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Golgi Apparatus: Introduction; The Golgi apparatus also called Golgi complex is usually located near the cell nucleus and In animal cells it is often close to the centrosome or cell center It consists of a collection of flattened, membrane-bound cisternae and Thus resembles a stake of a plates Each of these Golgi stakes usually consists of four to six cisternae Fig 13.4 Alberts 3rd Ed The number of Golgi stakes per cell varies greatly depending on the cell type; • Some animal cells contain one large stake, while certain plant cells contain hundreds of small ones • Groups of small vesicles are associated with the Golgi stakes, clustered on the side next to the ER and along the dilated rims of each cisterna These Golgi vesicles are thought to transport proteins and lipids both to and from the Golgi apparatus and between the Golgi cisternae During their passage through the Golgi apparatus, the transported molecules undergo an ordered series of covalent modifications Each Golgi stack has two distinct faces: • a cis face (entry face) and • a trans face (or exit face) Both cis and trans faces are closely connected to special compartments Which are composed of a network of interconnected tubular and cisternal structures These are the cis Golgi network also called the intermediary or salvage compartment and The trans Golgi network, respectively Proteins and lipids • enter the cis Golgi network in transport vesicles from the ER and • exit from the trans Golgi network in transport vesicles destined for the cell surface or another compartment Both networks are thought to be important for protein sorting: • Proteins entering the cis Golgi network can either move onward in the Golgi apparatus or be returned to the ER • Proteins existing the trans Golgi network are sorted according to whether they are destined for lysosomes, secretory vesicles or the cell face • The Golgi apparatus is specially prominent in cells that are specialized for secretion. Such as The globlet cells of the intestinal epithelium which secrete large amounts of polysaccharide-rich mucus in to the gut In such cells, unusually large vesicles are found on the trans side of Golgi apparatus Which faces the plasma membrane domain where secretion occurs Fig 13.5 Alberts 3rd Ed ER-Resident Proteins are Selectively Retrieved from the Cis Golgi Network; Vesicles destined for the Golgi apparatus bud from a specialized region of the ER called the transitional elements • Whose membrane lacks bound ribosomes and • is often located between the rough ER and the Golgi apparatus Fig 13.6 Alberts 3rd Ed Vesicles budding from the transitional elements of the ER are thought to be non-selective They will transport any protein in the ER to the Golgi apparatus, although it remains possible that there are signals which accelerate the process However, there is one strict requirement for the exit of a protein from the ER • It must be correctly folded and assembled • Proteins that misfolded or incompletely assembled in to their protein complexes are retained in the ER, either Bound to the special binding protein BiP or In aggregates that can not be packaged and are eventually degraded with in ER • Thus exit from the ER can be regarded as a quality check point means: Protein would be discarded unless folding and subunit assembly are successfully completed • In fact, the ER seems to be one of the main sites in the cell where proteins are degraded while other is lysosomes Therefore, correctly folded proteins do not need a special signal to be transported out of the ER But those that are resident in the ER lumen such as BiP do need such a signal to be retained there Retention of soluble ER-resident proteins is mediated by a short, four amino acid sorting signal, identified as KDEL (Lys-Asp-Glu-Lue) or a similar sequence Table 12.3 Alberts 3rd Ed By genetic engineering, if this retention signal has been removed from BiP, then BiP is secreted from the cell Similarly, if signal is transferred to a protein that is normally secreted, the protein is now retained in the lumen of ER The retention signal works not by anchoring resident proteins in the lumen of the ER But by selective retrieval of ER-resident proteins after they have escaped in transport vesicles and been delivered to the cis Golgi network In the cis Golgi network, a specific membrane-bound receptor protein binds to the ER retention signal and Packages any proteins displaying the signal into special transport vesicles that return the proteins to the ER Thus for these resident proteins the ER is like an open prison: there is nothing to stop them leaving, but if they leave, they are brought back Fig 13.7 Alberts 3rd Ed Golgi Proteins Return to the ER When Cells are Treated with the Drug; The continuous retrieval of ER-resident proteins from cis-Golgi network means that transport between these two organelles occurs in both directions As mentioned in Fig 13.7, receptors for the ER retention signal are also found in the later Golgi compartments, suggesting that a return pathway from these compartments to the ER exists The importance of return pathway from Golgi to ER is dramatically illustrated by studies using the drug brefeldin A • Which blocks protein secretion by disrupting the Golgi apparatus In brefeldin A treated cells, the Golgi apparatus largely disappears and the Golgi proteins end up in the ER • where they intermix with ER proteins When the drug is removed, the normal Golgi apparatus reforms and the Golgi proteins return to their proper Golgi compartments Fig 13.8 Alberts 3rd Ed Fig 13.9 Alberts 3rd Ed Oligosaccharide Chains are Processed in the Golgi Apparatus; As discussed earlier, a single species of N-linked oligosaccharide is attached en block to many protein in the ER This oligosaccharide is then trimmed while protein is still in the ER Fig 12.48 Alberts 3rd Ed Fig 12.49 Alberts 3rd Ed Further modifications and additions occur in the Golgi apparatus, depending on the protein The outcome is that two broad classes of N-linked oligosaccharides are found attached to mammalian glycoproteins: • The complex oligosaccharides and • The high mannose oligosaccharides The complex oligosaccharides can contain: • more than the original two N-acetylglucosamines as well as • a variable number of galactose and • sialic acid residues and in some cases, fucose • Sialic acid is of special importance because it is the only sugar in glycoproteins that contains a net negative charge Fig 13.10 Alberts 3rd Ed High-mannose oligosaccharides have • no new sugars added to them in the Golgi apparatus • they contain just two N-acetylglucosamine and many mannose residues The complex oligosaccharides are generated by a combination of