Mitochondria, Chloroplasts, and Peroxisomes PDF

12 Mitochondria, Chloroplasts, and Peroxisomes 12 Mitochondria, Chloroplasts, and Peroxisomes Mitochondria Chloroplasts and Other Plastids Peroxisomes Introduction The generation of metabolic energy is a major activity of all cells. Mitochondria gener...

12 Mitochondria, Chloroplasts, and Peroxisomes 12 Mitochondria, Chloroplasts, and Peroxisomes Mitochondria Chloroplasts and Other Plastids Peroxisomes Introduction The generation of metabolic energy is a major activity of all cells. Mitochondria generate energy from breakdown of lipids and carbohydrates. Chloroplasts use sunlight energy to generate ATP, and reducing power to synthesize carbohydrates from CO2 and H2O. Introduction Peroxisomes contain enzymes involved in a variety of metabolic pathways. Proteins destined for these organelles are synthesized on free ribosomes and imported to the organelles as completed polypeptides. Introduction Mitochondria and chloroplasts also have their own genomes, including some genes that are transcribed and translated within the organelle. Mitochondria Mitochondria are surrounded by a double-membrane system. Inner and outer membranes are separated by an intermembrane space. The inner membrane has numerous folds (cristae), which extend into the interior (matrix). Figure 12.1 Structure of a mitochondrion (Part 1) Figure 12.1 Structure of a mitochondrion (Part 2) Mitochondria The matrix contains the genetic system and enzymes for oxidative metabolism. Glycolysis occurs in the cytosol; pyruvate is transported into mitochondria, where its complete oxidation to CO2 yields the bulk of usable energy (ATP) obtained from glucose metabolism. Figure 12.2 Oxidative metabolism in mitochondria Mitochondria Enzymes of the citric acid cycle are in the mitochondrial matrix. Most of the energy is produced by oxidative phosphorylation, which takes place in the inner mitochondrial membrane. Mitochondria High-energy electrons from NADH and FADH2 are transferred through a series of carriers in the membrane to molecular oxygen. The energy from this is converted to potential energy stored in a proton gradient, which drives ATP synthesis. Mitochondria The inner membrane is thus the principal site of ATP generation. The surface area is increased by folding into cristae. It is impermeable to most ions and small molecules—a property critical to maintaining the proton gradient. Mitochondria The outer mitochondrial membrane is highly permeable to small molecules. Porins form channels that allow the free diffusion of small molecules. Composition of the intermembrane space is similar to the cytosol. Mitochondria Mitochondria are often positioned near locations of high-energy use, such as synapses in nerve cells. In most cells mitochondria form an interconnected network, and are constantly fusing and dividing to remodel this network, modifying mitochondrial morphology and function. Figure 12.3 A mitochondrial network Mitochondria Mitochondrial fusion allows exchange of genetic material. Fission is important in distribution of mitochondria between daughter cells at cell division, and in facilitating transport of mitochondria to areas of high energy demand. Mitochondria Mitochondria are thought to have evolved from bacteria that began living inside larger cells (endosymbiosis). Free-living α-proteobacteria have genomes similar to mitochondria. Mitochondria The intracellular parasite Rickettsia prowazekii (an α-proteobacteria) can reproduce only within eukaryotic cells, like mitochondria, but it transcribes and translates most of its own genes. Mitochondria Mitochondrial genomes are usually circular DNA molecules, present in multiple copies. Most encode only a few proteins that are essential for oxidative phosphorylation. They also encode all the rRNAs and most of the tRNAs needed for translating the protein-coding sequences. Mitochondria Mitochondrial genomes vary in size between different species. The human mitochondrial genome encodes 13 proteins involved in electron transport and oxidative phosphorylation. Plus 16S and 12S rRNAs; and 22 tRNAs, which are required for translation of the proteins. Figure 12.4 The human mitochondrial genome Mitochondria The mitochondrial genetic code is different from the universal code. U in the tRNA anticodon can pair with any of the bases in the third codon position of mRNA; thus four codons are recognized by a single tRNA. Some codons specify different amino acids in mitochondria than in the universal code. Table 12.1 Differences between the Universal and Mitochondrial Genetic Codes Mitochondria Mitochondrial DNA can be altered by mutations. Almost all the mitochondria of fertilized eggs are contributed by the oocyte, so germ-line mutations are transmitted to the next generation by the mother. Mitochondria Mutations in mitochondrial genes are associated with several diseases. Leber’s hereditary optic neuropathy, which leads to blindness, is caused by mutations in mitochondrial genes that encode components of the electron transport chain. Molecular Medicine, Ch. 12, p. 452 Mitochondria