KAMPALA INTERNATIONAL UNIVERSITY

Cell Organelles
Cell Biology Course Work
Hellen Wambui Kinyi 7/26/2011

CELL ORGANELLES
1.0 INTRODUCTION
The cell is the functional basic unit of life. It is the smallest unit of life that is classified as a living thing, and is often called the living unit of life. The word cell comes from the Latin cellula, meaning a small room. It was coined by Robert Hooke in 1665, when comparing the cork cells he saw through his microscope to the small rooms monks lived in. The cell theory, first developed in 1839 by Matthias Jakob Schleiden, states that all organisms are composed of one or more cells, that all cells come from preexisting cells, that vital functions of an organism occur within the cell, and that all the cells contain the hereditary information necessary for regulating cell functions and for transmitting information to the next generation. There are two types of cells; Eukaryotic and Prokaryotic. The prokaryotic cell is smaller and simpler than the eukaryotic cell; it lacks a nucleus and most of the intracellular organelles. There are two types of prokaryotes, Bacteria and Archea and they both share similar structural features. Eukaryotic cells are about 15 times greater than a typical prokaryotic cell and can be as much as 1000 times greater in volume. The eukaryotic cell contains membrane bound organelles in which most metabolic activities take place. These include the nucleus, mitochondria, endoplasmic reticulum, Golgi apparatus, mitochondria, lysosomes and peroxisomes. These organelles perform diverse functions and will be examined in great detail in the following discussion.

Figure 2: Prokaryotic Cell

Figure 1 : Eukaryotic Cell

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2:0 THE NUCLEUS

The term Nucleus is derived from Latin Nuculeus meaning Kernel. It is a membrane bound organelle found in eukaryotic cells. It contains most of the cells genetic material organized in multiple long linear DNA molecules in complex with a complex of proteins (histones) to form chromosomes. The nucleus is the largest organelle. In mammalian cells it has an average diameter of 6 micrometers and occupies 10% of the total cell volume.

2:1 HISTORY
The Nucleus was the first organelle to be discovered. The oldest preserved drawing dates back to the early microscopist Antoine van Leeuwenhoek (1632-1723). He observed the nucleus in the red blood cells of salmon. (Unlike mammalian cells those of other vertebrates retain the nucleus). The nucleus was also described by Franz Bayer in 1831 and in 1835 in more detail by a Scottish Botanist Robert Brown in a talk at the Linnean Society of London. He was studying orchids when he observed the nucleus but did not suggest a probable function. In 1838 Matthias Schleiden proposed that the nucleus plays a role in generating cells, but was strongly opposed by Franz Meyen who believed that cells multiplied by division and that cells had no nuclei. The idea that cells can be generated denovo contradicted work by Robert Remark (1852) and Rudolf Virchow (1855) who propagated the paradigm that cells are solely generated by cells, Omnis cellula e cellula. The function of the cell remained unclear until much later when mitosis was discovered and the Mendelian Rules were rediscovered at the beginning of the 20th Century.

Figure 3: the nucleus

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2:2 STRUCTURES
The nuclear envelope: a double membrane that encloses the entire organelle and separates its contents from the cellular cytoplasm. The nuclear lamina: a meshwork within the nucleus that adds mechanical support much like the cytoskeleton supports the cell as a whole. Nuclear pores: These are required to allow movement of molecules across the envelope. Nucleolus: Mainly involved in the assembly of ribosomes.

Although the interior of the nucleus does not contain any membrane bound sub compartments, its contents are not uniform and a number of sub nuclear bodies exist, made up of unique proteins, RNA molecules and particular parts of the chromosome. Viscous fluid within the nucleus is called nucleoplasm and is similar in composition to the cytosol. Microscopically, the nucleus appears as a dense roughly spherical organelle. 2:2:1 NUCLEAR ENVELOPE AND PORES The nuclear envelope consists of two cellular membranes: an outer and inner membrane arranged parallel to one another and separated by 10-50nm. Although the membranes are continuous they maintain distinct protein compositions. The inner nuclear membrane contains specific proteins that act as binding sites for chromatin and for the protein meshwork of the nuclear lamina that provides structural support for this membrane. The outer membrane is continuous with the membrane of the RER and similarly the outer nuclear membrane is studded with ribosomes engaged in protein synthesis. The proteins made on these ribosomes are transported into the perinuclear space which is continuous with the ER lumen. The nuclear envelope is perforated by nuclear pore complexes. In animal cells each complex is about 125x106 MW and is composed of more than 50 different proteins called nucleoporins that are arranged in an octagonal symmetry. The more active the cell is in transcription, the greater the number of pore complexes its nucleus contains.

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Figure 4: The arrangement of nuclear pore complexes in the nuclear envelope The nuclear pore contains a doughnut shaped eight fold symmetric ring structure at a position where the inner and outer membranes fuse. Attached to the ring is a structure called the nuclear basket that extends into the nucleoplasm and a series of filamentous extensions that reach the cytoplasm. Both structures serve to mediate binding to nuclear transport proteins. Each pore complex contains one or more open aqueous channels through which small water soluble molecules can passively diffuse. Proteins made for the nucleus have Nuclear Localization Signals (NLS). In many nuclear proteins they consist of sequences that are rich in positively charged amino acids lysine and arginine, the exact sequence varying for different nucleoproteins. The nuclear pore opening can dilate up to 26nm in diameter during the transport process. A structure in the center of the nuclear pore complex seems to function as a close fitting diaphragm opening just enough to let substrates pass. The molecular mechanism of this gating mechanism is still unknown. The NLSs are recognized by nuclear import receptors which are encoded by a family of related genes. The Nuclear import receptors are soluble cytosolic proteins that bind both the NLS on the protein to be transported and to the nucleoporins. The export system relies on nuclear export signals on the macromolecules to be exported as well as on complementary nuclear export receptors. Transport through nucleopores is mediated through a family of transport factors known as KARYOPHERINS. They include: Importins that mediate transport into the nucleus and Exportins that mediate transport out of the nucleus. Karyopherins are part of importin-superfamily which share a similar 3 dimensional structure. 2:2:2 THE NUCLEAR LAMINA This is a meshwork of interconnected protein subunits called nuclear lamins. The lamins are a special class of intermediate filament proteins that polymerize into a two dimensional lattice. The lamins are synthesized in the cytoplasm and transported into the nucleus. The nuclear lamina gives shape and stability to the nuclear envelope to which it is anchored by attachment to both the nuclear pore complexes and integral membrane proteins of the inner nuclear membrane. When a nucleus disassembles during mitosis, the nuclear lamina depolymerizes. This is due to phosphorylation of the lamins by cAMP dependent kinase activated at onset of mitosis. Proteins of the inner nuclear membrane are phosphorylated and the nuclear pore complexes disassemble and disperse in the cytosol. In anaphase, the nuclear envelope reassembles on the surface of the chromosomes as inner nuclear proteins and dephosporylated lamins rebind to chromatin. 2:2:3 THE NUCLEOPLASM (Nuclear sap) This is the amorphous fluid-like material that contains the soluble material of the nucleus. It contains: Proteins which include: Enzymatic proteins involved in metabolic pathways, replication and transcription of DNA. Some of the proteins are involved in regulation of chromatin structure and function RNA and ribonucleotide complexes: Most of the RNA present are in the form of ribonucleotide granules. Small molecules including co-enzymes, metabolites and ions. Page | 5

2:2:4 THE NUCLEOLUS This is the site for the processing of rRNAs and their assembly into ribosomes. It is not a membrane bound organelle and instead it is an aggregate of macromolecules including the rRNA genes, precursor rRNAs, mature rRNAs, rRNA processing enzymes, snoRNPs, ribosomal protein subunits and partly assembled ribosomes. This close assembly allows ribosomal synthesis to occur rapidly and smoothly. A typical nucleolus consists of: Fibrils about 5nm in diameter which predominate the core of the nucleolar. The nucleolar cortex surrounds this core and contains ribonucleoprotein granules. These are embedded in the nucleolar matrix.