trimming the original oligosaccharide added in the ER and The addition of further sugars The processing that generates complex oligosaccharide chains follows the highly ordered pathway Fig 13.11 Alberts 3rd Ed Fig 13.12 Alberts 3rd Ed The Golgi Cisternae are Organized as a Series of Processing Compartments; Proteins exported from the ER enter the first of the Golgi processing compartments - the cis compartment - which is thought to be continuous with the cis Golgi network Then they moved to next compartment - the medial compartment, consisting of the central cisternae of stack and Finally to the trans compartment- where glycosylation is completed The lumen of the trans compartment is thought to be continuous with the trans Golgi network Where proteins are segregated in to different transport vesicles and dispatched to their final destinations i.e. • Plasma membrane • Lysosomes or • Secretory vesicles These oligosaccharide processing pathways occur in a correspondingly organized sequence in the Golgi stack, with each cisterna containing its own set of processing enzymes Proteins are modified in successive stages as they move from cisterna to cisterna across the stack So that the stack forms a multistage processing unit The processing occurs in a spatial as well as a biochemical sequence: • Enzymes catalyzing early processing steps are localized in cisternae toward the cis face of Golgi stake • Whereas enzymes catalyzing later processing steps are localized in cisternae toward the trans face The transport of proteins between different Golgi cisternae is thought to be mediated by transport vesicles • Which bud from one cisterna and fuse with the next Fig 13.13 Alberts 3rd Ed Fig 13.14 Alberts 3rd Ed Proteoglycanes are Assembled in the Golgi Apparatus; It is not only the N-linked oligosaccharide chains on proteins that are altered as the protein pass through the Golgi cisternae en route from the ER to their final destinations Many other proteins are also modified in other ways. For example: • Some have sugars added to the OH groups of selected serine or threorine side chains • This O-linked glycosylation is catalyzed by a series of glycosyl transferase enzymes • That use the sugar nucleotides in the lumen of the Golgi apparatus to add sugar residues to a protein one at a time • Usually, N-acetylgalatosamine is added first followed by a variable number of additional sugar residues ranging from just a few to 10 or more The Golgi apparatus confer the heaviest glycosylation of all on proteoglycan core proteins • which it modifies to produce proteoglycan • this process involves the polymerization of one or more glycosaminoglycan chains via a xylose link on to serines on the core protein • Many proteoglycanes are secreted and become components of the extracellular matrix while other remain anchored to the plasma membrane • Others form a major component of slimy materials such as the mucus that is secreted to form a protective coating over many epithelia • The sugars incorporated in to glycosaminoglycanes are heavily sulfated in the Golgi apparatus immediately after these polymers are made and • This helps to give proteoglycans their negative charge Fig 19.36 Alberts 3rd Ed The Carbohydrates in Cell Membrane Faces the Side of the Membrane That is Topologically Equivalent to the Outside of the Cell; Because all oligosaccharide chains are added on the luminal side of the ER and Golgi apparatus, the distribution of carbohydrate on membrane proteins and lipids is asymmetrical (Glycosaminoglycan – unbranched polysaccharide chains composed of repeating disaccharide units either Nacetylglucosamine or Nacetylgalactosamine – an amino sugar and second one is usually uronic acid) Similar to asymmetry of the lipid bilayer, the asymmetric orientation of these glycosylated molecules is maintained during their transport to the: • plasma membrane • secretory vesicles or • lysosomes As a result, the oligosaccharides of all of the glycoproteins and glycolipids in the corresponding intracellular membranes face the lumen While those in the plasma membrane face the outside of the cell Fig 13.15 Alberts 3rd Ed What is the Purpose of Glycosylation? There is an important difference between the construction of oligosaccharide and the synthesis of other macromolecules such as • DNA • RNA and • Proteins DNA and RNA and proteins are copied from a template in a repeated series of identical steps using the same enzyme(s) While complex carbohydrates require a different enzyme at each step • Each product being recognized as the exclusive substrate for the next enzyme in the series Since the complicated pathways have evolved to synthesize them, it seems likely that • The oligosaccharides on glycolipids and glycoproteins have important functions which are mostly unknown N-linked glycosylation is prevalent in all eukaryotes including yeast but absent from prokaryotes N-linked oligosaccharides also occur in a very similar form in archaeal cell wall proteins Suggesting that the whole machinery required for their synthesis is evolutionarily ancient N-linked glycosylation promotes protein folding in two ways: i. It had direct role in making folding intermediates more soluble, thereby preventing their aggregation ii. The sequential modifications of the N-linked oligosaccharides establish a “glyco code” that marks o the progression of protein folding and o mediates the binding of the protein to chaperones and lectins (carbohydrate binding proteins). For example: - In guiding ER to Golgi transport - Lectins also participate in protein sorting in the trans Golgi network Besides, because chains of sugar have limited flexibility, they can limit the approach of other macromolecules to the protein surface Fig 13.16 Alberts 3rd Ed • In this way, a glycoprotein more resistant to digestion by proteolytic enzymes • It may be that oligosaccharides on cell surface proteins originally provided an ancestral cell with protective coat • Compared to the rigid bacterial cell wall, a mucus coat has the advantage that it leaves the cell with the freedom to change shape and move. For example: Mucus coat of lung and intestinal cells protects against many pathogens The recognition of sugar chains by lectins in the extracellular space is important in many developmental processes and in cellcell recognition. For example: o Selectins (cell adhesion molecules) are lectins that function in cell-cell adhesion during lymphocyte migration • Presence of oligosaccharides may modify a protein’s antigenic properties, making glycosylation an important factor in the production of proteins (antibodies) for pharmaceutical purposes • Glycosylation can also have important regulatory roles. For example: Signaling through the cell-surface