Mitochondria and Peroxisomes Flashcards
what is the appearance of mitochondria?
-2 membranes: outside with outer membrane and inside with inner membrane
-inner membrane is folded into structures called cristae to increase surface area and accommodate many protein complexes
-in between the 2 membranes = intermembrane space
-inner membrane inside is called the matrix with mitochondrial DNA
many mitochondria in the cell and mtDNA in each cell
-2 copies of nuclear DNA but more than 100s of copies of mtDNA
synthesis of ATP
-sugars, fats, and proteins are the first used in the TCA cycle or citric acid cycle in the matrix to create high energy compounds like NADH and FADH2
-used to create electrochemical gradient across the inner membrane using these pumps that pump protons from the matrix to enter membrane space –> force of energy created by mitochondria against the natural [] of the proton
-b/c of the gradient, the proton goes back to matrix using the F1/Fo ATPase and ATP synthase –> this energy creates ATP (complex five)
-in this energy production process, mitochondria use O2 to create ATP –> make smoke reactive oxygen species as a byproduct of electron transport
-taken care by enzymes which detox by reactive oxygen species (ROS)
-when you create a proton gradient, can be used to make ATP but also you can use for other purposes
mitochondrial ATP production
-import ADP to the matrix and also phosphate then after you make ATP, it gets transported out (many transporters)
-ATP is transported using a proton gradient and phosphate is also transported using this gradient
what happens since mitochondrial IM is impermeable?
many special carriers are needed to transport necessary small molecules into/out of the matrix
what are other functions of the mitochondria?
-metabolism of lipids, amino acids, and fatty acids (creates energy and urea degradation), and creates heme
-control our body temp by generating heat
-involved in signalling using Ca2+ and ROS
-controls apoptosis
mitochondria and disease
-general outcome of mitochondrial defect- tissues which are highly demanding for energy are mainly or predominantly affected
-muscle and heart, metabolic disorders, brain and neurons like neurodegeneration
-some types of cancer may be impacted like mutations in complex II that cause pheochromocytoma (neuroendocrine tumor)
what is a method to study mitochondria?
isolation of organelles
isolation of organelles
-break up the cells or tissues mechanically for homogenization –> make mixture of cytosol and broken organelles
-use differential centrifugation to spin down specific organelles based on size and density
-low speed: pellet with big structures like nuclei
-intermediate speed: mitochondria, lysosomes, peroxisomes, and intermediate size density structures
-really high speed: spin down pellet into smaller structures like fragments of ER (microsomes)
-use ultracentrifugation you can start pelleting big protein complexes like ribosomes
fractionations of mitochondria:
- OM- numerous ‘pores’, permeable to small molecules and ions
- IMS: a few proteins and enzymes (cytochrome C)
- IM: much of the OXPHOS machinery like cardiolipin
- Matrix: TCA cycle enzymes, mtDNA
outer membrane (OM)
-have many big pores that are permeable to many small structures
-forming the porin with big pores in OM –> small molecules like ATP or ADP can freely diffuse in and out across the OM
-beta-barrel protein- these types of proteins are found in the mitochondria of the membrane and bacterial membrane –> suggesting link between mitochondria and bacteria in origin
intermembrane space (IMS)
-cytochrome C- protein with 2 major functions: life and death of cells
-functions as a protein subunit of the ETC complex (complex four) –> oxidative phosphorylation machinery that creates proton gradient and ATP
-depending on the conditions cells are in or stimulation they receive, cytochrome C can function in ATP production or cell death
cytochrome C + apoptosis
when cells decide to die, cytochrome C is released from complex four and leads into the cytosol from IMS and binds to different regulators of apoptosis and stimulates or induces apoptosis
inner membrane (IM)
-huge membrane area with many proteins or transporters
-5 different complexes: complex 1-4 generate proton gradient by pumping out protons from the matrix side to the intermembrane space using NADH and FADH2 then complex 5 (ATP synthase) uses this gradient to create ATP from ADP and phosphate
-once ATP is generated in the matrix, the molecule is transported out to intermembrane space and eventually cytosol through porin
matrix (inside IM)
-T cell cycle uses pyruvate (product of glycolysis) as primary source to create high energy compound NADH and FADH2
-high energy compounds are used to create proton gradient across the IM by the ETC
the ‘code’ in mtDNA differs from that in nuclear DNA
-uses different codons from nuclear DNA Ex. AUA as Leucine in nuclear DNA but mitochondria use it for Methionine
-patterns similar to pattern of codons in bacteria- link between mitochondria and bacteria
what are some links between mitochondria and bacteria?