Nucleoli are formed around specific genetic loci called nucleolar organizing regions (NORs) first described by Barbra McClintock. Due to this non-random association, the nucleolus is termed as a genetic defined element. A nucleolar organizing region is composed of tandem repeats of rRNA genes, which are found in several different chromosomes. The human genome contains more than 200 clustered copies of rRNA genes on 5 different chromosomes (13, 14, 15, 21 and 22). A typical RNA gene consists of a promoter, internal and external transcribed spacers, rRNA coding sequences and an external non-transcribed spacer. In RBA biogenesis RNA polymerase (pol I and II) are required which function in a co-ordinated manner. In addition to ribosome biogenesis the nucleolus is also the site where other RNAs are produced and other RNA-protein complexes are assembled e.g. U6 snRNP which functions in mRNA splicing, Telomerase and signal recognition particles. 2:2:5 SUB NUCLEAR STRUCTURES These include: Cajal bodies GEMS (Gemini of coiled bodies) Interchromatin granule clusters (speckles)

These structures are not organelles and are the result of tight association of proteins and RNA components involved in gene expression. Cajal bodies and GEMS resemble one another and are frequently paired in the nucleus. They are thought to be the sites where snRNAs and snoRNAs undergo their final modifications and assembly with proteins. The speckles are thought to be stock piles of fully matured snRNPs that are ready to be used in splicing of pre-mRNAs. GEMS contain the SMN (survival of motor neuron) protein. Certain mutations of the gene encoding this protein are the cause of inherited spinal muscular dystrophy, a human disease characterized by a wasting away of the muscles.

2:3 CHROMATIN FIBERS

Eukaryotic DNA complexes with a specific group of proteins to form a series of intertwined fibers known as chromatin. The chromatin fibers are generally dispersed throughout the nucleus and become condensed during cell division into larger more discrete structures known as chromosomes. Protein Page | 6

accounts for 60-70% of the total mass of chromatin, DNA 30-40% and RNA a few percent. There are two types of chromatin: Euchromatin: This is less compact DNA form and contains genes that are frequently expressed by the cell. Heterochromatin: A more compact form and contains DNA that are infrequently transcribed. o Facultative heterochromatin consists of genes that are organized as heterochromatin only in certain cell types or at certain stages of development. o Constitutive heterochromatin consists of chromosome structural components such as the telomeres and centromeres.

Chromatin proteins can be divided into: 2:3:1 HISTONES They are also referred to as DNA binding proteins. They are a group of basic proteins whose high content of lysine and arginine give them a strong positive charge at physiological pH. There are 5 major types: H1, H2A, H2B, H3 and H4. Chromatin contains roughly equal numbers of H2A, H2B, H3 and H4 molecules and about half that number of histone H1 molecules. The primary structure of histone molecules has been highly conserved during evolution. These minor evolutionary changes suggest that even a small change would have harmful repercussions on nuclear function. Histones are subject to enzymatic modification reactions such as acetylation, methylation and phosphorylation which alter amino acid side chains after the protein has been synthesized. These alterations are reversible and influence both the structural and functional properties of histone molecules e.g. acetylated histone proteins are associated with chromatin fibers that are being transcribed, while phosphorylated histone proteins have been implicated in the condensation of chromatin fibers in the chromosomes. In eukaryotic sperm cell, histones are replaced with another basic protein known as protamines. About two thirds of the amino acids in protamine are arginine and the rest are neutral amino acids. Ionic bonds between the positively charged arginines and negatively charged phosphate groups present in DNA allow protamine molecules to wrap tightly around the DNA double helix lying in the minor groove. This compact structure facilitates the packaging and protection of the DNA sperm cells, whose main function is to deliver an intact set of DNA molecules to the egg. 2:3:2 NON-HISTONE PROTEINS These proteins remain after histones have been removed and include: Scaffold proteins, DNA polymerase, Heterochromatin Protein 1 and Polycomb. The high mobility group or HMG proteins are associated with chromatin fibers that are actively transcribed.

2:4 NUCLEOSOMES
When interphase DNA is broken up gently and examined under the microscope, most of the DNA is in the form of a fiber with a diameter of about 30nm. If further unfolded the chromatin appears as a series of beads on a string. The string is DNA and each bead is a nucleosome core particle that consists of Page | 7

DNA wound around a protein core formed from histones. The beads on a string represent the first level of DNA packaging. The nucleosome core consists of a complex of eight histone proteins: two molecules each of H2A, H2B, H3 and H4 and double stranded DNA that is 146 nucleotide base pairs long. The histone octamer forms a protein core around which the double stranded DNA is wound. Each nucleosome core particle is separated from the next by a region of linker DNA which can vary in length from a few nucleotide pairs up to 80. This provides the first level of DNA packaging. The 10nm nucleosome undergoes further coiling into a helical chain or solenoid about 30nm. Histone H1 mediates the folding of the 10nm chain of nucleosomes into the 30nm fiber. During cell division the chromatin fibers are packaged into highly condensed chromosomes.

FIGURE 6: Nucleosomes. Regularly spaced nucleosomes consist of histone complexes bound to DNA.

2:5 CHROMOSOMES
A chromosome is an organized structure of DNA containing many genes, regulatory elements and other nucleotide sequences. The word chromosome comes from the greek chroma (color) and soma (body) due to their property of being very strongly stained by particular dyes. There are two main forms of chromosomal organization: Lampbrush chromosomes: These are the largest chromosome known and are clearly visible even in the light microscope, where they are seen to be organized into a series of large chromatin loops emanating from a linear chromosomal axis. These lampbrush chromosome discovered by Ruckert in 1892 are formed during the active synthesis of mRNA molecules for future use during cleavage when no synthesis of mRNA is possible due to active involvement of chromosomes in mitotic cell division. Found in amphibian eggs before meiosis takes place. Polytene chromosomes: these are formed when multiple rounds of replication produce many sister chromatids that remain synapsed together. In addition to increasing the volume of the cell nuclei and causing cell expansion, polytene cells may also have a metabolic advantage as multiple copies of genes permits a high level of gene expression e.g. in Drosophilia melanogaster, the chromosomes of the larval salivary glands undergo many rounds of endoreplication to produce large amounts of glue before pupation.

In humans the genome is organized in 23 pairs of chromosomes of which 22 pairs are autosomes and 1 pair the sex chromosome. These chromosomes are the essential unit of cellular division and must be replicated, divided and passed successfully to daughter cells so as to ensure the genetic diversity and survival of the progeny. Chromosomes may exist as single linear strands or duplicated during synthesis phase of the cell cycle to contain two copies joined by a centromeres. Compaction of the duplicated chromosomes results during mitosis and meiosis results in the classic form arm structure. Page | 8

FIGURE 7: EUKARYOTIC CHROMOSOME 2:6 FUNCTIONS OF THE NUCLEUS The Nucleus has been referred to as the brain of the cell. All the major functions of the cell are done under instructions of the nucleus. The major functions are: It is involved in cell division It stores all the information that is to be transferred to the next generation. Assembly of ribosomes takes place inside the nucleolus present inside the nucleus. DNA transcription and replication takes place inside the nucleus. Control of gene expression also occurs in the nucleus.