Mammalian mitochondria genomes encode only 13 proteins but contain about 1500 different proteins encoded by the nuclear genome. Mitochondria Proteins encoded by nuclear genes include: Proteins needed for replication and expression of mitochondrial DNA Most proteins needed for oxidative phosphorylation All the enzymes involved in mitochondrial metabolism Mitochondria Most of the proteins are synthesized on free ribosomes and imported to mitochondria as complete polypeptides. Because of the double-membrane structure of mitochondria, import of proteins is complex. Mitochondria Proteins are targeted to the matrix by amino-terminal sequences (presequences) that are removed by proteolytic cleavage after import. Presequences bind to receptors on the mitochondria that are part of a protein complex (translocase of the outer membrane; Tom complex). Mitochondria Proteins are then transferred to another complex in the inner membrane (translocase of the inner membrane; Tim complex). Some proteins cross to the matrix via Tim23; others exit laterally and are inserted into the inner membrane. Figure 12.5 Import of mitochondrial proteins with presequences Mitochondria Protein translocation requires the electrochemical potential established across the inner membrane during electron transport. Proteins must be unfolded, and require Hsp70 chaperones. Mitochondria Presequences are cleaved by matrix processing peptidase (MPP), and the polypeptide is bound by other Hsp70 chaperones that facilitate folding. Mitochondria Some proteins with multiple transmembrane domains have internal import signals instead of presequences. After translocation across the outer membrane, they are bound by Tim9- Tim10 chaperones, which bring them to Tim22. The protein is transferred laterally into the inner membrane. Figure 12.6 Protein targeting to the mitochondrial inner membrane Mitochondria Some inner membrane proteins are encoded by the mitochondrial genome. They are synthesized on ribosomes in the mitochondrial matrix and targeted to the Oxa1 translocase in the inner membrane. They exit Oxa1 laterally to insert into the inner membrane. Mitochondria Proteins destined for the outer membrane or intermembrane space also pass through the Tom complex. Proteins with single transmembrane domains are inserted via the outer membrane protein Mim1. Mitochondria β-barrel proteins pass through Tom, are bound by Tim9-Tim10 and carried to another translocon called the SAM (sorting and assembly machinery). SAM mediates their insertion into the outer membrane. Figure 12.7 Sorting of proteins to the outer membrane and intermembrane space Mitochondria Lipids are also imported from the cytosol. Mitochondria catalyze synthesis of the phospholipid cardiolipin. Cardiolipin improves efficiency of oxidative phosphorylation by restricting proton flow across the membrane. Figure 12.8 Structure of cardiolipin Mitochondria Lipid transfer between ER and mitochondria is mediated by phospholipid transfer proteins. The lipids are transported through the cytosol and released at a new membrane, such as that of mitochondria. Figure 12.9 Phospholipid transfer proteins Mitochondria Many small molecules, such as ATP, ADP, and Pi must be moved in and out of the mitochondria. Energy is provided by the electrochemical gradient generated by proton pumping across the inner mitochondrial membrane during oxidative phosphorylation. Figure 12.10 Transport of metabolites across the mitochondrial inner membrane Mitochondria Transport of ATP and ADP is mediated by an integral membrane protein, the adenine nucleotide translocator. One molecule of ADP moves into the mitochondrion in exchange for one molecule of ATP transferred to the cytosol. Mitochondria ATP carries a more negative charge than ADP (–4 compared to –3), so exchange is driven by the voltage component of the electrochemical gradient. The Mechanism of Oxidative Phosphorylation Pi is brought in as phosphate (H2PO4–) in exchange for hydroxyl ions (OH–). This exchange is electrically neutral, but is driven by the proton concentration gradient. Higher pH within mitochondria corresponds to a higher concentration of hydroxyl ions, favoring their translocation to the outside. Chloroplasts and Other Plastids Chloroplasts are similar to mitochondria in many ways: Both generate metabolic energy Both evolved by endosymbiosis Both contain their own genetic systems Both replicate by division Chloroplasts and Other Plastids Chloroplasts are larger and more complex. They convert CO2 to carbohydrates; plus synthesize amino acids, fatty acids, and lipid for their own membranes. Nitrite (NO2–) reduction to ammonia (NH3), which is essential for incorporation of N into organic compounds, also occurs here. Chloroplasts and Other Plastids Chloroplasts are bounded by a double membrane—the chloroplast envelope. An internal membrane system, the thylakoid membrane, forms a network of flattened discs (thylakoids), frequently arranged in stacks called grana. Figure 12.11 Structure of a chloroplast Chloroplasts and Other Plastids Chloroplasts have three internal