
Targeting
to the Mitochondria, Chloroplast, and Peroxisome
Post-translational
transport is transport of completed proteins from the cytosol into
mitochondria, chloroplasts, peroxisomes, or the nucleus.
Nuclear transport will not be covered in this material.
With the exception of a few proteins that are synthesized in
mitochondria or chloroplasts, nuclear genes encode all other proteins in
eukaryotic cells.
Therefore, organellar proteins must be targeted to the correct membrane
and translocated into the organelle. These proteins contain particular signal
sequences that direct them to the correct organelle, and compartment insertion
involves signal sequence recognition by receptors on the targeted organelles.
Figure III.1.a.
Protein Targeting in Post-translational Pathway (redrawn
from H. Lodish, A. Berk, S.L.
Zipursky, et.al., Molecular Cell Biology, 4th ed., copyright ã2000,
W.H. Freeman and Company)
MITOCHONDRIA :
Mitochondria likely developed by the incorporation of bacteria into ancestral eukaryotic cells more than a billion years ago and their ensuing replication in the cytoplasm (Figure III.2.a). Over eons of evolution much of the bacterial DNA in these endosymbionts moved to the nucleus, so that in modern cells, more than 98% of mitochondrial proteins are imported after synthesis in the cytosol.
Figure III.2.a. Evolutionary Origin of Mitochondria (from B. Alberts, D. Bray, J. Lewis, et.al., Molecular Biology of the Cell, 3rd ed., copyright ã1994, Garland Publishing, Inc.)
Most of the proteins targeted to the mitochondria are transported to the mitochondrial matrix, but other targeted areas within the mitochondria include the outer membrane, the inner membrane, and the inner membrane space. Some of the proteins transported to the mitochondria are listed in the table below.
Table III.1.a.
List of Some Proteins Targeted to the Mitochondria (from
H. Lodish, A. Berk, S.L. Zipursky, Molecular Cell Biology, 4th ed.,
copyright ã2000,
W.H. Freeman and Company)
Mitochondrial
Targeting Signals
The most common type of mitochondrial targeting signal is an N-terminus extension of the precursor protein called the presequence (Figure III.3.a.). Mitochondrial proteins begin as precursors containing these presequences that are eventually cleaved and are not present in the mature protein. Presequences are matrix targeting and are rich in positively charged amino acids (arginine and lysine), hydroxylated amino acids (serine and threonine), and hydrophobic residues. These presequences contain approximately 20-25 amino acid residues and presequences of different mitochondrial proteins do not show sequence identity. However, they all have the potential to from an amphiphilic a helix in which positively charged residues localize to one side of the helix and uncharged and hydrophobic residues localize to the opposite side.
Figure III.3.a. Representation of Mitochondrial Presequence
Other mitochondrial targeting signals that have been described include a carboxy-terminal sequence that mediates import in a reverse C-terminal to N-terminal direction and a segment of positively charged amino acids located within the protein sequence that act as an internal mitochondrial targeting signal.
Cytosolic chaperones
are necessary to maintain proteins in an import- competent state.
Mitochondrial precursor proteins bind to chaperones (cis-acting targeting factors) in the cytosol. Two chaperones have been shown to bind mitochondrial precursor proteins; these are Hsc 70 and MSF (mitochondrial import stimulation factor). These chaperones use energy released by ATP hydrolysis to keep the bound precursors in an unfolded or partially folded state. MSF has also been shown to separate protein aggregates. The chaperones deliver the mitochondrial-targeted proteins to receptors located on the outer membrane of the mitochondria that are part of the TOM (translocases of the mitochondrial outer membrane) complex (Figure III.4.a). The Tom complex recognizes, binds, and translocates precursor proteins across the outer membrane of the mitochondria.
Figure III.4.a. Cytosolic Chaperones MSF and Hsc 70 Bind Precursor Proteins and Deliver Them to Outer Membrane Receptors Some precursor proteins bind Hsp70 and others bind MSF. The cis-acting targeting factors direct the proteins to Tom receptors on the mitochondrial outer membrane. Tom stands for translocases of the mitochondrial outer membrane. The associated numbers correspond to the size of the proteins in kilodaltons (for example, Tom20 is a 20-kilodalton translocase of the outer membrane).
