Mitochondria are double membrane-bound cytoplasmic organelles found in all eukaryotic cells. They are able to respire to generate the energy-rich molecule, Adenosine triphosphate (ATP) from breakdown of carbohydrates and fatty acids utilizing various enzymes and electron transport system. The ATP molecule is crucial to full fil the cellular functions.
- Cytochrome-C is the marker protein of whole mitochondria.
- Monoamine oxidase is very important enzyme present in outer This enzyme has been considered as biomarker for outer membrane.
- Cytochrome oxidase is the marker enzyme for mitochondria inner membrane.
- Malate dehydrogenase is the marker enzyme of mitochondrial
- Adenylate kinase is the marker enzyme of inter membrane space
- Mitochondrial matrix contains several enzymes, these are very important in beta oxidation of fats.
- Cardiolipin (diphosphatidyl glycerol) is restricted to the inner mitochondrial membrane and bacterial plasma membrane.
- Mitochondria is considered as semi-autonomous organelles, since they can synthesize 10% of their proteins using their genome.
- Mitochondrial membrane has specialized function in which phosphorylated compounds (eg. ATP and ATP) can penetrate through it, but this feature is not found in any other membranes (plasma membrane or other organelle membranes).
- Cytochrome-C is a molecular chronometer because it is employed in evolutionary studies to determine the timing of evolutionary events.
Mainly two cellular organelles of eukaryotes such as mitochondria and chloroplasts originated by symbiogenesis as bacterial endosymbionts.
Mitochondria are power house of the eukaryotic cells and first discovered by kolliler from straighted muscle cells of insects, hence named as Sarcosomes. However, different scientists called the mitochondria in different names, Altman called them as bioplast, fleming called them as flia and finally cristian coined the term ‘Mitochondria’ (mito means thread and chondria means body).
Mitochondria contain various types of enzymes, which mainly involved in the respiration and energy transduction. Mitochondria present in all types of eukaryotic cells except red blood cells (erythrocytes), and primitive anaerobic eukaryotes such as Microsporidia and Giardia.
In evolutionary course mitochondria gave rise two organelles.
a). Mitosome/crypton/krypton (Mitochondrion-Derived Organelle), these are identified in Entamoeba histolytica
b). Hydrogenosomes, these are found in trichomonads, anaerobic ciliates and fungi. They can produce molecular hydrogen and ATP.
Mitochondria membrane-bound organelle and dynamic, plastic as well as mobile in nature. Each mitochondrion is a double-membrane structure and contains four compartments such as outer membrane, inner membrane, intermembrane space and matrix.
Mitochondria variable in shape, that may be spherical, filamentous, chain form, sausage or cylindrical. Interestingly in many cells’ mitochondria looks like continuous form as endoplasmic reticulum hence the name was mitochondrial reticulum. This continuous form undergoes constant organization and reorganization. Mitochondrial reticulum shows regular fission and fusion.
Fusion is mediated by mitofusin, FPZO proteins. Fission is mediated by dynamin-related proteins such as Drp-1 and Dnm-1. The morphology and distribution of mitochondrial reticulum is important for cellular differentiation and function. The mutation in fusion or fission results into developing the abnormities. For instant, in Drosophila, a mutant prevents mitochondrial fusion leading to extensive fragmentation of the reticulum. This mutation blocks the spermatogenesis and cause male sterility.
The degree of function of any cell mainly depends on mass-range of mitochondria. Mitochondria mass is present more at those cells where the energy demand is high.
Mitochondria mass is abundant in actively metabolizing cells, flight muscle cells of birds, cardiac muscle cells, liver cells, and sperm cells, etc. In muscle cells, more mitochondria mass present near myofibrils. In flagellated cells, more mass present beneath the basal body.
In sperm the more mass present as a sheath is found in mid piece or connecting piece. However mitochondrial mass in the cells is variable with physiological conditions.
Prolonged exercise increases mitochondrial mass in muscle cells. Hogeboom provided experimental evidence for the first time as mitochondria play role in respiration.
3. Mitochondrial compartments
a) Mitochondrial outer membrane
The mitochondrial outer and inner membrane are closer to each other at a particular site, which called as contact side or contact zones.
Monoamine oxidase is a very important enzyme present in the outer membrane. This enzyme has been considered as a biomarker for outer membrane.
Outer membrane also contains flavonoid, rotenone (pet poison) insensitive NADH dehydrogenase complex.