compartments: Intermembrane space between the membranes of the envelope Stroma: inside the envelope but outside the thylakoid membrane Thylakoid lumen Chloroplasts and Other Plastids Chloroplast membranes are functionally similar to those of mitochondria. Outer membrane: contains porins and is permeable to small molecules. Inner membrane: impermeable to ions and metabolites, which must move through specific transporters. Chloroplasts and Other Plastids The stroma is equivalent in function to the mitochondrial matrix. It contains the genetic system and metabolic enzymes, including those needed to convert CO2 to carbohydrates. Chloroplasts and Other Plastids Electron transport and ATP generation take place in the thylakoid membrane. Protons are pumped across this membrane from the stroma to the thylakoid lumen. The thylakoid membrane is thus equivalent to the inner membrane of mitochondria. Figure 12.12 Chemiosmotic generation of ATP in chloroplasts and mitochondria Chloroplasts and Other Plastids Chloroplast genetic systems reflect their evolutionary origin from photosynthetic bacteria. Circular DNA molecules are present in multiple copies, but are larger and more complex than those of mitochondria. Chloroplasts and Other Plastids Several chloroplast genomes have been sequenced. The genes encode both RNAs and proteins involved in gene expression and photosynthesis. Chloroplast tRNAs are sufficient to translate all the mRNA codons using the universal genetic code. Table 12.2 Genes Encoded by Chloroplast DNA Chloroplasts and Other Plastids One subunit of rubisco is encoded by chloroplast DNA. Rubisco catalyzes addition of CO2 to ribulose-1,5-bisphosphate in the Calvin cycle. Rubisco is critical for photosynthesis, and it is thought to be the single most abundant protein on Earth. Chloroplasts and Other Plastids Other proteins are synthesized on free ribosomes and imported into chloroplasts as completed polypeptides. N-terminal sequences (transit peptides), direct translocation across the two membranes of the envelope and are then removed by proteolytic cleavage. Chloroplasts and Other Plastids Transit peptides direct proteins to the translocase of the chloroplast outer member (the Toc complex). Hsp70 molecules keep the polypeptide in an unfolded state. Some also hydrolyze GTP, providing energy for translocation. Figure 12.13 Import of proteins into the chloroplast stroma Chloroplasts and Other Plastids Proteins then enter the Tic complex on the inner membrane and are transported to the stroma, drawn by action of an Hsp93 chaperone. In the stroma, the transit peptide is cleaved by stromal processing peptidase (SPP). Chloroplasts and Other Plastids Proteins that must cross the thylakoid membrane have a second signal sequence, exposed after cleavage of the transit peptide. Proteins are translocated into the thylakoid lumen by two different pathways: Chloroplasts and Other Plastids 1. Sec pathway: ATP-dependent; signal sequence is recognized by SecA protein. Protein is translocated as an unfolded protein through the Sec translocon. Chloroplasts and Other Plastids 2. Tat (twin-arginine translocation): Proteins have a twin-arginine signal sequence; translocated in fully- folded state. Energy comes from the proton gradient. Signal sequences are cleaved by thylakoid processing protease (TPP). Figure 12.14 Import of proteins into the thylakoid lumen Chloroplasts and Other Plastids Proteins are targeted to the thylakoid membrane by three pathways: 1. Sec Pathway: Proteins with a transmembrane sequence can exit the Sec translocon laterally. Chloroplasts and Other Plastids 2. SRP pathway: Proteins are recognized by the chloroplast signal recognition particle (cpSRP) and inserted into the thylakoid membrane by Alb3 translocase. Figure 12.15 Protein targeting to the thylakoid membrane Chloroplasts and Other Plastids 3. Insertion of proteins may also occur by a “spontaneous” mechanism that does not involve any known transport machinery. Chloroplasts and Other Plastids Chloroplasts are a type of plastid. Plastids have the same genome as chloroplasts, but differ in structure and function. Chloroplasts are specialized for photosynthesis; other plastids are involved in other aspects of plant metabolism. Chloroplasts and Other Plastids Other plastids have a double-membrane envelope but lack thylakoid membranes and other components of the photosynthetic apparatus. Chloroplasts and Other Plastids Plastids are classified based on the pigments they contain. Chloroplasts contain chlorophyll. Chromoplasts contain carotenoids, resulting in the yellow, orange, and red colors of flowers and fruits. Chloroplasts and Other Plastids Leucoplasts (nonpigmented) store a variety of energy sources in nonphotosynthetic tissues. Amyloplasts store starch Elaioplasts store lipids Figure 12.16 Electron micrographs of chromoplasts and amyloplasts Chloroplasts and Other Plastids All plastids develop from proplastids, small undifferentiated organelles in rapidly dividing cells. Some mature plastids can change from one type to another. Chromoplasts develop from chloroplasts