signaling receptor - Notch determines the cell’s fate in development Fig P 600 Alberts 3rd Ed Lysosomes: Introduction; Lysosomes are membranous bags of hydrolytic enzymes used for the controlled intracellular digestion of macromolecules They contain about 40 types of hydrolytic enzymes including: • Proteases • Nucleases • Glycosidases • Lipases • Phospholipases • Phophatases and • Sulfatases All are hydrolyses and for optimal activity they require an acid environment and Lysosome provides this by maintaining pH of about 5 in its interior In this way, the content of cytosol are doubly protected against attack by the cell’s own digestive system The membrane of lysosome normally keeps the digestive enzymes out of the cytosol But even if they leak out, they can do little damage at the cytosolic pH of about 7.2 Like all other intracellular organelles, the lysosome not only contains a unique collection of enzymes But also has a unique surrounding membrane Transport proteins in this membrane allow the final products of the digestion of macromolecules such as • amino acids • sugars and • nucleotides to be transported to the cytosol from where they can be excreted or reutilized by the cell • An H+ pump in the lysosomal membrane utilizes the energy of ATP hydrolysis to pump H+ into lysosome • Thereby maintaining the lumen at its acidic pH Fig 13.17Alberts 3rd Ed • Most of the lysosomal membrane proteins are unusually highly glycosylated • Which is thought to help in protecting them from the lysosomal proteases in the lumen • As discussed earlier, endocytosed materials are initially delivered to organelles called endosomes before being delivered to lysosomes • Endosomes also have H+ pumps that keep their lumen at a low pH though not as low as that of lysosomes Fig 13.18Alberts 3rd Ed • Lysosomes were initially discovered by biochemical fractionations of cell extracts • Later on they were seen clearly in the electron microscope • They are extraordinarily diverse in shape and size • but can be identified as members of a single family of organelles by histochemistry using the precipitate produces by the action of an acid hydrolase on its substrate to show which organelles contain the enzyme Fig 13.19 Alberts 3rd Ed • By this criterion, lysosomes are found in all eukaryotic cells • The heterogeneity of lysosomal morphology contrasts with the relatively uniform structures of most other cellular organelles • The diversity reflects the wide variety of digestive functions mediated by acid hydrolyses including: The break down of intra and extracellular debris The destruction of phagocytosed microorganisms and The production of nutrients for the cell Plant and Fungal Vacuoles are Remarkably Versatile Lysosomes; Most plant and fungal cells including yeasts contain one or several very large, fluid-filled vesicles called vacuoles They typically occupy more than 30% of the cell volume and as much as 90% in some cell types Fig 13.20 Alberts 3rd Ed Vacuoles are related to lysosomes of animal cells containing a variety of hydrolytic enzymes But their functions are remarkably diverse The plant vacuole can act: • as a storage organelle for nutrients and for waste products • as a degradative compartment • as an economical way of increasing cell size and as a controller of turgor pressure – the osmotic pressure that pushes outward on the cell wall and keeps the plant from wilting (loss of rigidity of non-woody parts of plants) Fig 13.21 Alberts 3rd Ed • Different vacuoles with distinct functions i.e. digestion and storage are often present in the same cell Vacuole is important as a homeostatic device enabling plant cells to withstand wide variations in their environment When the pH in the environment drops. For example: • The flex of H+ in to the cytosol is balanced, at least in part, by increased transport of H+ in to the vacuole so as to keep the pH in the cytosol constant • Similarly, many plant cells maintain an almost constant turgor pressure in the face of large changes in the tonicity (it is a measure of the osmotic pressure gradient of two solutions separated by a semi permeable membrane) of the fluid in their immediate environment • They do so by changing the osmotic pressure of the cytosol and vacuole - in part by controlled breakdown and re-synthesis of polymers such as polyphosphate in the vacuole and in part by altering rates of transport of sugars, amino acids and other metabolites across the plasma membrane and vacuolar membrane • Substances stored in the plant vacuoles in different species range from rubber to opium to the flavoring of garlic • Often stored products have a metabolic function. For example; Proteins can be preserved for years in the vacuoles of the storage cells of many seeds such as those of peas and beans When the seeds germinate, the proteins are hydrolyzed and the mobilized amino acids provide a food supply for the developing embryo Anthocyanin pigments that are stored in vacuoles, color the petals of many flowers to attract pollination insects While noxious molecules that are released from vacuoles when a plant is eaten or damaged provide a defense against predators Materials are Delivered to Lysosomes by Multiple Pathways; Lysosomes in general are meeting places in which several streams of intracellular traffic converge A route that leads outward from the ER via the Golgi apparatus delivers most digestive enzymes While at least three paths from different sources feed substances in to lysosomes for digestion i. The best studies is that followed by macromolecules taken up from the external medium by endocytosis ii. Degradation in lysosomes is used in all cell types for disposal of obsolete parts of the cell itself – autophagy iii. Provides materials to lysosomes for degradation occurs mainly in cells specialized for the phagocytosis of large particle and microorganisms Fig 12.22 Alberts 3rd Ed Some Cytosolic Proteins are Directly Transported in to Lysosomes for Degradation; There may be a fourth pathway for proteins to enter a lysosome for degradation: • Some proteins contain certain signal on their surface called KFERQ sequences where K for lysine F for phenylalanine E for glutamate R for arginine and Q for glutamine - In liver cell, an average mitochondrion has a lifetime of about 10 days The process seems to begin with the enclosure of an organelle by membrane derived from ER Because of this sequence, proteins will be delivered selectively to lysosomes for degradation Lysosomal Enzymes are Sorted from Other Proteins in the Trans Golgi Network by a Membrane-Bound Receptor Protein that Recognizes Mannose -6-Phosphate; We now consider the system that delivers the other half of the traffic in to lysosomes i.e. • Specialized lysosomal hydrolases and • Membrane proteins Both are synthesized in the rough ER and transported through the Golgi apparatus Transport vesicles bud from the trans Golgi network incorporating lysososmal proteins While excluding the many other proteins being packaged in to different transport vesicles for delivery elsewhere The question is how lysosomal proteins are recognized and selected with required accuracy? Fig 13.14 Alberts 3rd Ed Mannose -6-phosphate (M6P) groups are recognized by their receptor - For lysosomal hydrolases, the answer is known -They carry a unique marker in the form of mannose-6-PO4 -That is added exclusively to the Nlinked oligosaccharides of these soluble lysosomal enzymes Which are transmembrane proteins present in the trans Golgi network These proteins bind to the lysosomal enzymes and help package them in to specific transport vesicles That then bud from the Golgi network and subsequently fuse with a late endosome delivering their contents to the lumen of lysosomes The Mannose-6-Phospahte Receptor Shuttles Back and Forth Between Specific Membranes; The M6P receptor binds its specific oligosaccharide at pH 7 in the trans Golgi network and releases it at pH 6 Which is the pH in the interior of late endosomes Thus the lysosomal hydrolases dissociate from the M6P in the late endosomes and begin to digest the endocytosed material delivered from early endosomes Fig 13.23 Alberts 3rd Ed A Signal Patch in the Polypeptide Chain Provides the Clue for Tagging a Lysosomal Enzyme with Mannose 6-Phosphate; Lysosomal hydrolases not only contains N-linked oligosaccharides with terminal mannose residues which is the site for addition of M6P group but also a signal patch in its conformation Fig 13.25 Alberts 3rd Ed Two enzymes act sequentially to catalyze the addition of M6P groups to lysosomal hydrolases Fig 13.24 Alberts 3rd Ed Defects in the GlcNAc Phosphotransferase Cause a Lysosomal Storage Disease in Human; Lysosomal storage diseases are caused by genetic defects that affect one or more of the lysosomal hydrolases and Result in accumulation of their undigested substrates in lysosomes with sever pathological consequences. For example: • Hurler’s disease - the enzyme glycosaminoglycanes is missing required for break down So in these mutant individuals, the lysosomes accumulate massive quantities of glycosaminoglycanes as these can not be digested • Inclusion cell disease (I-cell disease) - almost all the hydrolytic enzymes are missing from the lysosomes of fibroblasts and their undigested substrates accumulate in lysosomes which consequently form large “inclusions” in the patients In these individuals all the hydrolases missing from lysosomes are found in the blood. Why? Because they failed to be sorted properly in the Golgi apparatus So hydrolases are secreted rather than transported to lysosomes Miss sorting was occurred due to the defect or missing GlcNAcphosphotranferase – a single gene defect and is like most genetic enzyme deficiencies, it is recessive Fig P 610 Alberts 3rd Ed Peroxisomes: Introduction; Peroxisomes differ from mitochondria and chloroplasts in many ways Most notably, they are surrounded by only a single membrane and Do not contain DNA or ribosomes In spite these differences, peroxisomes are thought to acquire their protein by the similar process of selective import from the cytosol Because peroxisomes have no genome therefore all of their proteins must be imported Thus peroxisomes resemble the ER in being self replicating membranebounded organelles that exist without genomes of their own Peroxisomes are found in all eukaryotic cells They contain oxidative enzymes such as • catalase and • urate oxidase at such high concentrations that in some cells of peroxisomes stand out in electron micrograph • Because of the presence of a crystalloid core, largely composed of urate oxidase Fig 12.27 Alberts 3rd Ed Like mitochondrion, peroxisome is a major site of oxygen utilization Peroxisomes Use Molecular Oxygen and Hydrogen Peroxide to Carry out Oxidative Reactions; Peroxisomes are so called because they usually contain one or more enzymes that use molecular oxygen to remove hydrogen atoms from specific organic substrates i.e. R in an oxidative reaction that produces hydrogen peroxide Catalase utilizes the H2O2 generated by the other enzymes in the organelle to oxidize a variety of other substrates including: • phenols • formic acids • formaldehyde and alcohol by the per oxidative reaction: • This type of oxidative reaction is particularly important in liver and kidney cells • Whose peroxisomes detoxify various toxic molecules that enter the bloodstream • About quarter of alcohol people drink is oxidized to acetaldehyde in this way • In addition, when excess H2O2 accumulated in the cell, catalase converts it to H2O A major function of the oxidative reactions carried out in peroxisomes is the breakdown of fatty acid molecules In a process called β-oxidation, the alkyl chains of fatty acids are shortened sequentially by blocks of two carbon atoms at a time That are converted to acetyl-CoA and exported from peroxisomes to cytoplasm for reuse in biosynthetic reactions In mammalian cells, β-oxidation occurs both in mitochondria and peroxisomes However, in yeast and plant cells, this essential reaction is exclusively found in peroxisomes An essential biosynthetic function of animal peroxisomes is to catalyze the first reactions in the formation of plasmalogens - most abundant class of phospholipids in myelin sheet that insulated the axon of nerve cells Fig 12.31 Alberts 5th Ed Plasmalogen deficiencies cause profound abnormalities in the myelination of nerve cell axons Which is why many peroxisomal disorders lead to neurological disease Peroxisomes are unusually diverse organelles and even in the different cells of a single organism may contain very different sets of enzymes They can also adapt remarkably to changing conditions. For example: • Yeast cells grown on sugar have small peroxisomes but • When some yeast cells are grown on methanol, they develop large peroxisomes that oxidize methanol and • When grown on fatty acids, they develop large peroxisomes that breakdown fatty acids to acetyl-CoA by β-oxidation Peroxisomes also have very important roles in plants Two very different types have been studies extensively: • One type is present in leaves where it is catalyzes the oxidation of a side product of the crucial reaction that fixes CO2 in carbohydrate Fig 12.28A Alberts 3rd Ed This process is called photorespiration because it uses up O2 and liberates CO2 • The other type of peroxisome is present in germinating seeds and in filamentous fungi Where it plays an essential role in converting the fatty acids stored in seed lipids in to sugars needed for