The outer membrane is permeable to several metabolites and molecules as well as some ions. These molecules can enter only if they are less than 5kd mass.
Prions are trimeric channels present in the outer membrane and involved in the permeability of above-mentioned molecules.
Structurally, prions are composed of beta plated sheet configuration and present on outer membrane of mitochondria as well as outer membrane of prokaryotic organisms such as gram-negative bacteria and chloroplast.
Outer membrane also contains cholesterol, which involved in regulation of membrane dynamics.
b) Mitochondrial inner membrane
The mitochondrial inner membrane is more complex form and folded into many times and separates the matrix and inter membrane space, known as cristae. These folds mainly formed to increase the surface area inside the organelle.
The inner membrane has an electric potential ranging from -160 to -120mv. The range of electrical potential in inner membrane is mainly due to proton electro chemical gradient. This energy potential can be generated when protons are shifted from matrix (lower) to inner membrane space (higher).
This membrane potential is an integral component of proton motive force which contributes to ATP synthesis.
Inner membrane is composition of 72 to 77% proteins and 28 to 23% lipids and 0% of carbohydrates. About 20% of the membrane lipids constitute of Cardiolipin (diphosphitidile glycerol).
Interestingly, cardiolipin also found on bacterial plasma membrane. Cardiolipin is involved in quaternary organization of complex-III and also involved in function of complex IV to promote ATP synthase.
Cardiolipin also involved in proton hopping and contributes to ATP synthesis. It also plays a crucial role in programmed cell death.
The inner membrane contains different types of transmembrane complexes (proteins). These complexes involved in electron transport.
NADH dehydrogenase complex (complex-I)
Succinate dehydrogenase complex (Complex-II)
Cytochrome bc complex (complex -III)
Cytochrome oxidase or terminal oxidase (complex -IV)
ATP synthase complex (complex -V)
Cytochrome oxidase is the marker enzyme for mitochondria inner membrane.
Cytochrome –C is the marker protein of mitochondria.
c) Mitochondrial intermembrane space
The double membranes (outer and inner membranes) of the mitochondria creats some space between them, called as intermembrane space. This space is a composition, which is similar to cytoplasm. The proteins present in this space distinct from the other part of the mitochondria. This intermembrane space is continuous into the core of the crests or cristae.
d) Mitochondrial matrix/mitosol
Mitochondrial matrix is liquid area contains a complex mixture of soluble proteins and enzymes and enclosed by the inner membrane. The enzymes present in the matrix are involved in the oxidation and reduction processes. Enzymes of Krebs cycle present in matrix. These enzymes are important to synthesize the ATP molecules, mitochondrial ribosomes, tRNAs, and mitochondrial DNA.
Mitochondrial matrix is alkaline (pH range between 7.4 to 8) compared to inter membrane space (pH is 6.8 which is slightly acidic).
Matrix contains enzymes involved in the conversion of Pyruvate to acetyl co.A and Krebs cycle.
The Krebs cycle enzyme succinate dehydrogenase is found on the inner membrane and is an integral component of electron transport system.
Malate dehydrogenase is the marker enzyme of the mitochondrial matrix.
Mitochondrial matrix contains several enzymes, these are very important in beta-oxidation of fats.
The mitochondrial matrix contains 70s ribosomes. The ribosome in mitochondria of human cell is 55s type, called Mitoribosomes. The 55s ribosome divide into two subunits such as large 25s and small 35s subunit. 25s contains 12s rRNA and 35s subunit contains 5s rRNA and 16s rRNA.
Matrix also contains genetic material which is anchored to inner membrane usually. Mitochondrion can synthesize 10% percent of its proteins using its protein-synthetic machinery, hence mitochondria considered as semi-autonomous organelles.
4. Mitochondria function in the cell
- The main role of mitochondria in the cell is to use the glucose and produce the energy in the form of chemical bonds with the ATP molecule this mechanism called cellular respiration. Overall three main steps involved in this process such as glycolysis, Krebs or citric acid cycle, and ATP synthesis. The produced ATP is readily available to broken down by other cellular organelles to use energy for their own activities.
- Mitochondria can also produce heat in brown fat (thermogenesis),
- Accumulate iron-containing pigments (Heme ferritin), ions of Ca2+ and HPO42- (or phosphate; e.g., osteoblasts of bones or yolk proteins).