during fruit ripening. Figure 12.17 Interconversions of plastids Chloroplasts and Other Plastids Development of plastids is controlled by both environmental signals and intrinsic developmental signals. In photosynthetic cells of leaves, proplastids develop into chloroplasts, but only in the presence of light. Chloroplasts and Other Plastids If kept in the dark, development of proplastids is arrested at an intermediate stage (etioplasts). An array of tubular internal membranes has formed, but chlorophyll has not been synthesized. If moved into the light, etioplasts develop into chloroplasts. Figure 12.18 Development of chloroplasts Figure 12.19 Electron micrograph of an etioplast Chloroplasts and Other Plastids Most of the proteins in plastids are encoded by nuclear genes. Plastids therefore send signals to the nucleus that regulate gene transcription for plastid proteins. Understanding the mechanisms responsible for plastid signaling is a challenging problem in plant molecular biology. Peroxisomes Peroxisomes: single-membrane organelles containing enzymes involved in many metabolic reactions. They do not have their own genomes. Most peroxisomal proteins are synthesized on free ribosomes and imported as completed polypeptides. Figure 12.20 Electron micrograph of peroxisomes Peroxisomes Peroxisomes can replicate by division but can also be regenerated even if entirely lost. Peroxisomal proteins are typical eukaryotic proteins (many mitochondrial and plastid proteins resemble those of prokaryotes). Peroxisomes Many substrates are broken down by oxidative reactions in peroxisomes, which leads to production of hydrogen peroxide. Catalase converts hydrogen peroxide to water or uses it to oxidize another organic compound. Figure 12.21 Fatty acid oxidation in peroxisomes Peroxisomes Peroxisomes are also involved in synthesis of lipids. In animal cells, cholesterol and dolichol are synthesized in peroxisomes and in the ER. In the liver, peroxisomes are involved in synthesis of bile acids from cholesterol. Peroxisomes Peroxisomes have enzymes for synthesis of plasmalogens— phospholipids with one hydrocarbon chain joined to glycerol by an ether bond rather than an ester bond. Plasmalogens are important membrane components in some tissues. Figure 12.22 Structure of a plasmalogen Peroxisomes In seeds, peroxisomes convert fatty acids to carbohydrates via the glyoxylate cycle (variant of the citric acid cycle). This provides energy and raw materials for the germinating plant. The peroxisomes are sometimes called glyoxysomes. Figure 12.23 The glyoxylate cycle Peroxisomes In leaves, peroxisomes are involved in photorespiration. In photosynthesis, CO2 is first added to the sugar ribulose-1,5-bisphosphate by rubisco. But sometimes O2 is added instead of CO2, resulting in production of glycolate. Peroxisomes Glycolate is transferred to peroxisomes and oxidized to glycine. Glycine goes to mitochondria: two glycine are converted to serine, with CO2 released. Serine returns to peroxisomes, where it is converted to glycerate. Finally, glycerate goes back to chloroplasts, and reenters the Calvin cycle. Figure 12.24 Role of peroxisomes in photorespiration Peroxisomes Occasional utilization of O2 in place of CO2 is an inherent property of rubisco. Peroxisomes allow most of the carbon in glycolate to be recovered and utilized. Peroxisomes Peroxisome transmembrane proteins are transported from the ER, including peroxins or Pex proteins involved in peroxisome assembly. Mutations in peroxins are associated with peroxisome biogenesis disorders that result from defective peroxisome assembly. Peroxisomes The recessive genetic disorders are RCDP type 1 and Zellweger spectrum disorders. Mutations that completely destroy Pex protein function result in severe disease; mutations that only reduce function of the mutated Pex protein cause less severe forms. Molecular Medicine, Ch. 12, p. 472 Peroxisomes Peroxisome transmembrane proteins are translocated into the ER and inserted into the ER membrane. These proteins then bud in vesicles. Two different types of vesicles containing different classes of peroxins fuse to form a functional peroxisome. Figure 12.25 Assembly of peroxisomes Peroxisomes Peroxisome matrix proteins are synthesized on free ribosomes and imported as folded polypeptides. Most are targeted to peroxisomes by a PTS1 signal, recognized by a Pex5 receptor. The Pex5/cargo complex binds to a docking complex on the peroxisome. Figure 12.26 Import of peroxisomal matrix proteins Peroxisomes Some peroxisome membrane proteins are also synthesized on cytosolic ribosomes. They have a targeting signal (mPTS) that is recognized by Pex19. Pex19/cargo complexes are recognized by Pex3 and Pex16 in the peroxisome membrane. Figure 12.27 Import of peroxisomal membrane proteins from the cytosol Peroxisomes Peroxisomes can be formed by two distinct mechanisms: Vesicle budding from the ER (peroxisomes have new content) Growth and division of existing peroxisomes (more rapid) Allows response to different metabolic needs. Figure 12.28 Formation of new peroxisomes