Transport
of precursor proteins across the mitochondrial outer membrane
Some precursor proteins bind MSF, which results in binding to a set of receptors on the outer membrane called Tom37 and Tom70. Many of the precursor proteins that bind MSF seem to contain internal targeting sequences that direct them to the mitochondria. Such precursor proteins include members of the inner-membrane located carrier family such as the ADP/ATP carrier. Tom37/Tom70 transfer the precursors to Tom 20/Tom22 with release of MSF.
Most precursor proteins bind to Hsc 70 in the cytosol and are delivered to Tom20. Tom20 as well as many of the Tom receptors have a negatively charged patch, and these patches have been considered important for directing the transfer of the positively charged presequences from receptor subunits to other receptor subunits and into the mitochondria as a result of ionic interactions. This is called the acid chain hypothesis. However, a hydrophobic interaction between precursor proteins and Tom20 has also been discovered. Four alpha helices in the middle of the Tom20 domain form a stable structure and three of the helices form a binding groove made up of mainly hydrophobic amino acids, while hydrophilic residues are found in the periphery of the groove. An amphiphilic alpha helical structure of presequences is critical for binding to Tom20. This receptor recognizes the hydrophobic surface of the amphiphilic presequences (Figure III.5.a.) Affinity of Tom20 for presequences is low and this flexibility could explain the ability of Tom20 to bind many different presequences.
Figure III.5.a. Interaction of a Mitochondrial Presequence with the Hydrophobic Binding Groove of the Import Receptor Tom20 (from N. Pfanner, Current Biology 10:R414, 2000) The carboxy-terminal half of the presequence binds to Tom20. The leucine residues of the presequence interact with the hydrophobic binding groove of Tom20. The boundary between the presequence and the mature protein is indicated by the triangle.
Presequences binding to Tom20 also bind to receptor Tom22, and this seems to be mediated by ionic interactions between the basic residues of the presequence and the acidic residues of the Tom22 receptor. It is likely that Tom20 and Tom22 recognize opposite sides of the same presequence, Tom20 recognizing the hydrophobic surface and Tom22 the hydrophilic surface of the amphiphilic alpha helix. Tom22 seems to be involved in regulating the gating activity of the Tom40 channel.
Tom40 is the major pore-forming subunit of the TOM complex and is predicted to be predominantly composed of beta strands. This protein behaves like a beta-barrel porin. This characteristic distinguishes this translocon from the alpha helical translocons found in other eukaryotic membranes except plastids, which also have a beta-barrel translocon. The porins serve as a major class of transporters in the outer membrane of gram-negative bacteria. Tom40 is structurally analogous to bacterial porins and is probably a relic of a gram-negative prokaryote incorporated into ancestral eukaryotes for mitochondrial development. Precursor proteins pass through the mitochondrial outer membrane by passing through the Tom40 channel. Other small Tom subunits identified in the TOM complex include Tom 5, Tom6, and Tom7. Tom5 is tightly associated with Tom40 and is involved in precursor protein transfer from receptors to the channel. Tom6 and Tom7 are also tightly associated with Tom40 but seem to play indirect roles in preprotein translocation in which they modulate the dynamics of the other TOM subunits with Tom40. Tom6 is involved in the assembly of Tom22 with Tom40 but is not necessary for maintaining this interaction. Tom7 maybe associated with insertion of proteins into the mitochondrial outer membrane (Figure III.6.a.).
Figure III.6.a. The Tom Complex for Preprotein Import (from M.T. Ryan, R. Wagner, and N. Pfanner, Int. J. Biochem. Cell Bio. 32:16, 2000) Tom20 recognizes precursor proteins containing presequences and Tom70 preferentially recognizes precursor proteins containing internal targeting sequences or proteins bound to cytosolic chaperones (Cpn) such as MSF. The proteins are transferred from these receptors to Tom22 and are transferred into the Tom40 channel with the aid of Tom5.
Transport
of proteins into the mitochondrial outer membrane
Two models for insertion of proteins into the mitochondrial outer membrane have been described, and evidence supports a role for Tom40 in both models. One suggestion is that the protein is inserted into the translocation channel of Tom40 and then the protein moves laterally through a break in the channel into the phospholipid bilayer (Figure III.7.a- model a). Alternatively, the protein does not enter the translocation channel, but instead is inserted directly into the membrane, facilitated by the membrane surface of Tom40 (Figure III.7.a- model b). Currently, integration of proteins into the mitochondrial outer membrane phospholipid bilayer is poorly understood.