- Involved in cellular respiration or oxidation (not in photosynthesis)
- Involved in the conversion of pyruvate to acetyl co. A
- Involved in Krebs cycle which occurs in the matrix
- The inner membrane of mitochondria involved in electron transport system and oxidative phosphorylation.
- Involved in regulation of redox status of the cell.
- Involved in beta-oxidation of fats.
- Some steps of the steroid hormone (aldosterone and cortisol) biosynthesis occur in mitochondria.
- Involved in the urea
- Involved in few steps of heme biosynthesis.
- Involved in detoxification of ammonia in liver.
- Involved in degradation of neurotransmitter which is the function of outer
- Involved in the degradation of tryptophan.
- Involved in phosphorylation of nucleotides.
- Involved in non-shivering thermogenesis in hibernating animal neonates.
- Mitochondria are involved in PCD or Apoptosis.
- The release of cytochrome -C from mitochondria leads to PCD.
- The brown adipose tissue contains a specialized mitochondrion, which have natural uncoupler called Thermogenin or Uncoupling protein-I (UCP-I).
5. How do heat generate during a cold condition of the body?
Coldà HypothalamusàNor epinephrineàGPCR (present on brown adipose cell plasma membrane)àcAMPàFatty acidsàFatty acids enter mitochondriaàstimulation of thermogenesis (Thermogenesis).
6. Mitochondria genome
Mitochondrial genome is multiple copy number. A few mitochondrial DNA molecules from cluster called mitochondrial nucleoid.
Mitochondrial genome is circular in configuration. Some mitochondrial genomes are linear in configuration.
Example: Plasmodium, Tetrahymena, Amoebedium, and clamidomonas.
In trypanosome, a disc shaped mass of circular DNA molecules are found and which are called as kinetoplast. The mitochondrial genome size is various in some organisms.
6 kb of genome size present in mitochondria of plasmodium (smallest mitochondrial genome).
Human have 16.8 kb mitochondrial genome.
Animals have less than 30 kb, yeast cells have 78 kb and plants have 100 kb to 2mb mitochondrial genome.
The majority portion of plant-mitochondrial genome is composed of junk DNA. 90% of the genome has repeat sequences and also contains introns, pseudogenes, insertion elements, viral genes, chloroplast genes, and nuclear genes.
Fungal mitochondrial genome also contains introns; however, mammalian mitochondrial genome does not contain introns.
7. Laboratory techniques for mitochondrial study
a) Microscopic observation of mitochondria
Micheles was the first person to stain mitochondria using Janus green-B.
Mitochondria can be observed by phase contrast microscope and interference microscope.
Rhodamine is a fluorescent dye which is more sensitive than other dyes and has been used in mitochondria isolating methods of intact cultured cells. These types of stains are more appropriate for in situ mitochondrial metabolic studies.
Mitochondria can also be observed by Green Florescence Protein (GFP)-tagging of mitochondrial proteins, by immune florescence technique employing a mitochondrial specific dye called DIOC-6.
The dynamic nature of the mitochondrial reticulum can be observed by using Time-lopse florescence microscopy and Time-lopse microsinamatic microscopy.
Digitanin is a detergent, by using this we can remove the outer membrane of mitochondria and remaining parts such as inner membrane and matrix jointly termed as Mitoplast.
b) Cytochemical Marking of Mitochondrial Enzymes
Mitochondria have distinct marker enzymes in their different compartments distinctly for histochemical markings, such as monoamine oxidase for outer membrane, cytochrome oxidase for inner membrane, adenylate kinase for outer chamber, and malate dehydrogenase for matrix.
Mitochondria can be easily isolated from cell fractionations using differential centrifugation.
Homogeneous fractions of mitochondria have been obtained from heart, liver, skeletal muscle, and some other tissues. In differential centrifugation mitochondria sediment at 5000 to 24000 g, while in living cells at the ultracentrifugation (20,000 to 400,000 g) mitochondria are deposited intact at the centrifugal pole.
7. Scientific tip for membrane protein
Mostly detergents such as lubrol or some other organic solvents may be used in laboratory to inner membrane in order to get membrane fragments into vesicles, called submitochondrial vesicles.
These vesicles are produced in inverted position, hence F0-F1 particles are seen outer surface of the vesicles.
In most of these vesicles the complexes of electron transport and synthase have a reverse orientation. ATP synthase in this orientation exhibits ATPase activity.
These vesicles enable as to manipulate the levels of small molecules such as NADH and FADH2 and study the energetic and mechanics of electron transport, which is not possible in intact mitochondria.
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