the growth of the young plant Because this conversion of fats to sugars is accomplished by a series of reactions known as the glyoxylate cycle - a variation of the tricarboxylic acid cycle and these peroxisomes are also called glyoxysomes Fig 12.28B Alberts 3rd Ed In glyoxylate cycle two molecules of acetyl-CoA produced by fatty acid breakdown in the peroxisome are used to make succinate Which leave the peroxisome and converted in to glucose The glyoxylate cycle, as mentioned earlier a variation of the tricarboxylic acid cycle, is an anabolic pathway occurring in plants, bacteria, protists and fungi It does not occur in animal cells and animals are unable to convert the fatty acids in fats into carbohydrates Fig down loaded from Web site The glyoxylate cycle centers on the conversion of acetyl-CoA to succinate for the synthesis of carbohydrates and allows the conversion of acetyl-CoA to result in net increase in malate or oxaloacetate, which is not possible with the TCA cycle alone Two acetyl-CoA are input per cycle with no loss of CO2, making possible net synthesis of a 4-carbon product. The two additional enzymes of the glyoxylate cycle are isocitrate lyase and malate synthase In microorganisms, the glyoxylate cycle allows cells to utilize simple carbon compounds as a carbon source when complex sources such as glucose are not available 1 x 2 x A Short Signal Sequence Directs the Import of Proteins into Peroxisomes; A specific sequence of three amino acids (Ser-Lys-Leu) located at the Cterminus of many peroxisomal proteins functions as an import signal Table 12.3 Alberts 3rd Ed Other peroxisomal proteins contain a signal sequence near the Nterminus If either sequence attached to a cytosolic protein, the protein is imported in to peroxisomes The import process is still poorly understood, although it is known to involve both soluble receptor proteins in cytosol • Which recognize the targeting signals and • Docking proteins in the cytosol on the cytosolic surface of the peroxisomes At least 23 distinct proteins called peroxines participate in the import process which is driven by ATP hydrolysis A complex of at least six different peroxines forms a membrane translocator Even oligomeric proteins do not have to unfold to be imported in to peroxisomes, therefore, the mechanism differs from that used by mitochondria and protoplast At least one soluble import receptor, the peroxin - Pex5, accompanies its cargo all the way into peroxisomes and after cargo release, cycles back to cytosol These aspects of peroxisomal protein import resemble protein transport in to the nucleus The importance of this import process and of peroxisomes is demonstrated by the inherited human disease Zellweger syndrome • in which a defect in importing proteins in to peroxisomes lead to a profound peroxisomal deficiencies • These individuals, whose cells contain empty peroxisomes have severe abnormalities in their brain, liver and kidney and they die soon after birth • A mutation in the gen encoding peroxin Pes2, a peroxisomal integral membrane protein involved in protien import, causes one form of disease • Similarly, a defective receptor for N-terminal import signal causes a milder inherited peroxisomal disease It has long been debated whether new peroxisomes arise from preexisting ones by organelle growth and fission and therefore, replicate in an autonomous way like mitochondria and plastids or they derive as a specialized compartment from the ER Aspects of both view may be true Figure 12.33 Alberts 5th Ed Most peroxisomal membrane proteins are made in cytosol and insert in to the membrane of preexisting ones Yet other are first integrated in to the ER membrane from where they may bud in specialized peroxisomal precursor vesicle New precursor vesicles may then fuse with one another and begin importing additional peoxisomal proteins using their own protein import machinery to grow in to mature peroxisomes Which can enter in to a cycle of growth and fission Page 574 Albert 3rd Ed CELL EXTERIOR Mitochondria Introduction; Mitochondria and chloroplasts are double-membrane enclosed organelles They specialize in ATP synthesis using energy derived from electron transport and oxidative phosphorylation in mitochondria and from photosynthetic phosphorylation in chloroplasts Although both contains its own DNA, ribosomes and other compartments required for protein, most of their proteins are encoded in the nucleus and imported from the cytosol Each imported protein must reach the particular organelle subcompartment in which it functions There are two sub-compartments in mitochondria: • The internal matrix space and the inter-membrane space These compartments are formed by the two concentric mitochondrial membrane: • The inner membrane - which encloses the matrix space and forms extensive invaginations called cristae and • Outer membrane – which is in contact with cytoplasm Fig 12.20A Alberts 3rd Ed Chloroplasts have the same two sub-compartments plus an additional sub-compartment – the thylakoid membrane Fig 12.20B Alberts 3rd Ed Each of the sub-compartments contains a distinct set of proteins The growth of mitochondria and chloroplasts by import of proteins from the cytosol is therefore a major feat, requiring that proteins be translocated across a number of membranes in succession and end up in the appropriate place The relatively few proteins encoded by the genome of these organelles are located mostly in the inner membrane in mitochondria and in the thyakoid membrane in chloroplasts These organelle encoded polypeptides generally from subunits of protein complexes Whose other subunits are encoded by nuclear genes and are imported from the cytosol The formation of such hybrid protein complexes requires a balanced synthesis of the two types of subunits How protein synthesis is coordinated on different types of ribosomes located two membranes apart is still largely a mystery? Translocation in to the Mitochondrial Matrix Depends on a Matrix Targeting Signal; Proteins imported in to mitochondria are usually taken up from the cytosol within seconds or minutes of their release from ribosomes Thus mitochondrial proteins are first fully synthesized as mitochondrial precursor proteins in the cytosol and then translocated in to mitochondria by a post translational mechanism One or more signal sequences direct all mitochondrial precursor proteins to their approximate mitochondrial sub-compartment Table 12.3 Alberts 3rd Ed Many proteins entering the matrix space contain a signal sequence at Nterminus • Which is removed by signal peptidase rapidly after import Others including outer membrane and many inner membrane and intermediate space proteins have an internal signal sequence that is not removed