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3:0 THE MITOCHONDRIA

FIGURE 8: The Mitochondria Mitochondrion (plural mitochondria) is a membrane-enclosed organelle found in most eukaryotic cells. These organelles range from 0.5 to 10 micrometers in diameter. Mitochondria are sometimes described as "cellular power plants" because they generate most of the cell's supply of adenosine triphosphate (ATP), used as a source of chemical energy. In addition to supplying cellular energy, mitochondria are involved in a range of other processes, such as signaling, cellular differentiation, cell death, as well as the control of the cell cycle and cell growth. Mitochondria have been implicated in several human diseases, including mitochondrial disorders and cardiac dysfunction,and may play a role in the aging process. The word mitochondrion comes from the Greek mitos, thread and chondrion, granule. Several characteristics make mitochondria unique. The number of mitochondria in a cell varies widely by organism and tissue type. Many cells have only a single mitochondrion, whereas others can contain several thousand mitochondria. The organelle is composed of compartments that carry out specialized functions. These compartments or regions include the outer membrane, the intermembrane space, the inner membrane, and the cristae and matrix. Mitochondrial proteins vary depending on the tissue and the species. In humans, 615 distinct types of proteins have been identified from cardiac mitochondria. Although most of a cell's DNA is contained in the cell nucleus, the mitochondrion has its own independent genome. Further, its DNA shows substantial similarity to bacterial genomes.

3:1 HISTORY
The discovery of mitochondria is not accredited to a single scientist as such, but is accredited to the several scientists who contributed to the discovery of mitochondrion and identification of its structure and functions. The earliest accounts of description of mitochondria go back to 1840. However, Richard Altmann was the first one to recognize the occurrence of these organelles and called them bioblasts. The name mitochondrion was coined by Carl Benda in 1898 and the word mitochondrion has its origin in Page | 10

the Greek language where mitos means thread and chondros means granule, which describes the appearance of mitochondrion during spermatogenesis. Mitochondria have many features in common with prokaryotes. As a result, they are believed to be originally derived from endosymbiotic prokaryotes. A mitochondrion contains DNA, which is organized as several copies of a single, circular chromosome. The circular structure is also found in prokaryotes, and the similarity is extended by the fact that mitochondrial DNA is organized with a variant genetic code similar to that of Proteobacteria. This suggests that their ancestor, the so-called protomitochondrion, was a member of the Proteobacteria. In particular, the proto-mitochondrion was probably closely related to the rickettsia. The ribosomes coded for by the mitochondrial DNA are similar to those from bacteria in size and structure. They closely resemble the bacterial 70S ribosome and not the 80S cytoplasmic ribosomes, which are coded for by nuclear DNA. The endosymbiotic hypothesis suggests that mitochondria descended from bacteria that somehow survived endocytosis by another cell, and became incorporated into the cytoplasm. The ability of these bacteria to conduct respiration in host cells that had relied on glycolysis and fermentation would have provided a considerable evolutionary advantage. In a similar manner, host cells with symbiotic bacteria capable of photosynthesis would have had an advantage. The incorporation of symbiotes would have increased the number of environments in which the cells could survive. This symbiotic relationship probably developed 1.7 billion years ago. 3:2 STRUCTURES A mitochondrion contains outer and inner membranes composed of phospholipid bilayers and proteins. The two membranes, however, have different properties. Because of this double-membraned organization, there are five distinct compartments within the mitochondrion. There is the outer mitochondrial membrane, the intermembrane space (the space between the outer and inner membranes), the inner mitochondrial membrane, the crista space (formed by infoldings of the inner membrane), and the matrix (space within the inner membrane). 3:2:1 THE OUTER MEMBRANE The outer mitochondrial membrane, which encloses the entire organelle, has a protein-to-phospholipid ratio similar to that of the eukaryotic plasma membrane (about 1:1 by weight). It contains large numbers of integral proteins called porins. These porins form channels that allow molecules 5000 Daltons or less in molecular weight to freely diffuse from one side of the membrane to the other. Larger proteins can enter the mitochondrion if a signaling sequence at their N-terminus binds to a large multisubunit protein called translocase of the outer membrane, which then actively moves them across the membrane. Disruption of the outer membrane permits proteins in the intermembrane space to leak into the cytosol, leading to certain cell death. The mitochondrial outer membrane can associate with the endoplasmic reticulum (ER) membrane, in a structure called MAM (mitochondria-associated ERmembrane). This is important in ER-mitochondria calcium signaling and involved in the transfer of lipids between the ER and mitochondria.

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3:2:2 THE INTERMEMBRANE SPACE The intermembrane space is the space between the outer membrane and the inner membrane. Because the outer membrane is freely permeable to small molecules, the concentrations of small molecules such as ions and sugars in the intermembrane space are the same as the cytosol. However, large proteins must have a specific signaling sequence to be transported across the outer membrane, so the protein composition of this space is different from the protein composition of the cytosol. One protein that is localized to the intermembrane space in this way is cytochrome c. 3:2:3 THE INNER MEMBRANE The inner membrane is more complex in structure than the outer membrane as it contains the complexes of the electron transport chain and the ATP synthetase complex. It is permeable only to oxygen, carbon dioxide and water. The inner mitochondrial membrane contains proteins with five types of functions: 1. 2. 3. 4. 5. Those that perform the redox reactions of oxidative phosphorylation ATP synthase, which generates ATP in the matrix Specific transport proteins that regulate metabolite passage into and out of the matrix Protein import machinery. Mitochondria fusion and fission protein

It contains more than 151 different polypeptides, and has a very high protein-to-phospholipid ratio (more than 3:1 by weight, which is about 1 protein for 15 phospholipids). The inner membrane is home to around 1/5 of the total protein in a mitochondrion. In addition, the inner membrane is rich in an unusual phospholipid, cardiolipin. This phospholipid was originally discovered in beef hearts in 1942, and is usually characteristic of mitochondrial and bacterial plasma membranes. Cardiolipin contains four fatty acids rather than two and may help to make the inner membrane impermeable. Unlike the outer membrane, the inner membrane doesn't contain porins and is highly impermeable to all molecules. Almost all ions and molecules require special membrane transporters to enter or exit the matrix. Proteins are ferried into the matrix via the translocase of the inner membrane (TIM) complex or via Oxa1. In addition, there is a membrane potential across the inner membrane formed by the action of the enzymes of the electron transport chain. 3:2:4 CRISTAE The inner mitochondrial membrane is compartmentalized into numerous cristae, which expand the surface area of the inner mitochondrial membrane, enhancing its ability to produce ATP. For typical liver mitochondria the area of the inner membrane is about five times greater than the outer membrane. This ratio is variable and mitochondria from cells that have a greater demand for ATP, such as muscle cells, contain even more cristae. These folds are studded with small round bodies known as F1 particles or oxysomes. These are not simple random folds but rather invaginations of the inner membrane, which can affect overall chemiosmotic function.

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3:2:5 THE MATRIX The matrix is the space enclosed by the inner membrane. It contains about 2/3 of the total protein in a mitochondrion. The matrix is important in the production of ATP with the aid of the ATP synthase contained in the inner membrane. The matrix contains a highly-concentrated mixture of hundreds of enzymes, special mitochondrial ribosomes, tRNA, and several copies of the mitochondrial DNA genome. Of the enzymes, the major functions include oxidation of pyruvate and fatty acids, and the citric acid cycle. Mitochondrial DNA, is organized as several copies of a single, circular chromosome. This mitochondrial chromosome contains genes for redox proteins such as those of the respiratory chain. The CoRR hypothesis proposes that this co-location is required for redox regulation. The mitochondrial genome codes for some RNAs of ribosomes, and the twenty-two tRNAs necessary for the translation of messenger RNAs into protein.