Figure III.7.a. Models for Insertion of Proteins into the Mitochondrial Outer Membrane (from K. Gabriel, S.K. Buchanan, and T. Lithgow, TIBS 26:39, 2001) Two models for insertion of proteins into mitochondrial outer membrane: (a) lateral movement of proteins into the phospholipid bilayer through the Tom40 channel; (b) direct insertion of proteins into the phospholipid bilayer.
Translocation into the Mitochondrial Matrix Depends on a Matrix Targeting Signal
The mitochondria depends on the coordinated import of precursor proteins from the cystol coupled with the export of mitochondrially coded proteins from the matrix to the inner membrane. The translocase of the outer membrane (TOM) consists of protein import receptors and the import channel.
After passage through the TOM complex, proteins are sorted via a number of mechanisms either directly to the outer membrane, the intermembrane space, or the translocase of the inner membrane (TIM)
TIM23 machinery mediated protein translocation into the matrix and the TIM22 machinery mediated insertion to the inner membrane.
Figure III8a. Import of proteins across the inner membrane into the matrix. This pathway is mediated by the TIM23 complex and an associated ATP driven protein tansport motor on the inner face of the inner membrane. In the matrix, the matrix processing protease removes the matrix-targeting sequence and a battery of chaperones may aid in the folding to generate the mature protein.
Precursors with an amino-terminal targeting presequence follow the general import pathway. The import is mediated by the Tim17/Tim23 complex and the translocation motor consisting of Tim44, mitochondrial heat shock protein hsp70 and the nucleotide exchange factor mGrpE.
Translocation into the Mitochondrial Matrix Requires Both the Electrochemial Gradient Across the Inner Membrane and ATP Hydrolysis
Matrix side components - Tim44, mhsp70 and mGrpE- function as the ATP-dependent translocation motor.
Export Pathways
Another pathway is mediated by the OXA1 translocase,(nuclear-coded inner membrane protein that mediates export of N- and C-tails of the mitochondrially coded precursor cytochrome c oxidase subunit (Cox2) and also plays a role in ATP synthase formation) - OXA1 interacts directly with nascent mitochondrially synthesized polypeptides. Examples of protiens that utilize this pathway are those whose amino termini face the intermembrane space. (See Figure Below)
Figure III 9a. Protein import and export pathways in the mitochondria. Cytosolic proteins are imported through the TOM and then, depending upon their destination, remain in the outer membrane, intermembrane space, or engage the translocase of the inner membrane. Mitochondrial encoded proteins may be exported to the inner membrane via Oxa1 and Pnt1.
A second export component - Pnt1. Pnt1 is an integral inner membrane protein facing into the matrix that mediates export of the C-terminus of Cox2. (Note: Its precise role in export has not been determined.)
1- Conservative Pathway
2- Nonconservative Pathway
Figure III 10a. (redrawn
from H. Lodish, A. Berk, S.L.
Zipursky, et.al., Molecular Cell Biology, 4th ed., copyright ã2000,
W.H. Freeman and Company)
Nonconservative Pathway: The matrix-targeting sequence moves across both the outer and inner membranes, but the hydrophobic intermembrane space-targeting sequence becomes anchored in the inner membrane. This prevents translocation of the C-terminus of the protein through the inner membrane and apparently causes disassembly of the transport channel. The targeting sequence anchored in the inner membrane diffuses away from the translocation site (step 1b), as the rest of the protein traverses the outer membrane into the intermembrane space, and the matrix-targeting sequence is cleaved. Cleavage of the intermembrane space-targeting sequence by a specific protease releases the protein to which heme is added, followed by folding of the cytochrome into its mature conformation (step 2b).
"Tiny Tims" of the Intermembrane Space
A family of small proteins in the mitochondrial intermembrane space mediates import of inner membrane proteins across the intermembrane space.. Five proteins, Tim8, Tim9, Tim10, Tim12, Tim13 have been identified in the yeast intermembrane space, while similar complements are present in other metazons.
