Lecture 3 (mitochondria)
Describe the structure and function of mitochondria*
functions: ATP synthesis Citric acid cycle Oxidation of pyruvate and fatty acids Heme biosynthesis Amino acid metabolism Role in apoptotic cell death structure: 2 membranes (inner, outer) two aqueous compartments (matrix, inter membrane space)
high energy demand
mitochondria provide energy Localization of mitochondria near sites of high ATP demand in cardiac muscle and a sperm tail. (A) Cardiac muscle in the wall of the heart is the most heavily used muscle in the body, and its continual contractions require a reliable energy supply. It has limited built-in energy stores and has to depend on a steady supply of ATP from the copious mitochondria aligned close to the contractile myofibrils (see Figure 16-32). (B) During sperm development, microtubules wind helically around the flagellar axoneme, where they are thought to help localize the mitochondria in the tail to produce the structure shown.
Distinguish between outer and inner membrane*
outer: Contains porins (aqueous pores) Channels comprised of identical protein trimers β-strands form a sheet that twists into a β-barrel Permeable to molecules less than 5000 D is freely permeable to ions and to small molecules because it has porins envelops the inner boundary membrane. Also include proteins for: Lipid synthesis Mitochondrial protein import Mitochondria fission and fusion Intermembrane space (between outer and inner) is chemically equivalent to cytosol in pH inner: Surrounds the internal mitochondrial matrix compartment Is highly folded to form invaginations known as cristae ( which contain in their membranes the proteins of the electron-transport chain; increases surface area) Membrane is a diffusion barrier to ions and small molecules (selected ions, most notably protons and phosphate, as well as essential metabolites such as ATP and ADP, can pass through it by means of special transport proteins) is highly folded into tubular or lamellar cristae, which crisscross the matrix Major functions: -Electron transport chain -ATP synthesis -Metabolite transport Has high cardiolipin: allows membrane potential to be established -"Double" phospholipid -Decreases membrane permeability -Results in a chemically distinct matrix Is highly differentiated into functionally distinct regions with different protein compositions.
mitochondrial dynamics & aging
role mitochondrial fusion: most likely to ensure an even distribution of mitochondrial DNA throughout the mitochondrial reticulum, and to prevent the accumulation of damaged DNA in one part of the network. When the fusion machinery is defective, DNA is lost from a subset of the mitochondria in the cell. Loss of mitochondrial DNA leads to a loss of respiratory-chain function, and it can cause disease. Fusion-fission cycle: fusion: increase in mass of mitochondria; preceded by biogenesis (synthesis); leads to fused mitochondria, then fission fission: increase in number of mitochondria; preceded by mitophagy (degradation); leads to fissioned mito. then fusion when balanced, create healthy mitochondria imbalance= aging, leads to split between -fragmented mito (fusion<fission; decreases: complementation, mtDNA integrity, biogenesis (mass); increases: oxidative stress) -enlarged mito (fusion>fission; decreases: mitophagy, biogenesis (mass); increases: damaged mito, oxidative stress) Results in mito crisis (decrease: Ca2+ regulation and respiration) and eventually apoptosis
mitochondria life cycle
starts with growth and division of pre-existing organelles (biogenesis) and ends with degradation of impaired or surplus organelles by mitophagy (turnover). In between, mitochondria undergo frequent cycles of fusion and fission that allow the cell to generate multiple heterogeneous mitochondria or interconnected mitochondrial networks, depending on the physiological conditions. Fusion and fission of mitochondria are important for many biological functions. Division is required for inheritance and partitioning of organelles during cell division, for the release of pro-apoptotic factors from the intermembrane space, for intracellular distribution by cytoskeleton-mediated transport and for turnover of damaged organelles by mitophagy. Fused mitochondrial networks are important for the dissipation of metabolic energy through transmission of membrane potential along mitochondrial filaments and for the complementation of mitochondrial DNA (mtDNA) gene products in heteroplasmic cells to counteract decline of respiratory functions in ageing (X and Y depict alleles of different mitochondrial genes).