The signal sequences are both necessary and sufficient for the import and correct localization of the proteins When genetic engineering techniques are used to link these signals to a cytosolic protein, the signals direct the protein to the correct mitochondrial sub-compartment The signal sequences that direct precursor proteins in to the mitochondrial matrix space are best understood They all form an amphiphilic α helix in which: • Positively charged residues cluster on one side of the helix • While uncharged hydrophobic residues cluster on the opposite side Specific receptor proteins that initiate protein translocation recognize this configuration rather than the precise amino acid sequence of the signal sequence Fig 12.23 Alberts 5th Ed Fig 12.21 Alberts 3rd Ed Multisubunit protein complexes that function as protein translocators mediate protein translocation across mitochondrial membranes The translocator of outer membrane (TOM) complex transfers proteins across the outer membrane and two (transporter of inner membrane (TIM complexes i.e. TIM23 and TIM 22) transfer proteins across the inner membrane These complexes contain some components; • that act as receptors for mitochondrial precursor proteins and • other components that form the translocator channels The TOM complex is required for the import of all nucleus-encoded mitochondrial proteins It initially transports their signal sequences into the inter-membrane space and helps to insert transmembrane proteins in to the outer membrane β-barrel proteins – particularly abundant in outer membrane, are then passed on to an additional translocator, the SAM complex • which help them to fold properly in the outer membrane The TIM23 complex • transports some soluble proteins into the matrix space and • helps to insert transmembrane proteins into the inner membrane TIM22 complex mediates the insertion of a subclass of inner membrane proteins including the transporters that • moves ADP, ATP and phosphates in and out of mitochondria The OXA complex - an other protein translocator located in inner mitochondrial membrane mediates • the insertion of those inner membrane proteins that are synthesized within mitochondria • It also help to insert some imported inner membrane proteins that are initially transported into the matrix space by the other complexes Fig 12.23 Alberts 5th Ed The molecular mechanism of protein import into mitochondria has been done by analyses of • cell free, reconstituted transport systems in which mitochondria in a test tube import radiolabel mitochondrial precursor proteins Mitochondrial Precursor Polypeptide Chains; Proteins are Imported as Unfolded Mitochondrial precursor proteins do not fold in to their native structures after they are synthesized rather They remain unfolded in the cytosol through interactions with other proteins Some of these interacting proteins are: • general chaperone proteins of the Hsp family whereas • Others are dedicated to mitochondrial precursor proteins and bind directly to their signal sequences All interacting proteins help to prevent the precursor proteins from aggregating or folding up spontaneously before they engage with the TOM complex • As a first step in the import process, the import receptors of TOM complex bind the signal sequence of the mitochondrial precursor protein • the interacting proteins are then stripped off and • unfolded polypeptide chain is fed - first with signal sequence into the translocation channel Fig 12.22 Alberts 3rd Ed Fig 12.25 Alberts 5th Ed ATP Hydrolysis and a Membrane Potential Driven Protein Import into the Matrix Space; Directional transport requires energy which in most biological systems is supplied by ATP hydrolysis ATP hydrolysis fuels mitochondrial protein import at two discrete sites i.e. i. One outside the mitochondria and ii. One in the matrix space In addition to this, protein import requires another energy source • which is the membrane potential across the inner mitochondrial membrane Fig 12.26 Alberts 5th Ed Bacteria and Mitochondria use Similar Mechanisms to Insert Porins into their Membrane; Outer mitochondrial membrane, like the outer membrane of Gramnegative bacteria, contains abundant pore forming proteins – porins Fig 10.18 Alberts 5th Ed Thus it is freely permeable to inorganic ions and metabolites but not to most proteins Porins are β-barrel proteins and are first imported through the TOM complex Fig 10.26 Alberts 5th Ed In contrast to other outer membrane proteins • Which are anchored in the membrane through α-helical regions, the TOM complex can not integrate porins into lipid bilayer Instead, porins are first transported into inter-membrane space • where they transiently bind specialized chaperone proteins • which keep the porins from aggregating They then bind to the Sam complex in the outer membrane, which • insert them to into the outer membrane and • help them to fold correctly Fig 12.27 Alberts 5th Ed One of the central subunit of SAM complex is homologous to a bacterial outer membrane protein that • helps insert β-barrel proteins in to the bacterial outer membrane from periplasmic space – the topological equivalent of the inter-membrane space of mitochondria • This conversed pathway is further evidence for the endo-symbiotic origin of mitochondria Transport into the Inner Mitochondrial Membrane and Intermembrane Space Occurs via Several Routes; The same mechanism that transports proteins into the matrix space, using the TOM and TIM23 translocators, also mediates the initial translocation of many proteins Fig 12.28 Alberts 5th Ed Page 568 Alberts 3rd Ed Function: Mitochondria and chloroplasts, membrane bound organelles convert energy to forms that can be used to drive cellular reactions These membrane has a crucial role in the function of this energyconverting organelles by providing a frame work for electron-transport processes Although mitochondria covert energy derived from chemical fuels whereas chloroplasts convert energy derived from sunlight The two types of organelles are organized similarly, moreover, both produce large amounts of ATP by the same mechanism The common pathway by which mitochondria, chloroplasts and even bacteria harness energy for biological purposes operates by a process known as chemiosmotic coupling-reflecting a link between the chemical bond-forming reactions • That generate ATP (chemi) and membrane-transport processes (osmotic) The energy from oxidation of foodstuffs or from sunlight is used to drive membrane bound proton pump (H+ pump) That transfer H+ from one side of the membrane to the other These pumps generate an electrochemical protons gradient across the membrane Which is used to drive various energy-requiring reaction when the protons flow back “downhill” through membrane-embedded protein machines Fig 14.1 Alberts 5th Ed Fig 14.2 Alberts 5th Ed / Fig 14.1 Alberts 3rd Ed Fig 14.3 Alberts 5th Ed / Fig 14.2 Alberts 3rd Ed The Citric Acid Cycle Generates High-Energy Electrons; • Mitochondria can use both pyruvate and fatty acids as fuel • Pyruvate come from glucose and other sugars, whereas fatty acids come from fats • Both of these fuel molecules are transported across the inner mitochondrial membrane and are • then converted to the crucial metabolic intermediate acetyl CoA by enzymes located in the mitochondrial matrix • The acetyl groups in acetyl CoA are then oxidized in the matrix via the citric acid cycle • The cycle converts the carbon atoms in acetyl CoA to CO2 which the cell releases as a water product • Most importantly, this oxidation generates high-energy electrons carried by the activated carrier molecules NADH and FADH2 Fig 14.9 Alberts 5th Ed • These high-energy electrons are than transferred to the inner mitochondria membrane, where they enter the electron-transport chain The loss of electrons from NADH and FADH2 also generates the NAD+ and FAD that is needed for continued oxidative metabolism Fig 14.10 Alberts 5th Ed A Chemiosmotic Process Coverts Oxidation Energy in ATP; • Although the citric acid cycle is considered to be part of aerobic metabolism, it does not itself use oxygen • Only in the final, catabolic reactions that take place on the inner mitochondrial membrane is molecular oxygen (O2) directly consumed • Nearly all energy available from burning carbohydrates, fat and other food stuffs in the earlier stages of their oxidation initially saved in the form of high-energy electrons removed from substrates by NAD+ and FAD • These electrons carried by NADH and FAD2 then combine with O2 by means of respiratory chain embedded in the inner mitochondrial membrane harness the large amount of energy released to derive the conversion of ADP + Pi to ATP • For this reason, the term oxidative phosphorylation is used to describe this last series of reactions Fig14.11 Alberts 5th Ed NADH Transfers its Electron to Oxygen Through Three Large Respiratory Enzyme Complexes; • Although the respiratory chain harvests energy by a different mechanism than that used in other catabolic reactions, the principle is the same • The energetically favorable reaction H2 + 1/2O2 occur in many small steps H2O is made to • So that most of the energy released can be stored instead of being lost to the environment as heat • The hydrogen atoms are first separated in to protons and electrons • The electrons pass through a series of electron carriers in the inner mitochondrial membrane • At several steps along the way, protons and electrons are transiently recombined • But only at the end of the electron-transport chain are the protons returned permanently • When they are used to neutralize the negative charges created by the final addition of the electrons to oxygen molecule Fig 14.12 Alberts 5th Ed As Electron Move Along the Respiratory Chain, Energy is Stored as an Electrochemical Proton Gradient Across the Inner Membrane; • Oxidative phosphorylation is made possible by the close association of the electron carriers with protein molecules • The proteins guide the electrons along the respiratory chain so that the electron move sequentially from one enzyme complex to another - no short circuits • Most important, the transfer of electrons is coupled to oriented H+ uptake and release and to allosteric changes in selected protein molecules • The net result is that the energetically favorable flow of electron pumps H+ across the inner membrane, from the matrix space to the inter membrane space • The movement of H+ has two major consequences: It generates a pH gradient across the inner mitochondrial membrane, with the higher in the matrix than in the cytosol, where the pH is generally close to 7 It generates a voltage gradient (membrane potential) across the inner mitochondrial membrane with inside negative and out side positive As a result of the net outflow of positive ions • The pH gradient (ΔpH) drives H+ back in to matrix, thereby reinforcing the effect of membrane potential (ΔV) • Which acts to attract any positive ion in to the matrix and to push any negative ion out • Together, the ΔpH and the ΔV are said to constitute an electrochemical proton gradient • The electrochemical proton gradient exerts a proton-motive force which can be measured in units of milli volts (mV) Fig 14.13 Alberts 5th Ed / Fig 14.19 Alberts 3rd Ed The Proton Gradient Drives ATP Synthesis; • The electrochemical proton gradient across the inner mitochondrial membrane derives ATP synthesis in the critical process of oxidative phosphorylation • This is made possible by the membrane-bound enzyme ATP synthetase - plays a role of a turbine, permitting the protons gradient to drive the production of ATP Fig 14.14 Alberts 5th Ed / Fig 14.20 Alberts 3rd Ed Fig 2.27 Alberts 5th Ed The Proton Gradient Drives Coupled Transport Across the Inner Membrane; • The electrochemical proton gradient drives other processes besides ATP synthesis • In mitochondria many charged small molecules. Such as: Pyruvate ADP and Pi are pumped in to the matrix from the cytosol, while others such as: ATP must be moved in the opposite direction NADH dehydrogenase complex Cytochrome b-c1 complex Cytochrome oxidase • Transporters that bind these molecules can couple their transport to the energetically favorable flow of H+ in to mitochondrial matrix • Thus, for example, pyruvate and inorganic phosphate (Pi) are cotransported in ward with H+ as the H+ moves into the matrix • ADP and ATP are co-transported in opposite directions by a single transporter protein • Since an ATP molecule has one more negative charge than ADP, each nucleotide exchange results in a total of one negative charge being moved out of mitochondrion • Thus the voltage difference across the membrane drives this ADPATP co-tranporter Fig 14.16 Alberts 5th Ed / Fig 14.21 Alberts 3rd Ed Proton Gradients Produce Most of the Cell’s ATP; • Glycolysis alone produces a net yield of 2 molecules of ATP for every molecule of glucose that is metabolized and • This is the total energy yield for the fermentation processes that occur in the absence of O2 Table 14.1 Alberts 5th Ed • In conclusion, the vast majority of the ATP produces from the oxidation of glucose in an animal cells is produced by chemiosmotic mechanisms in the mitochondrion also produces a large amount of ATP from the NADH and FAD2 that is derive from the oxidation of Proteins can Move Between Compartments in Different Ways; The synthesis of all proteins begins on ribosomes in the cytosol except few that are synthesized on the ribosomes of mitochondria and