3:3 REPLICATION AND INHERITANCE

Mitochondria divide by binary fission similar to bacterial cell division; unlike bacteria, however, mitochondria can also fuse with other mitochondria. The regulation of this division differs between eukaryotes. In many single-celled eukaryotes, their growth and division is linked to the cell cycle. For example, a single mitochondrion may divide synchronously with the nucleus. This division and segregation process must be tightly controlled so that each daughter cell receives at least one mitochondrion. In other eukaryotes (in mammals for example), mitochondria may replicate their DNA and divide mainly in response to the energy needs of the cell, rather than in phase with the cell cycle. When the energy needs of a cell are high, mitochondria grow and divide. When the energy use is low, mitochondria are destroyed or become inactive. In such examples, and in contrast to the situation in many single celled eukaryotes, mitochondria are apparently randomly distributed to the daughter cells during the division of the cytoplasm. An individual's mitochondrial genes are not inherited by the same mechanism as nuclear genes. At fertilization the mitochondria, and therefore the mitochondrial DNA, usually comes from the egg only. The sperm's mitochondria enter the egg but do not contribute genetic information to the embryo. Instead, paternal mitochondria are marked with ubiquitin to select them for later destruction inside the embryo. The egg cell contains relatively few mitochondria, but it is these mitochondria that survive and divide to populate the cells of the adult organism. Mitochondria are, therefore, in most cases inherited down the female line, known as maternal inheritance. This mode is seen in most organisms including all animals.

3:4 MITOCHONDRIAL FUNCTIONS

The most prominent roles of mitochondria are to produce ATP through respiration, and to regulate cellular metabolism. The central sets of reactions involved in ATP production are collectively known as the citric acid cycle, or the Krebs Cycle. However, the mitochondrion has many other functions in addition to the production of ATP.

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3:4:1 ENERGY CONVERSION A dominant role for the mitochondria is the production of ATP, as reflected by the large number of proteins in the inner membrane for this task. This is done by oxidizing the major products of glucose, pyruvate, and NADH, which are produced in the cytosol. This process of cellular respiration, also known as aerobic respiration, is dependent on the presence of oxygen. When oxygen is limited, the glycolytic products will be metabolized by anaerobic respiration, a process that is independent of the mitochondria. The production of ATP from glucose has an approximately 13-fold higher yield during aerobic respiration compared to anaerobic respiration. Some of the pathways involved include: 1. Pyruvate Dehydrogenase Complex: Pyruvate, the end product of aerobic glycolysis, must be transported into the mitochondrion before it can enter the TCA cycle. This is accomplished by a specific pyruvate transporter that helps pyruvate cross the inner mitochondrial membrane. Once in the matrix, pyruvate is converted to acetyl CoA by the pyruvate dehydrogenase complex, which is a multienzyme complex. 2. Tricarboxylic Acid Cycle (TCA): The citric acid cycle (Krebs cycle) is a series of reactions in mitochondria that oxidize acetyl residues and reduce coenzymes that upon re-oxidation are linked to the formation of ATP. The citric acid cycle is the final common pathway for the aerobic oxidation of carbohydrate, lipid, and protein because glucose, fatty acids, and most amino acids are metabolized to acetyl-CoA or intermediates of the cycle. It also has a central role in gluconeogenesis, lipogenesis, and interconversion of amino acids. The enzymes of the citric acid cycle are located in the mitochondrial matrix, with the exception of succinate dehydrogenase, which is bound to the inner mitochondrial membrane as part of Complex II. The citric acid cycle oxidizes the acetyl-CoA to carbon dioxide, and, in the process, produces reduced cofactors (three molecules of NADH and one molecule of FADH2) that are a source of electrons for the electron transport chain, and a molecule of GTP (that is readily converted to an ATP). 3. ELECTRON TRANSPORT CHAIN AND OXIDATIVE PHOSPHORYLATION: These processes re-oxidize NADH and FADH2 and trap the energy released as ATP. In eukaryotes this oxidative phosphorylation occurs in the inner mitochondrial membranes. The redox energy from NADH and FADH2 is transferred to oxygen (O2) in several steps via the electron transport chain. These energy-rich molecules are produced within the matrix via the citric acid cycle but are also produced in the cytoplasm by glycolysis. Reducing equivalents from the cytoplasm can be imported via the malate-aspartate shuttle system of antiporter proteins or feed into the electron transport chain using a glycerol phosphate shuttle. Protein complexes in the inner membrane (NADH dehydrogenase, cytochrome c reductase, and cytochrome c oxidase) perform the transfer and the incremental release of energy is used to pump protons (H+) into the intermembrane space. As the proton concentration increases in the intermembrane space, a strong electrochemical gradient is established across the inner membrane. The protons can return to the matrix through the ATP synthase complex, and their potential energy is used to synthesize ATP from ADP and inorganic phosphate (Pi). This process is called chemiosmosis, and was first described by Peter Mitchell who was awarded the 1978 Nobel Prize in Chemistry for his work.

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Figure 9: Diagram of ETC and Oxidative Phosphorylation in the Inner Mitochondrial Membrane

3:4:2 PRODUCTION HEAT Under certain conditions, protons can re-enter the mitochondrial matrix without contributing to ATP synthesis. This process is known as proton leak or mitochondrial uncoupling and is due to the facilitated diffusion of protons into the matrix. The process results in the unharnessed potential energy of the proton electrochemical gradient being released as heat. The process is mediated by a proton channel called thermogenin, or UCP1, a 33kDa protein first discovered in 1973. Thermogenin is primarily found in brown adipose tissue, or brown fat, and is responsible for non-shivering thermogenesis. Brown adipose tissue is found in mammals, and is at its highest levels in early life and in hibernating animals. In humans, brown adipose tissue is present at birth and decreases with age. 3:4:3 STORAGE OF CALCIUM The concentrations of free calcium in the cell can regulate an array of reactions and is important for signal transduction in the cell. Mitochondria can transiently store calcium, a contributing process for the cell's homeostasis of calcium. In fact, their ability to rapidly take in calcium for later release makes them very good "cytosolic buffers" for calcium. The endoplasmic reticulum (ER) is the most significant storage site of calcium, and there is a significant interplay between the mitochondrion and ER with regard to calcium. The calcium is taken up into the matrix by a calcium uniporter on the inner mitochondrial membrane. It is primarily driven by the mitochondrial membrane potential. Release of this calcium back into the cell's interior can occur via a sodium-calcium exchange protein or via "calcium-induced-calciumrelease" pathways. This can initiate calcium spikes or calcium waves with large changes in the membrane potential. These can activate a series of second messenger system proteins that can coordinate processes such as neurotransmitter release in nerve cells and release of hormones in endocrine cells.

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Mitochondria play a central role in many other metabolic tasks, such as: Regulation of the membrane potential, Cellular proliferation regulation, Regulation of cellular metabolism, Certain heme synthesis reactions and Steroid synthesis. Some mitochondrial functions are performed only in specific types of cells. For example, mitochondria in liver cells contain enzymes that allow them to detoxify ammonia, a waste product of protein metabolism. A mutation in the genes regulating any of these functions can result in mitochondrial diseases.