adv of mitochondria
Acquisition of mitochondria was a prerequisite for evolution of complex animals. Generates > 15-times more ATP energy than anaerobic glycolysis 1 molecule of glucose--> 2 molecules of ATP (anaerobic glycolysis) OR--> 36 molecules of ATP (aerobic oxidation) Are inherited from the mother Occupy ~ 20% of cytoplasmic volume in the cell Varying numbers; 2-2500 per human cell Dynamic and plastic: -Move around the cell -Change shape -Divide -Fuse together The mitochondria can reproduce by growing larger and then dividing, when the cell needs more energy If the cell needs less energy, the mitochondria will die
Cancer cells
Cancer cells have altered metabolism in mito: -Suppressed oxidative phosphorylation -Enhanced glycolysis Mitochondria reprogramming in cancer: -Suppressed apoptosis -Overproduction of Reactive Oxygen Species ROS Cancer cells reprogram metabolic state of mitochondria by acidifying the environment, resulting in increased: blood volume and turbulence, and decreased: blood flow and O2 tension
Disease
Damage of mtDNA may induce diverse diseases MtDNA damage and mutation can be induced by cell stress from environmental particulates and/or DNA abnormality. MtDNA damage/mutation cause mitochondrial dysfunction reducing bioenergeneric metabolism that can promote degenerative diseases, metabolic dysfunction, aging, apoptosis and cancer. Cell stress + nDNA/mtDNA abnormality-->mito damage/mutation--> mito dysfunction (decrease bioenergetic metabolism)--> degenerative diseases (kidney and heart failure), aging/apoptosis, abnormal inflammation/immunity, cancer, metabolic dysfunction (diabetes, obesity) Due to mitotic segregation, some cells will accumulate higher levels of faulty mitochondrial DNA than others. Above some threshold, serious deficiencies in respiratory-chain function will develop, producing cells that are senescent (aging) these diseases are recognized by their passage from affected mothers to both their daughters and their sons, with the daughters but not the sons producing children with the disease. A factor in this inevitable process is the accumulation of deletions and point mutations in mitochondrial DNA. Oxidative damage to the cell by reactive oxygen species (ROS) such as H2O2, superoxide, or hydroxyl radicals also increases with age. The mitochondrial respiratory chain is the main source of ROS in animal cells, and animals in which mitochondrial superoxide dismutase—the main ROS scavenger—has been knocked out, die prematurely. If there is a mutation in the genes that code for mitochondrial proteins, decreased ATP production leads to energy failure of the cell and, eventually, to the organ. Many different organs may be involved. In general, the organs that require the most ATP are the ones with symptoms. These include: colon, inner ear, eye, kidney, pancreas, blood, liver, heart, skeletal muscle, brain, nuclear DNA
human mitochondrial genome
Densely packed with genes -Cellular respiration: ATP synthase, NADH dehydrogenase, and cytochrome oxidase -Gene expression: rRNA and tRNA Only 13 protein coding genes in genome 16S and 12S are not protein coding book: The human mitochondrial genome of ≈16,600 nucleotide pairs contains 2 rRNA genes, 22 tRNA genes, and 13 protein-coding sequences. There are two transcriptional promoters, one for each strand of the mitochondrial DNA (mtDNA). The DNAs of many other animal mitochondrial genomes have been completely sequenced. Most of these animal mitochondrial DNAs encode precisely the same genes as humans, with the gene order being identical for animals ranging from fish to mammals.
origin of mitochondria
Evolution of mitochondria ~1.6 billions years ago Endosymbiont theory: Mitochondria and chloroplasts evolved from bacteria receives strong support from the observation that the genetic systems of mitochondria and chloroplasts are similar to those of present-day bacteria. In response to the rising oxygen levels, nonphotosynthetic oxygen-consuming organisms evolved, and the concentration of oxygen in the atmosphere equilibrated at its present-day level. it is thought that eukaryotic cells originated through a symbiotic relationship between an archaeon and an aerobic bacterium (a proteobacterium). the two organisms are postulated to have merged to form the ancestor of all nucleated cells, with the archeaon providing the nucleus and the proteobacterium serving as a respiring, ATP-producing endosymbiont—one that would eventually evolve into the mitochondrion. Chloroplasts arrived later. evidence: mitochondria have own DNA, ribosomes; have a double membrane; reproduce by fission; have similar shape to bacteria
Describe key features of mitochondrial genome and highlight differences between nuclear and mitochondrial genome*
Found in nucleoid region of matrix (diff) Usually small circular molecules (diff) No associated histone proteins (diff) •The loss of mtDNA in mammals is incompatible with life. • Broad variations in mtDNA copy number are observed. •Mitochondria can dispose of severely damaged mtDNA and resynthesize new. •mtDNA released into the cytosol or extracellularly plays an important role in innate immunity. Genome: size of mitochondrial genomes does not correlate well with the number of proteins encoded in them; genetic code in different organisms is not the same Multiple copies per mitochondrion Replication occurs throughout cell cycle Size varies between organisms -lots of noncoding DNA in others -Approximately 16,500 bp in mammals Less complex mitochondrial genomes encode subsets of the proteins and ribosomal RNAs that are encoded by larger mitochondrial genomes unique features: Dense gene packing (little noncoding in humans) Relaxed codon usage (less tRNA needed) Variant genetic code (4 different codons)
DNA repair mechanisms
Mitochondria lack efficient DNA repair mechanisms Mitochondria have a major contribution to aging Mitochondrial DNA accumulates mutations at higher rate as compared to nuclear DNA (DNA lacks histones, have less efficient DNA repair) Mitochondria are exposed to higher levels of free radicals* which oxidate and damage proteins and DNA. The less complex DNA replication and repair systems in mitochondria mean that accidents are corrected less efficiently. This results in a 100-fold higher occurrence of deletions and point mutations than in nuclear DNA.