Figure III 11a. Import of proteins into the mitochondrial inner membrane. As the precursor emerges for the TOM complex, it binds to the Tim9/Tim10 or Tim8/Tim13 complex of the intermembrane space. The bound precursor is then usually delivered to an insertion complex composed of Tim10, Tim12, Tim18, Tim22, and Tim54 that catalyzes the membrane potential dependent insertion of the precursor into the inner membrane.
The specific route taken by the substrate to reach the inner membrane is still uncertain. The possibility is the small Tim complexes act as chaperone-like molecules to guide the precursor across the aqueous intermembrane space, yielding a soluble intermediate in which the precursor is bound to the 70kDa complexes in the intermembrane space.
References:
Alberts B, Bray D, Lewis J, et al. Molecular Biology of the
Cell, 3rd edition. New
York: Garland Publishing, Inc. 1994;714-715.
Gabriel K, Buchanan SK, Lithgow T. The alpha and the beta: protein
translocation across mitochondrial and plastid outer membranes. TIBS
2001;26:36-40.
Herrmann JM, Neupert W. Protein transport into mitochondria. Curr
Opin Microbiol 2000;3:210-214.
Lodish H, Berk A, Zipursky SL, et al. Molecular Cell
Biology, 4th edition. New
York: WH Freeman and Company 2000;675-685.
Mihara K. Targeting and insertion of
nuclear-encoded preproteins into
the mitochondrial outer membrane. Bioessays 2000;22:364-371.
Pfanner N. Protein sorting: recognizing mitochondiral presequences.
Curr Biol 2000;10:R412-415.
Ryan MT, Wagner R, Pfanner N. The transport machinery for the import of
preproteins across the outer mitochondrial membrane. Int J Biochem Cell
Biol 2000;32:13-21.
MITO
1. Rapaport D, Kunkele KP, Dembowski M, Ahting U, Nargang FE, Neupert W, Lill R.
Dynamics of the TOM complex of mitochondria during binding and tanslocation of preproteins. Mol Cell Bio. 1998 Sep;18(9) : 5256-62.
2. Sepuri NB, Schulke N, Pain D.
GTP hydrolysis is essential for protein import into the miochondrial matrix. J Biol Chem. 1998 Jan 16;273(3) : 1420-4.
3. Stuart RA, Gruhler A, van der Klei I, Guiard B, Koll H, Neupert W.
The requirement of matrix ATP for the import of precursor proteins into the mitochondrial matrix and intermembrane space. Eur J Biochem. 1994 Feb 15; 220 (1) : 9-18.
Web Sites:
1. http://cellbio.utmb.edu/cellbio/mitoch1.htm
2. http://www.protocol-online.net/cellbio/organe
3. http://www.cellsalive.com/cells/mitochon.htm
CHLOROPLASTS :
The chloroplasts has three membranes; outer membrane, inner membrane and the thylakoid membrane. Therefore the chloroplasts has three membrane spaces. The intermembranous space, the stroma and the thylakoid membrane space. Proteins need to be able to go through each of these membranes and into their respected compartments.
Protein targeting to Chloroplasts:
Figure III.12.a.: Proteins synthesized on free ribosomes either remain in the cytosol or are targeted to the nucleus, mitochondria, plastids or peroxisomes. (Biochemistry and the molecular biology of plants)
Figure III.13.a.: Proteins synthesized on membrane-bound ribosomes are first translocated into the lumen of the ER and transported to the Golgi. These proteins may subsequently be targeted to the plasma membrane or the tonoplast, secreted or sent to the vacuole. Some proteins remain in the ER or Golgi because they have special recognition signals. (Biochemistry and the molecular biology of plants)
Figure III.14.a.: Biosynthesis of chloroplast proteins and their targeting to five different compartments within the chloroplast. Chloroplast proteins may be encoded by nuclear DNA or chloroplast DNA; the respective mRNAs are translated by ribosomes in the cytosol (80s ribosome) or in the chloroplast stroma (70s ribosome). Proteins made as a precursor polypeptide in the cytosol may be targeted to the outer membrane or may enter the chloroplast stroma. Once across the envelope membranes, proteins may remain in the stroma compartment or may be targeted to the thylakoid membrane, thylakoid lumen, or inner envelope membrane. (Biochemistry and the molecular biology of plants)