3:5 DISEASES OF THE MITOCHONDRIA

Mitochondrial disorders often present as neurological disorders, but can manifest as myopathy, diabetes, multiple endocrinopathy, or a variety of other systemic manifestations. Diseases caused by mutation in the mtDNA include Kearns-Sayre syndrome, MELAS syndrome and Leber's hereditary optic neuropathy. In the vast majority of cases, these diseases are transmitted by a female to her children, as the zygote derives its mitochondria and hence its mtDNA from the ovum. Diseases such as Kearns-Sayre syndrome, Pearson's syndrome, and progressive external ophthalmoplegia are thought to be due to large-scale mtDNA rearrangements, whereas other diseases such as MELAS syndrome, Leber's hereditary optic neuropathy, myoclonic epilepsy with ragged red fibers (MERRF), and others are due to point mutations in mtDNA. In other diseases, defects in nuclear genes lead to dysfunction of mitochondrial proteins. This is the case in Friedreich's ataxia, hereditary spastic paraplegia, and Wilson's disease. These diseases are inherited in a dominance relationship, as applies to most other genetic diseases. A variety of disorders can be caused by nuclear mutations of oxidative phosphorylation enzymes, such as coenzyme Q10 deficiency and Barth syndrome. Environmental influences may interact with hereditary predispositions and cause mitochondrial disease. For example, there may be a link between pesticide exposure and the later onset of Parkinson's disease.

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4:0 THE ENDOPLASMIC RETICULUM

The endoplasmic reticulum (ER) is an eukaryotic organelle that forms an interconnected network of tubules, vesicles, and cisternae within cells. The tubules and sacs are thought to interconnect, so that the ER membrane forms a continuous sheet enclosing a single internal space. This highly convoluted space is called the ER lumen or the ER cisternal space, which is continuous with the perinuclear space and occupies more than 10% of the total cell volume. The ER membrane separates the ER lumen from the cytosol, and it mediates the selective transfer of molecules between these two compartments. The lacey membranes of the endoplasmic reticulum were first seen by Keith R. Porter, Albert Claude, and Ernest F. Fullam in 1945. The functions of the endoplasmic reticulum vary greatly depending on the exact type of endoplasmic reticulum and the type of cell in which it resides. The three varieties are called rough endoplasmic reticulum, smooth endoplasmic reticulum and sarcoplasmic reticulum. The quantity of RER and SER in a cell can quickly interchange from one type to the other, depending on changing metabolic needs: one type will undergo numerous changes including new proteins embedded in the membranes in order to transform. Also, massive changes in the protein content can occur without any noticeable structural changes, depending on the enzymatic needs of the cell.

4:1 THE ROUGH ENDOPLASMIC RETICULUM (RER)

The surface of the rough endoplasmic reticulum (RER) is studded with protein-manufacturing ribosomes giving it a "rough" appearance hence its name. However, the ribosomes bound to the RER at any one time are not a stable part of this organelle's structure as ribosomes are constantly being bound and released from the membrane. The RER captures selected proteins from the cytosol as they are being synthesized. All these proteins are directed to the ER membrane by the same kind of signal sequence and are translocated across it by similar mechanisms. Here, a ribosome in the cytosol begins synthesizing a protein until a signal recognition particle recognizes the pre-piece of 5-15 hydrophobic amino acids preceded by a positively charged amino acid. This signal sequence allows the recognition particle to bind to the ribosome, causing the ribosome to bind to the RER and pass the new protein through the ER membrane. The pre-piece is then cleaved off within the lumen of the ER and the ribosome released back into the cytosol. The membrane of the RER is continuous with the outer layer of the nuclear envelope. Although there is no continuous membrane between the RER and the Golgi apparatus, membrane-bound vesicles shuttle proteins between these two compartments. Vesicles are surrounded by coating proteins called COPI and COPII. COPII targets vesicles to the golgi and COPI marks them to be brought back to the RER. The RER works in concert with the Golgi complex to target new proteins to their proper destinations. A second method of transport out of the ER are areas called membrane contact sites, where the membranes of the ER and other organelles are held closely together, allowing the transfer of lipids and other small molecules. The functions of the RER include:

Synthesis of lysosomal enzymes with a mannose-6-phosphate marker added in the cis-Golgi network

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Synthesis of secreted proteins, either secreted constitutively with no tag, or regulated secretion involving clathrin and paired basic amino acids in the signal peptide. Synthesis of integral membrane proteins that stay imbedded in the membrane as vesicles exit and bind to new membranes. Rab proteins are key in targeting the membrane, SNAP and SNARE proteins are key in the fusion event. Initial glycosylation of proteins as assembly continues. This is N-linked (O-linking occurs in the Golgi).

4:2 SMOOTH ENDOPLASMIC RETICULUM

Regions of the ER that lack ribosomes are called the smooth endoplasmic reticulum. In the great majority of cells, these regions are often scanty and are often partly smooth and partly rough. They are sometimes called transitional endoplasmic reticulum because they contain ER exit sites from which transport vesicles carrying newly synthesized proteins and lipids bud off for transport to the golgi apparatus. In certain cells however, the smooth ER is abundant and has additional functions. It is usually prominent in cells that specialize in lipid metabolism. Cells such as the Leydig cells of the testis that synthesize steroid hormones from cholesterol, have an expanded Smooth ER compartment to accommodate the enzymes needed to make cholesterol and to modify it to form the hormones. The hepatocyte is another cell with abundant smooth ER. It is the principal site of the production of lipoprotein particles, which carry lipids via the bloodstream to other parts of the body. The enzymes that synthesize the lipid components of the lipoproteins are located in the membrane of the smooth ER, which also contains enzymes that catalyze a series of reactions to detoxify both lipid soluble drugs and various harmful compounds produced by metabolism.

4:3 THE SARCOPLASMIC RETICULUM

The sarcoplasmic reticulum (SR), from the Greek sarx, ("flesh"), is a special type of smooth ER found in smooth and striated muscle. The only structural difference between this organelle and the SER is the medley of proteins they have, both bound to their membranes and drifting within the confines of their lumens. This fundamental difference is indicative of their functions: the SER synthesizes molecules while the SR stores and pumps calcium ions. The SR contains large stores of calcium, which it sequesters and then releases when the muscle cell is stimulated. The SR's release of calcium upon electrical stimulation of the cell plays a major role in excitation-contraction coupling.