mitochondria & cytoskeleton
Mitochondria tend to be aligned along microtubules, associate with cytoskeleton mitochondria are mobile and can move long distances (up to a meter or more in the extended axons of neurons), being propelled along the tracks of the microtubular cytoskeleton; can be fixed during high energy demand
Mitochondrial proteins
Most are encoded in the nuclear genomes -Must be imported into mitochondria Gene transfer occurred during eukaryote evolution -Some genes may have been "untransferrable" book: Proteins imported into mitochondria are usually taken up from the cytosol within seconds or minutes of their release from ribosomes. thus, in contrast to protein translocation into the ER, which often takes place simultaneously with translation by a ribosome docked on the rough ER membrane, mitochondrial proteins are rst fully synthesized as mitochondrial precursor proteins in the cytosol and then translocated into mitochondria by a post-translational mechanism. note: Why do mitochondria and chloroplasts require their own separate genetic systems, when other organelles that share the same cytoplasm, such as peroxisomes and lysosomes, do not? the highly hydrophobic nature of the nonribosomal proteins encoded by organelle genes. is may make their production in and import from the cytoplasm simply too difficult and energy-consuming.
mitochondrial protein
Most of mitochondrial proteins are encoded by nucleus book: Most of the protein components of the mitochondrial respiratory chain are encoded by nuclear DNA, with only a small number encoded by mitochondrial DNA (mtDNA). Transcription of mtDNA produces 13 mRNAs, all of which encode subunits of the oxidative phosphorylation system, and the 24 RNAs (22 transfer RNAs and 2 ribosomal RNAs) needed for translation of these mRNAs on the mitochondrial ribosomes (brown). The mRNAs produced by transcription of nuclear genes are translated on cytoplasmic ribosomes (green), which are distinct from the mitochondrial ribosomes. The nuclear-encoded mitochondrial proteins (dark green) are imported into mitochondria through two protein translocases called TOM and TIM, and constitute the vast majority of the approximately 1000 different protein species present in mammalian mitochondria. The nuclear-encoded mitochondrial proteins in humans include the majority of the oxidative phosphorylation system subunits, all proteins needed for expression and maintenance of mtDNA, and all proteins of the mitochondrial ribosomes. The mtDNA-encoded subunits (orange) assemble together with the nuclear subunits to form a functional oxidative phosphorylation system. (slide 26)
Disease prevention, IVF and Mitochondrial replacement
One of the therapies that is being investigated includes "donor mitochondria". If a woman has an mtDNA mutation, she has the possibly of passing any amount of mutated mitochondria to her future children. Using an in vitro fertilization technique, the nucleus of the egg (with the mother's DNA) can be placed into a donor egg that has had its nucleus removed. In this way, a woman can have her own biological children with the exception of her own mtDNA. This would reduce the transmission of mutated mtDNA. Replace damaged mitochondria with healthy one of donor Donor provides eggs w/ healthy mitochondria 1. 2 eggs (one from mother w faulty mito and one from donor w healthy mito) are fertilized w sperm; form pronuclei 2. nuclei from donor is removed leaving cells w healthy mito 3. nuclei from mother is put into healthy egg 4. 1-2% of cases diseased mito is carried over
Parkinson's Disease
PD involves progressive degeneration of dopaminergic neurons leading to lack of dopamine in the brain. Mutations in Parkin and PINK1, mitochondria-associated proteins, are frequent causes of PD. Loss of Parkin and PINK1 lead to abnormal mitochondria, decrease in mitochondrial respiration PINK1 is an outer membrane protein that acts as a sensor of mitochondrial membrane potential
PINK1 and PARK2
PINK1 and PARK2 mediate recognition of damaged mitochondria Damaged mitochondria (e.g. with low inner membrane potential) accumulate PINK1 in the OMM PINK1 autophosphorylates and is recognized by parkin (PARK2) Parkin mediates ubiquitination of mitochondrial OM proteins (incl Mfn1 and 2) which prevents their fusion and promotes mitophagy PINK1 and PARK2 mutations are a major cause of Parkinson's disease book: when mitochondria function normally, the inner mitochondrial membrane is energized by an electrochemical H+ gradient that drives ATP synthesis and the import of mitochondrial precursor proteins and metabolites. Damaged mitochondria cannot maintain the gradient, so protein import is blocked. As a consequence, a protein kinase called Pink1, which is normally imported into mitochondria, is instead retained on the mitochondrial surface where it recruits the ubiquitin ligase Parkin from the cytosol. Parkin ubiquitylates mitochondrial outer membrane proteins, which mark the organelle for selective destruction in autophagosomes. Mutations in Pink1 or Parkin cause a form of early-onset Parkinson's disease, a degenerative disorder of the central nervous system. It is not known why the neurons that die prematurely in this disease are particularly reliant on mitophagy.
mitochondrial gene expression
Transcription: Use one nuclear-encoded RNA polymerase One promoter for transcription of both strands Translation: Use fMet-tRNA for protein synthesis Ribosomes sensitive to bacterial inhibitors *only a small % of proteins encoded by mtDNA