Figure III.15.a. Mechanism of protein import into the chloroplast. At top of figure, chaperones in the cytosol hold a protein with its transit peptide exposed in the unfolded configuration. The protein on the left has a stromal targeting domain only, whereas the protein on the right has a bipartite transit peptide with both stromal and lumenal domains. Whether these two types of proteins enter through distinct types of pores or through the same pore is not known. The transit peptide binds first to the lipids and proteins of the outer membrane of the chloroplast envelope and is then engaged by the import apparatus that forms the proteinaceous pore. The apparatus consists of outer membrane proteins and inner membrane proteins. The compartment of this complex may be dynamic, changing during the translocation process. After translocation to the inside, the protein can have three different destinations. Proteins that remain in the stroma lose their transit peptides and are folded and assembled in a process that requires ATP and a large chaperone containing Hsp60. Proteins that function in the thylakoid membrane are engaged by a chloroplast signal recognition particle; insertion into the thylakoid membrane requires GTP. Proteins destined for the lumen of the thylakoid interact with one of two different import complexes in the thylakoid membrane. These proteins lose their lumenal transit domain after passage into the thylakoid. Depending on the protein being transported, this translocation process only requires a pH differential or pH differential and ATP. (Biochemistry and the molecular biology of plants)
Figure III.15.b.:
figure III.16.a.: Step 1; After a S-subunit precursor is fabricated in the cytosol, it presumably binds to the cytosolic chaperones and keep in the unfolded state. The receptor and channel proteins between the outer and inner membranes mediates the translocation from the cytosol and into the stroma. The Toc86 receptor binds to the stromal-import sequence, and the precursor then passes through an associated transport channel. This transport channel is composed mainly of Toc75 protein and then moves into the intermembrane space. The binding of GTP by Toc34 maybe causes a conformational change that affects the gating properties of the Toc75 channel. As the protein enters into the stroma, it binds to the Hsc70 chaperone. (Molecular Biology, 4th Edition)
Figure III.17.a.: The proteins enter through the membrane and into the stroma as described in figure 1D. Once in the stroma the proteins need to move into the thylakoid membrane. Steps 2a and 3a: Plastocyanin are kept unfolded in the stromal space by chaperones. The ~25 residue thylakoid signal peptide has a hydrophobic amino acids. It directs the unfolded stromal precursor into the thylakoid lumen by binding to specific receptor and channel-transport proteins on the thylakoid membrane. The thylakoid signal peptide is removed in the lumen by an endonuclease. Once the peptide is removed the protein folds into the mature state. Steps 2b and 3b: Metal binding proteins fold in the stroma and complex redox cofactors are added (like FeS). These proteins contain a ~25 residue thylakoid transfer peptide that has two arginine residues at the N-terminus followed by hydrophobic amino acids. The translocation of the large globular proteins into the thylakoid membrane are powered by a pH gradient maintained across the membrane. Once inside the membrane the thylakoid targeting sequence is removed and the protein folds into its mature state. (Molecular Biology, 4th Edition)
References:
Books:
1. Molecular Cell Biology, 4th Edition, Lodish, Berk, Zipursky, Matsudaira, Baltimore, and Darnell.2001
2. Biochemistry & Molecular Biology of Plants, Buchanan, Gruissem, Jones. 2000
Journal articles:
1. Toc, Tic and chloroplast protein import; P. Jarvis, J. Soll; Biochimica et Biophysica Acta 1541 (2001) 64-79.
2. Translocation of proteins across the multiple membranes of complex plastids, G. Dooren, S. Schwartzbach, T. Osafune, G. McFadden. Biochimica et Biophyscia Acta 1541 (2001) 34-53.
3. Import and Routing of Nucleus-Encoded Cholorplasts Proteins. K. Cline, R. Henry. Annual Review of Cell Biology, 1996. 12:1-26.
Web Sites:
1. http://opbs.okstate.edu/~melcher/MG/MGW2/MG254.html
2. http://carnegiedpb.stanford.edu/hoffman/hoffman.html
Synthesis and Targeting of Peroxisomal Proteins
Peroxisome morphology. (A) Electron micrograph of wild-type S. cerevisiae grown on oleic acid to induce peroxisomes. Bar = 1 µM. P, peroxisome; M, mitochondrion; N, nucleus; L, lipid droplet; V, vacuole; ER, endoplasmic reticulum (micrograph from V. Protopopov). (B–D) Normal human fibroblasts. (B) Electron microscopic cytochemistry for catalase. (C) Immuno-gold labeling with antibodies against peroxisomal membrane proteins. (B,C) Bar = 250 nm (from Santos et al. 1998a). (D) Immunofluorescence of chloramphenicol acetyltransferase, which had been sent into the peroxisome with a human PTS2 fused at the N terminus (PTS2-ChAT). (E) Fibroblast from a rhizomelic chondrodysplasia punctata (RCDP) patient. Immunofluorescence of PTS2-ChAT as in (D). (D) Bar = 10 µM (from Purdue et al. 1997).