4:4 FUNCTIONS OF THE ENDOPLASMIC RETICULUM

The endoplasmic reticulum serves many general functions, including the facilitation of protein folding and the transport of synthesized proteins in sacs called cisternae. Correct folding of newly-made proteins is made possible by several endoplasmic reticulum chaperone proteins, including protein disulfide isomerase (PDI), ERp29, the Hsp70 family member Grp78, calnexin, calreticulin, and the peptidylpropyl isomerase family. Only properly-folded proteins are transported from the rough ER to the Golgi complex. TRANSPORT OF PROTEINS Secretory proteins, mostly glycoproteins, are moved across the endoplasmic reticulum membrane. Proteins that are transported by the endoplasmic reticulum and from there throughout the cell are Page | 18

marked with an address tag called a signal sequence. The N-terminus (one end) of a polypeptide chain contains a few amino acids that work as an address tag, which are removed when the polypeptide reaches its destination. Proteins that are destined for places outside the endoplasmic reticulum are packed into transport vesicles and moved along the cytoskeleton toward their destination. The endoplasmic reticulum is also part of a protein sorting pathway. It is, in essence, the transportation system of the eukaryotic cell. The majority of endoplasmic reticulum resident proteins are retained in the endoplasmic reticulum through a retention motif. This motif is composed of four amino acids at the end of the protein sequence. The most common retention sequence is KDEL (lys-asp-glu-leu). However, variation on KDEL does occur and other sequences can also give rise to endoplasmic reticulum retention. It is not known if such variation can lead to sub-endoplasmic reticulum localizations. There are three KDEL receptors in mammalian cells, and they have a very high degree of sequence identity. The functional differences between these receptors remain to be established. Other functions

Insertion of proteins into the endoplasmic reticulum membrane: Integral membrane proteins are inserted into the endoplasmic reticulum membrane as they are being synthesized (cotranslational translocation). Insertion into the endoplasmic reticulum membrane requires the correct topogenic signal sequences in the protein. Glycosylation: Glycosylation involves the attachment of oligosaccharides. Disulfide bond formation and rearrangement: Disulfide bonds stabilize the tertiary and quaternary structure of many proteins. Drug Metabolism: The smooth ER is the site at which some drugs are modified by microsomal enzymes which include the cytochrome P450 enzymes.

Figure 10: The endoplasmic reticulum Page | 19

5:0 THE RIBOSOME

The ribosome is one of the most abundant of subcellular organelles. Due to its abundance and relatively easy purification, ribosomes especially those of microorganisms and rat liver have been studied since the 1950s. Ribosomes are components of the cell that make proteins from all amino acids.

5:1 HISTORY
Ribosomes were first observed in the mid 1950s by a Romanian cell biologist, George Palade using an electron microscope as dense particles or granules for which he won a Nobel Prize. The term ribosome was proposed by the scientist Richard B. Roberts in 1958. The term was adopted in a symposium to designate ribonucleotide particles in sizes ranging from 35s to 100s. The structure and function of ribosomes and associated molecules known as the translational apparatus has been of research interest since the mid twentieth century and is a very active field of study today.

5:2 THE ISOLATION OF RIBOSOMES

Ribosomes are recovered from E.coli cells by disrupting the cells in buffered 5mM Magnesium Chloride solution and subjecting the extract to high speed centrifugation. Given that the particles isolated by centrifugation, forms the basis of their naming at the rate at which they sediment in a centrifugal field, Svedberg Unit.

5:3 THE MORPHOLOGY OF RIBOSOMES

The ribosomal subunit of prokaryotes and eukaryotes are quite similar. Prokaryotes have 70s ribosomes each consisting of a small 30s subunit and a large 60s subunit. Their large subunit is composed of 5s RNA subunit, a 23s subunit and 34 proteins. The 30s subunit has a 16s RNA subunit bound to 21 proteins. Eukaryotes have 80s ribosomes each consisting of a small 40s and a large 80s subunit. Their large subunit is composed of a 5s RNA, a 28s RNA, a 5.8s subunit and about 49 proteins. The 40s subunit has 18s RNA and about 33 proteins. The various ribosomes share a core structure which is quite similar despite the large differences in size. Much of the RNA is highly organized into various tertiary motifs such as pseudo knots that exhibit coaxial stacking. The extra RNA in the larger ribosome is in several continuous insertions, such that they form loops out of the core structure without disrupting or changing it. All the catalytic activity of the ribosome is carried out by RNA. The ribosomal proteins are generally located on the surface and fill in the gaps and crevices of the folded RNA. Some of these proteins contain globular domains on the ribosome surface that send out extended regions of polypeptide chain that penetrate short distances into holes in the RNA core. The main role of ribosomal proteins seems to be the stabilization of the RNA core, while permitting the changes in the rRNA conformation that are necessary for this RNA to catalyse efficient protein synthesis.

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Figure 11: The Ribosome

5:4 BIOGENESIS
In bacterial cells, ribosomes are synthesized in the cytoplasm through the transcription of multiple ribosome gene operons. In eukaryotes, the process takes place both in the cell cytoplasm and in the nucleolus. The assembly process involves the coordinated function of over 200 proteins in the synthesis and processing of the four rRNAs as well as assembly of the rRNAs with the ribosomal proteins.

5:5 LOCATIONS OF RIBOSOMES

Ribosomes are classified as being either free or membrane bound. These groups differ only in their spatial distribution but have identical structure. Whether the ribosome exists in a free or membrane bound state depends on the presence of an ER targeting sequence on the protein being synthesized, so an individual ribosome might be membrane bound when it is making one protein but free in the cytosol when it is making another. Free ribosomes move about in the cytosol but are excluded from the cell nucleus and other organelles. Proteins that are formed by free ribosomes are released into the cytosol and are used within the cells. Since the cell contains high concentration of glutathione and is thus a reducing environment, proteins containing disulfide bonds which are formed from oxidized cysteine residues cannot be produced in this compartment. Membrane bound ribosomes: When a ribosome begins to synthesize proteins that are needed in some organelles, the ribosome making this protein becomes membrane bound. In eukaryotic cells this happens in the RER. The newly produced polypeptide chains are inserted directly into the ER by the ribosome and are then transported to their destinations through the secretory pathway. Bound ribosomes usually produce proteins that are used within the plasma membrane or are expelled from the cell via exocytosis.

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5:6 THE FUNCTION OF RIBOSOMES

The main function of ribosomes is protein synthesis (translation). A ribosome binds to a mRNA molecule and reads the nucleotide sequence from the 5 to 3 direction, synthesizing the corresponding protein from amino acids in an N-terminal to C-terminal direction. The amino acids used are covalently bound to tRNA molecules to form aminoacyl-tRNAs. Each aminoacyl-tRNA bears a triplet of bases, called an anticodon. The ribosome reads each triplet codon of the mRNA in turn and an aminoacyl-tRNA molecule with an anticodon that is complementary to the codon binds to it via hydrogen bonding. A peptide bond is then formed between the incoming amino acid and the growing end of the polypeptide chain. Usually at any one time, many ribosomes are translating an mRNA simultaneously, forming a structure called polyribosome or polysome.

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6:0 GOLGI APPARATUS

Figure 12: The Golgi apparatus The Golgi apparatus also known as Golgi body or the Golgi complex is an organelle found in most eukaryotic cells. The Golgi apparatus processes and packages macromolecules, such as proteins and lipids, after their synthesis and before they make their way to their destination; it is particularly important in the processing of proteins for secretion. The Golgi apparatus forms a part of the cellular endomembrane system. Due to its fairly large size, the Golgi apparatus was one of the first organelles to be discovered and observed in detail. The apparatus was discovered in 1897 by Italian physician Camillo Golgi during an investigation of the nervous system After first observing it under his microscope, he termed the structure the internal reticular apparatus. The structure was then renamed after Golgi not long after the announcement of his discovery in 1898

6:1 MORPHOLOGY OF GOLGI APPARATUS

The Golgi is composed of stacks of membrane-bound structures known as cisternae. An individual stack is sometimes called a dictyosome especially in plant cells. A mammalian cell typically contains 40 to 100 stacks between four and eight cisternae are usually present in a stack; however, in some protists as many as sixty have been observed. Each cisterna comprises a flat, membrane enclosed disc that includes special Golgi enzymes which modify or help to modify cargo proteins that travel through it. They are found in both plant and animal cells. The cisternae stack has four functional regions: the cis-Golgi network, medial-Golgi, endo-Golgi, and trans-Golgi network. Vesicles from the endoplasmic reticulum fuse with the network and subsequently progress through the stack to the trans Golgi network, where they are packaged and sent to the required destination. Each region contains different enzymes which Page | 23

selectively modify the contents depending on where they reside. The cisternae also carry structural proteins important for their maintenance as flattened membranes which stack upon each other. In animal cell the apparatus is usually located near the nucleus and close to the centrosome. This localization is dependent on the microtubules.