plastids Their subsequent fate depends on their amino acid sequence, which can contain sorting signals that direct their delivery to locations outside the cytosol Most proteins do not have a sorting signal and consequently remain in the cytosol as a permanent residents However, many others have specific sorting signals that direct their transport from the cytosol in to • the nucleus • the ER • mitochondria • plastids or • Peroxisomes Sorting signals can also direct the transport of proteins from the ER to other destinations in the cell To understand the general principles by which sorting signals operate, it is important to distinguish • three fundamentally different ways by which proteins move from one compartment to another i. The protein traffic between the cytosol and nucleus occurs between topologically equivalent spaces, which are in continuity through the nuclear pore complexes - The process is called gated transport because the nuclear pore complexes function as selective gates - that can actively transport specific macromolecules and macromolecular assemblies - although they also allow free diffusion of smaller molecules ii. In transmembrane transporter, membrane-bound protein translocators directly transport specific proteins across a membrane from the cytosol into a space that is topologically distinct - The transported protein molecule usually must unfold in order to sneak through the membrane. For example: The initial transport of selected proteins from the cytosol into the - ER or - Mitochondria occurs in this way iii. In vesicular transport, transport vesicles carry proteins from one compartment to another The vesicles become loaded with a cargo of molecules derived from the luman of one compartment as they pinch off from its membrane They discharge their cargo in to a second compartment by fusing with its membrane. For example: - The transfer of soluble proteins from ER to the Golgi apparatus occurs in this way - Because transported protein do not cross a membrane, they move only between the compartments - that are topologically equivalent Fig 12.7 Alberts 5th Ed / Fig 12.6 Alberts 3rd Ed Fig 12.7 Alberts 3rd Ed / 12.6 Alberts 5th Ed * Each of three modes of protein transfer is usually selectively guided by sorting signals in the transported protein That are recognized by complementary receptor proteins in the target organelle. For example: If a large protein to be imported into the nucleus, it must possess a sorting signal that is recognized by receptor proteins associated with the nuclear pore complex If a protein to be transferred directly across a membrane, it must possess a sorting signal that is recognized by the translocator in the membrane to be crossed Similarly, if a protein is to be incorporated into certain types of transport vesicles or to be retained in certain organelles, its sorting signal must be recognized by a complementary receptor in the appropriate membrane Signal Peptides and Signal Patches Direct Proteins to the Correct Cellular Address; There are at least two types of sorting signals on proteins i. This resides in a continuous stretch of amino acid sequence, typically 15-69 residues long - This signal peptide is often but not always removed from the finished protein by a specialized signal peptidase once the sorting process has been completed ii. This consists of a specific three-dimensional arrangement of atoms on the protein’s surface that forms when the proteins fold up The amino acids residues that comprise this signal patch can be distant from one another in the linear amino acid sequences and they are generally remain in the finished protein Fig 12.8 Alberts 3rd Ed Table 12.3 Alberts 3rd Ed Cells can not Construct their Membrane-bounded Organelles de novo: They require Information in the Organelle itself; When a cell reproduces by division, it has to duplicate its membranebounded organelles In general, cells do this by enlarging the existing organelles by incorporating new molecules in to them The enlarge organelles then divide and are distributed to the two daughter cells Thus each daughter cell inherits from its mother a completer set of specialized cell membranes This heritance is essential because a cell could not make such membranes de novo i.e. from scratch. For example: • If the ER were completely removed from a cell, how could the cell reconstruct it? • The membrane proteins that define the ER and carry out many of its functions are themselves products of the ER • A new ER could not be made without as existing ER or • At the very least, a membrane that specially contains the translocators required to import selected proteins in to ER from the cytosol including the ER specific transporters themselves • The same is true for mitochondria and plastids • Thus it seems that the information required to construct an organelle does not reside exclusively in the DNA that specifies the organelles proteins • Information in the form of at least one distinct protein that preexists in the organelle membrane is also required and • This information is passed from parent cell to progeny cell in the form of the organelle itself • Presumably, such information is essential for the propagation of the cell’s compartmental organization • Just as the information in DNA is essential for the propagation of the cell’s nucleotide and amino acid sequences Evolutionary Origins Explain the Topological Relationships of Organelles; To understand the relationships between the compartments of the cell • It is helpful to consider how they might have evolved? • The precursors of first eukaryotic cells are thought to have been simple organisms that resembled bacteria • Which generally have plasma membrane but no internal membranes • The plasma membrane in such cells, therefore provides all membrane-dependent functions including: the pumping of ions ATP synthesis protein secretion and lipid synthesis • Typical present day eukaryotic cells are 10-30 times larger in linear dimension and 1000-10,000 times greater in volume than a typical bacterium such as E. coli • The evolution of internal membranes evidently accompanied by specialization of membrane function. For example: Consider the generation of thylakoid vesicles in chloroplasts Fig 12.3 Alberts 5th Ed • Other compartments in eukaryotic cells may have originated in a conceptually similar way • The invagination and pinching off of specialized intracellular membrane structures from the plasma membrane creates organelles with an interior that is topologically equivalent to the exterior of the cell Fig 12.3 Alberts 5th Ed Fig 12.4 Alberts 5th Ed Fig 12.5 Alberts 5th Ed / Fig 12.4 Alberts 3rd Ed x --------------------------- x---------------------------- x ----------------------------x