Peroxisomes are organelles that are very small in diameter and lack DNA and ribosomes. The peroxisome is also lined only by a single membrane. With no DNA of its own, peroxisomal proteins are encoded by nuclear genes, synthesized on free ribosomes in the cytoplasm, and then incorporated into pre-existing peroxisomes. As an increased amount of protein adds up, the peroxisome will divide and form new peroxisomes (see below). This division is very similar to the way mitochondria and chloroplasts divide.
Peroxisome biogenesis. Upper Half. The steady-state biogenesis of the organelle: Proteins synthesized on free polyribosomes are posttranslationally incorporated into pre-existing peroxisomes (both into the membrane and into the internal matrix space), and phospholipids synthesized in the ER are transported to the membrane. Thus peroxisomes grow. They divide to form daughter peroxisomes. Although one often speaks of the daughters as new peroxisomes, this is something of a misnomer: Daughter peroxisomes contain a mixture of proteins that were synthesized recently and proteins that were made longer ago, with a stochastic distribution of protein ages. In a tissue in steady state, such as liver, the peroxisomes are degraded randomly by autophagy; in rapidly growing yeast, they are diluted by cell growth and division. This model implies continuity in space and time of the peroxisome compartment. It explains the observed biochemical remodeling of peroxisomes in response to changed physiological requirements, without making a new organelle (e.g., in plants during the transition at germination from peroxisomes rich in glyoxylate cycle enzymes to peroxisomes that catalyze photorespiration). Enzymes synthesized in large amounts in response to transcriptional up-regulation are steadily added to the existing peroxisomes; older enzymes that are down-regulated gradually disappear as the organelle undergoes autophagy or is diluted out by cell division. Top Center. Peroxisomes are induced when needed in many cell types and are strongly repressed when not required in certain cell types such as glucose-grown yeast. The size of individual peroxisomes and the total volume of the peroxisome compartment may easily change by a factor of 10 and sometimes by a factor of 100 or more. Protein abundance is transcriptionally regulated. Bottom Half. Peroxisome biogenesis may be impaired by mutations, either naturally occurring in genetic diseases or scientist-induced. Reintroduction of a wild-type gene leads to re-formation of normal peroxisomes. In the case of mutations that impair the import of matrix proteins, membrane ghosts of the organelle persist and may be refilled with matrix enzymes. In the case of mutations that affect membrane assembly or maintenance, recovery of peroxisomes is a two-step process: First the membrane ghosts reappear and subsequently the matrix proteins are imported into them. In some instances, small peroxisomal vesicles or tubules have been detected, which are the structural basis to reform the compartment. We suspect, based on hints described in the text, that such protoperoxisomes may exist in other mutants as well. Alternatively, peroxisomes would have to form de novo, a heretic concept suggested by some, for which there is no compelling evidence at present. Not shown: The model predicts that in animals, the peroxisome is transmitted to offspring via a germ cell, presumably the egg. Numbers indicate the PEX gene products that are required for various aspects of peroxisome biogenesis. PTS1 and PTS2 are targeting sequences for peroxisomal matrix proteins and mPTS is a targeting sequence for peroxisomal membrane proteins.
All peroxisomes contain enzymes that use oxygen to oxidize various substrates, forming hydrogen peroxide. One example is oxidation of fatty acids in the peroxisome (see figure below).
Catalase, a peroxisome-localized enzyme, efficiently decomposes hydrogen peroxide to water. Peroxisomes are most abundant in liver cells, and constitute about two percent of the cell volume of the liver.