6:2 FUNCTIONS OF THE GOLGI APPARATUS

The Golgi apparatus is integral in modifying, sorting, and packaging of macromolecules for cell secretion (exocytosis) or use within the cell. It primarily modifies proteins delivered from the rough endoplasmic reticulum but is also involved in the transport of lipids around the cell, and the creation of lysosomes. Enzymes within the cisternae are able to modify substances by the addition of carbohydrates (glycosylation) and phosphates (phosphorylation). In order to do so, the Golgi imports substances such as nucleotide sugars from the cytosol. These modifications may also form a signal sequence which determines their final destination. For example, the Golgi apparatus adds a mannose-6-phosphate label to proteins destined for lysosomes. The Golgi plays an important role in the synthesis of proteoglycans, which are molecules present in the extracellular matrix of animals. It is also a major site of carbohydrate synthesis. This includes the production of glycosaminoglycans (GAGs), long unbranched polysaccharides which the Golgi then attaches to a protein synthesised in the endoplasmic reticulum to form proteoglycans. Another task of the Golgi involves the sulfation of certain molecules passing through its lumen via sulphotranferases that gain their sulphur molecule from a donor called PAPs. This process occurs on the GAGs of proteoglycans as well as on the core protein. The level of sulfation is very important to the proteoglycans' signalling abilities as well as giving the proteoglycan its overall negative charge. The phosphorylation of molecules requires that ATP is imported into the lumen of the Golgi and then utilised by resident kinases such as casein kinase 1 and casein kinase 2. One molecule that is phosphorylated in the Golgi is Apolipoprotein, which forms a molecule known as VLDL that is a constituent of blood serum. It is thought that the phosphorylation of these molecules is important to help aid in their sorting for secretion into the blood serum. The Golgi has a putative role in apoptosis, with several Bcl-2 family members localized there, as well as to the mitochondria. A newly characterized protein, GAAP (Golgi anti-apoptotic protein), almost exclusively resides in the Golgi and protects cells from apoptosis.

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7:0 LYSOSOMES
Lysosomes are membrane enclosed organelles filled with hydrolytic enzymes that are used for controlled intracellular digestion of macromolecules. They contain about 40 types of hydrolytic enzymes, including proteases, nucleases, glycosidases, lipases, phospholipases, phosphatases and sulfatases. All are acid hydrolases that require an acidic environment for activity. The lysosome provides this by maintaining a pH of about 5.0 in its interior. In this way the contents of the cytosol are doubly protected against attack by the cells own digestive system. This low pH is maintained by an H+ pump located on its surrounding membrane which uses the energy of ATP hydrolysis to pump H+ into the lysosome, thereby maintaining the lumen at its acidic pH. The lysosomal membrane also contains transport proteins that allow the final products of digestion of macromolecules such as amino acids, sugars and nucleotides to be transported to the cytosol from where they can be excreted or be reutilized by the cell. Most lysosomal membrane proteins are unusually highly glycosylated, which helps to protect them from the lysosomal proteases in the lumen. The morphology of lysosomes is heterogeneous reflecting the wide variety of digestive functions mediated by acid hydrolases, including the breakdown of intra and extracellular debris, the destruction of phagocytosed organisms and production of nutrients for the cell.

7:1 PATHWAYS LEADING TO LYSOSOMES

Figure 13: Pathways leading to formation of lysosomes

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Lysosomes are usually meeting places where several streams of intracellular traffic converge. Digestive enzymes are delivered to them by a route that leads outward from the endoplasmic reticulum via the Golgi apparatus, while substances to be digested are fed in by at least three paths depending on their source: 1. Endocytosed molecules are initially delivered in vesicles to small, irregularly shaped intracellular organelles called early endosomes. Some of these ingested molecules are selectively retrieved and recycled to the plasma membrane, while others pass onto late endosomes. It is here that endocytosed molecules first meet lysosomal hydolases, which are delivered to the endosome from the golgi apparatus. The interior of the late endosome is mildly acidic, pH 6, and it is the site where the hydrolytic digestion of endocytosed molecules begins. Mature lysosomes form from the late endosomes, accompanied by a further decrease in internal pH. Lysosomes are thought to be produced by a gradual maturation process, during which endosomal membrane proteins are selectively retrieved from the developing lysosome by transport vesicles that deliver these proteins back to endosomes or the trans golgi network. 2. A second pathway to degradation of lysosomes is used in all cell types for disposal of obsolete parts of the cell called autophagy. This process begins with the enclosure of an organelle by membranes of unknown origin, creating an autophagosome, which then fuses with a lysosome. This process is highly regulated, and selected cell components can somehow be marked for lysosomal destruction during cell remodeling. Example the smooth endoplasmic reticulum that proliferates in a liver in response to the drug Phenobarbital is selectively removed by autophagy after the drug is withdrawn. 3. The third pathway that brings materials to lysosomes for degradation is found mainly in cells specialized for the phagocytosis of large particles and microorganisms. Such phagocytes engulf objects to form a phagosome which is then converted to a lysosome in a mechanism similar to that of the autophagosome. Lysosomal hydrolases are synthesized in the rough endoplasmic reticulum and transported through the Golgi apparatus to the trans-Golgi network. The transport vesicles that deliver these proteins to late endosomes bud from the trans-Golgi network. The lysosomal proteins are recognized and selected in the trans Golgi network as they carry a unique marker in the form of mannose-6-phosphate, which is added exclusively to the N-linked oligosaccharides of these soluble lysosomal enzymes as they pass through the lumen of the cis-golgi network. The mannose-6-phosphates are recognized by transmembrane mannose-6-phosphate receptor proteins, which are present in the trans-Golgi network. The receptor proteins bind to lysosomal hydrolases on the luminal side of the membrane and to adaptins in assembling clathrin coats on the cytosolic side. In this way the hydrolases are packaged into clathrin coated vesicles that bud from the trans-golgi network and are delivered to the late endosome. The mannose-6- phosphate receptor protein binds its specific oligosaccharide at pH 6.5-6.7 in the trans Golgi network and it releases it at pH 6, which is the interior of the late endosomes. Having released their bound enzymes, the mannose-6-phosphate receptors are retrieved into transport vesicles that bud

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from late endosomes; the receptors are then returned to the membrane of the trans Golgi network for reuse. The hydrolases have a specific recognition patch which marks them for the addition of mannose-6phosphate groups. This recognition signal is a cluster of neighboring amino acids on each proteins surface. Two enzymes act sequentially to catalyze addition of mannose-6-phosphate groups to lysosomal hydrolases. The first is a GlcNAc-phosphotransferase that specifically binds the hydrolases and adds GlcNAc phosphate to one or two of the mannose residues on each oligosaccharide chain. A second enzyme then cleaves off the GlcNac residue, leaving behind a newly created Mannose-6-phosphate marker. Since most lysosomal hydrolases contain multiple oligosaccharides they acquire many M6P residues, providing a high affinity signal for the M6P receptor.