After synthesis and release from the cytosolic ribosomes, new peroxisomal proteins generally fold into their mature conformation in the cytosol before import into the peroxisome, unlike mitochondrial and chloroplast proteins. The import of catalase, for example, into liver peroxisomes requires ATP hydrolysis (see end of section), but no electrochemical gradient exists across the peroxisomal membrane. The targeting signal for the uptake of catalase is located at the C-terminus of the protein and consists of a Ser-Lys-Leu sequence (SKL). Synthesis of catalase and its incorporation into peroxisomes is diagrammed in the figure below. It is believed 4 major steps take place in catalase assembly. The first is the formation of the Catalase tetramer by four apo-catalase monomers and attachment of heme to each. In the second and third step, the cytosolic peroxisome receptor protein PST1R binds an SKL signal sequence and escorts the catalase tetramer to the PEX 14 receptor on the peroxisomal membrane. In the model below, the catalase-PTS1R complex is transported across the membrane and into the lumen of the peroxisome. It should be noted that there is some debate as to whether or not PTS1R actually enters the lumen. It is also believed that uncharacterized proteins probably form part of the receptor transport channel. Finally, PTS1R is back in the cytoplasm to pick up another peroxisome protein.
Recent News: ATPase activity localized at the peroxisomal plasma membrane
It was demonstrated recently that there is neutral Mg-ATPase activity in human peroxisomal membranes. To establish the precise experimental conditions for detection of this ATPase, both cytochemical and biochemical characterizations were first carried out in liver peroxisomes from control and cipofibrate-treated rats. The results demonstrated an Mg-ATPase reaction in both normal and proliferated peroxisomes. The nucleotidase activity, with marked preference for ATP, was sensitive to the inhibitors N-ethylmaleimide and 7-chloro-4-nitro-benzo-2-oxadiazole (NBDCl). An ultrastructural cytochemical analysis was developed to evaluate the peroxisomal localization, which localized the reaction product to the peroxisomal membrane. These characteristics can help to differentiate the peroxisomal ATPase from the activity found in mitochondria and endoplasmic reticulum. The conditions established for detecting the rat peroxisomal ATPase were then applied to human peroxisomes isolated from liver and skin fibroblasts in culture. A similar Mg-ATPase activity was readily shown, both cytochemically and biochemically, in the membranes of human peroxisomes. These results, together with previous evidence, strongly support the presence of a specific ATPase in the human peroxisomal membrane. This ATPase may play a crucial role in peroxisome biogenesis. This summary was written by Cecilia Koeniga, Claudia Arayaa, Cetna Skorina, Claudio Valenciaa, Andrés Toroa, Federico Leightona, and Manuel J. Santosa in the J Histochem Cytochem 50:405–414, 2002.
Cytochemical reactions in purified peroxisomal fractions from ciprofibrate-treated rats. (A) Fraction incubated for catalase with DAB. The reaction product is confined to the peroxisomal matrix, which in the small peroxisomes shows partial extraction and in the bigger ones a higher degree of extraction. (B) Fraction incubated for Mg-ATPase. The reaction product is associated with the peroxisomal membranes. In perpendicular sections, the deposits of reaction product appear localized on the cytoplasmic side of the membrane. A positive Mg-ATPase reaction is also present in the few membranous profiles present in this fraction. (C) Glucose-6-phosphatase reaction medium. The membranous profiles (endoplasmic reticulum) are prominently stained, whereas peroxisomes are free of reaction product. (D) Mg-ATPase standard medium in the presence of NBDCl. The reaction product is confined to the endoplasmic reticulum membranous profiles, whereas peroxisomal membranes are negative. Bar = 0.5 µm
Cytochemical ATPase reaction in an L-fraction from normal rat liver. The reaction product is associated with the peroxisomal membranes. In perpendicular sections, the reaction product appears to be located on the cytoplasmic side of the peroxisomal membrane. A positive Mg-ATPase reaction is also seen in the membranous profiles present in this fraction. (Inset) Distribution of the reaction in a peroxisome sectioned through the nucleoid. Bar = 0.5 µm.
References:
Books:
1. Molecular Cell Biology, 4th Edition, Lodish, Berk, Zipursky, Matsudaira, Baltimore, and Darnell.2001
2. Biochemistry & Molecular Biology of Plants, Buchanan, Gruissem, Jones. 2000
Journal Articles:
J Histochem Cytochem 50:405–414, 2002.
Annu. Rev. Cell Dev. Biol. 2001 17:701-752.