7:2 LYSOSOMAL STORAGE DISEASES

These are caused by genetic defects that affect one or more of the lysosomal hydrolases. The defects result in the accumulation of the undigested substrates in lysosomes, with severe pathological consequences, most often in the nervous system. In most cases there is a mutation in a structural gene that codes for an individual lysosomal hydrolases. Sphingolipidoses: characterized by the accumulation of various form of sphingolipids, which are intermediates in glycolipid degradation. DISEASE Gauchers disease ENZYME DEFECT Glucocerebroside-glucosidase STORED SUBSTANCE Glucocerebroside

Niemann-Pick disease Types A and B Types C and D GM1 gangliosidosis GM2 gangliosidosis Tay-Sachs disease Sandhoff disease Fabrys disease Farbers disease Krabbes leucodystrophy Hexosaminidase A Hexosaminidase A and B -Galactosidase Acid ceramidase Galactocerebroside-GM2 gangliosides and related glycolipids; oligosaccharides also in Sandhoff disease Ceramide trihexosides Ceramide Galactocerebroside Page | 27 Sphingomyelinase Uncertain -Galactosidase Sphingomyelin, and glycolipid GM1 gangliosides cholesterol

galactosidase

The mucopolysaccharidoses (MPS): This is a group of disorder characterized by the intracellular deposition and increased urinary excretion of mucopolysaccharides DISEASE ENZYME DEFECT STORAGE/URINARY PRODUCT

I-H (Hurler) I-S (Scheie) II (Hunter) linked) III (Sanfilippo) -types A to D

-L-Iduronidase Dermatan sulphate, Heparan sulphate

(X- Iduronate sulphamidase

A)Heparan sulphate sulphamidase B) -N-acetylglucosaminidase C)Acetyl CoA:aminodeoxyglucoside N- Heparan sulphate acetyltransferase D)N-acetylglucosamine-6-sulphatase

IV (Morquio) -types A and D

A) N-acetylgalactosamine-6-sulphatase B)-Galactosidase

Keratan sulphate

VI (Maroteaux- N-acetylgalactosamine-4-sulphatase Lamy VII (Sly) -glucuronidase

Dermatan sulphate

Dermatan sulphate and heparin sulphate

Mucolipidoses (ML): Here a defect prevents addition of the mannose-6-phosphate recognition marker to lysosomal enzymes in the rough endoplasmic reticulum; without this marker, the enzyme cannot be packaged into lysosomes and so there is an enzyme lack. Inclusion cell disease (ML-II): This is a rare disorder in which almost all hydrolytic enzymes are missing from the lysosomes of fibroblasts and their undigested substrates accumulate in lysosomes, which consequently form large inclusions in the patients cells. This is due to a recessive form of a single gene defect, causing all the hydrolases to be

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secreted rather than transported to lysosomes. This is due to a defective or missing GlcNAcphosphotransferase, causing the hydrolases not to be phosphorylated. DISEASE II (I-cell disease) ENZYME DEFECT CLINICAL FEATURES

Uridine diphosphate-N- Hurler-like acetylglucosamine: lysosomal III (Pseudo-Hurler enzyme precursor N- Hurler-like; polydystrophy) acetlyglucosamine phosphate than II transferase IV Uncertain; sialidase possibly

much

milder

ganglioside Psychomotor retardation; severe visual impairment

The glycoproteinoses: this is a group of lesions characterized by deficiencies of enzymes involved in the degradation of the oligosaccharide side chains of glycoproteins. They include sialidosis, mannosidosis, fucosidosis and aspartylglycosaminuria. The clinical features include abnormal facies, mental retardation, and hepatosplenomegaly and in most types, vacuolated lymphocytes are present in circulation.

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8:0 REFERENCES
1. Pavelk M, Mironov AA (2008). The Golgi Apparatus: State of the art 110 years after Camillo Golgi's discovery. Berlin: Springer. ISBN 3-211-76309-0. 2. Davidson MW (2004-12-13). "The Golgi Apparatus". Molecular Expressions. Florida State University. http://micro.magnet.fsu.edu/cells/golgi/golgiapparatus.html. Retrieved 2010-09-20. 3. Wolfe SA (1993). Molecular and Cellular Biology. Belmont, CA: Wadsworth Pub. Co. p. 828. ISBN 0-534-12408-9. 4. Krieger M, Scott MP, Matsudaira PT, Lodish HF, Darnell JE. Lawrence Z, Kaiser C, Arnold B (2004). Molecular cell biology (5th edn ed.). New York: W.H. Freeman and CO. 5. Prydz K, Dalen KT (January 2000). "Synthesis and sorting of proteoglycans". J. Cell. Sci. 113 Pt 2: 193205. PMID 10633071. 6. Lodish, H; Berk A, Matsudaira P, Kaiser CA, Krieger M, Scott MP, Zipursky SL, Darnell J. (2004). Molecular Cell Biology (5th ed.). New York: WH Freeman. ISBN 0716726726. 7. Bruce Alberts, Alexander Johnson, Julian Lewis, Martin Raff, Keith Roberts, Peter Walter, ed (2002). Molecular Biology of the Cell, Chapter 4, pages 191-234 (4th ed.). Garland Science. 8. Lamond, Angus I.; William C. Earnshaw (1998-04-24). "Structure and Function in the Nucleus". Science 280: 547553. 9. Kurz, A; Lampel, S; Nickolenko, JE; Bradl, J; Benner, A; Zirbel, RM; Cremer, T; Lichter, P (1996). "Active and inactive genes localize preferentially in the periphery of chromosome territories". The Journal of Cell Biology (The Rockefeller University Press) 135 (5): 11951205. 10. Porter KR, Claude A, Fullam EF (March 1945). "A study of tissue culture cells by electron microscopy". J Exp Med. 81 (3): 233246. doi:10.1084/jem.81.3.233. PMID 19871454 11. Campbell, Neil A. (1996) Biology Fourth Edition. Benjamin/Cummings Publishing, pp. 120-121 12. Lodish, Harvey, et al. (2003) Molecular Cell Biology 5th Edition. W. H. Freeman, pp. 659-666 13. Levine T, Loewen C (August 2006). "Inter-organelle membrane contact sites: through a glass, darkly". Curr. Opin. Cell Biol. 18 (4): 3718. 14. Maxfield FR, Wstner D (October 2002). "Intracellular cholesterol transport". J. Clin. Invest. 110 (7): 8918. 15. Toyoshima C, Nakasako M, Nomura H, Ogawa H (2000). "Crystal structure of the calcium pumps of sarcoplasmic reticulum at 2.6 A resolution". Nature 405 (6787): 64755. 16. Henze K, Martin W (2003). "Evolutionary biology: essence of mitochondria". Nature 426 (6963): 1278. 17. McBride HM, Neuspiel M, Wasiak S (2006). "Mitochondria: more than just a powerhouse". Curr. Biol. 16 (14): R551. 18. Gardner A, Boles RG (2005). "Is a "Mitochondrial Psychiatry" in the Future? A Review". Curr. Psychiatry Review 1 (3): 255271. 19. Alberts, Bruce; Alexander Johnson, Julian Lewis, Martin Raff, Keith Roberts, Peter Walter (1994). Molecular Biology of the Cell. New York: Garland Publishing Inc. 20. Voet, Donald; Judith G. Voet, Charlotte W. Pratt (2006). Fundamentals of Biochemistry, 2nd Edition. John Wiley and Sons, Inc. pp. 547. 21. Taylor SW, Fahy E, Zhang B, Glenn GM, Warnock DE, Wiley S, Murphy AN, Gaucher SP, Capaldi RA, Gibson BW, Ghosh SS (2003 March). "Characterization of the human heart mitochondrial proteome". Nat Biotechnol. 21 (3): 2816.

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