- presence of beta-barrel protein in the mitochondrial membrane and bacterial membrane
- codon patterns of mitochondria are similar to those of bacteria
- both have circular DNA structures
- CL and mitochondrial-specific lipids are also found in bacteria suggesting that precursor of mitochondria during evolution
- mitochondria have two membranes suggesting one from bacteria and one from the host cell plasma membrane
mitochondria have their own DNA (mtDNA) but it encodes only a few proteins
-13 proteins in humans –> all of them are subunits for different ETC complexes
-nuclear DNA is a linear structure but mtDNA is a circular structure
-mtDNA also encode ribosomal RNA and tRNA for protein synthesis in the matrix
-mtDNA tend to accumulate more mutations at increased rate compared to nuclear DNA –> due to inefficient repair and possibly ROS exposure, which can damage DNA
many diseases caused by mutations in mtDNA
-LHON- mutation in the subunit of complex I, ND1, 4, and 6
–> creates defects in protein synthesis, which affects muscles
-if you mutate the gene encoding F1 for ATPase five then that will lead to disease affecting nervous system
-many mitochondrial proteins are encoded by nuclear DNA
mtDNA is inherited differently from nuclear DNA
-mtDNA is only inherited from an oocyte but not sperm or father’s side
-mitochondria and mtDNA are degraded once fertilized and then fusion between oocyte and sperm happens –> only maintain mitochondria and mtDNA derived from the oocyte
-mutations in the oocyte can be inherited but mutation in mtDNA of sperm is not
-maternal inheritance and not 100% penetrance –> since we have many mtDNA with 100s of copies in cells, depending on ratio of mutant mtDNA to WT mtDNA the symptoms and defect outcome can be different
-people found that over 60% of mtDNA had to be mutated to actually start seeing the defects in the body
-WT mtDNA can dominate mutant mtDNA
how do you treat mitochondrial disease caused by a mutation in mtDNA?
-repress bad mitochondria with good mitochondria
-experiment showed you can replace affected mitochondria with healthy mitochondria using patient nuclear DNA –> remove the disease-causing mutation
can eukaryotic cells ever lose all of their mtDNA?
-yes- mtDNA encodes subunit of oxidative phosphorylation but you can generate ATP in other ways to bypass this requirement like through glycolysis
-cells can make mitochondria without mtDNA but cannot do oxidative phosphorylation
although mitochondria contain their own genome, the vast majority of mitochondrial proteins are encoded in the nucleus
-mammalian mitochondria have ~1500 proteins
-mitochondrial proteins are encoded in the nuclear genome and need to be imported into the mitochondria
multi-subunit complexes often contain subunits encoded in mtDNA and others encoded in the nucleus
-Complex I is a huge complex with 46 subunits
-some generated in the matrix and have to be transported to inner membrane
-some are synthesized in the cytosol and transported to the mitochondria and inserted into the inner membrane –> these proteins work together to make a functional complex
nuclear-encoded mitochondrial proteins are translated in the cytosol, what happens after synthesis?
- protein needs to find the mitochondria- targeting signal that allows the precursor proteins to send to mitochondrial surface
- once it reaches the mitochondria, needs to cross the IM and OM- transporters are pores that help protein to cross
- proteins need to be sorted into different compartments
- if in a complex, need to interact with other proteins and assemble
import pathway
-precursor protein begins in spaghetti form in the cytosol with chaperones that help keep it unfolded
-uses a targeting sequence that gets recognized by receptor protein to help the precursor protein find the mitochondria
-once it gets to the mitochondrial surface, the precursor protein needs to cross the OM through the TOM complex
-translocates across the IM through the TIM complex
-precursor is ‘pulled’ through the TOM-TIM complexes by matrix chaperone (PAM or Hsc70) comlex
-presequence is removed by processing protease in the matrix
-imported protein folds with help of matrix chaperones
targeting sequence is the pre-sequence
-many mitochondrial proteins carry N-terminal presequences that are cleaved off after their import
-amino acid sequences need to form alpha helix and one side is hydrophobic while the other side is positively charged
-pattern is important for being recognized by receptor proteins
TOM and TIM machinery
-TOM- translocates outer membrane
-TIM- translocates inner membrane
-many other proteins help –> one of them is a receptor protein- different receptor proteins to recognize slightly different presequence but channels are coupled to receptor protein
-TOM and TIM complexes form large, aqueous pores in the outer and inner membrane
-size of pore is very small- size of unfolded amino acid chain and means the proteins need to go through this pore unfolded and not folded –> helped by chaperone proteins
chaperones play roles both inside and outside organelles
-cytoplasmic chaperones keep precursor protein unfolded after translation
-during translocation across the OM and IM through TOM and TIM, chaperone keep the precursors straight
-once they reach the matrix, another type of chaperone (matrix chaperone) helps protein to be properly folded into functional structures
-both cytosolic and mitochondria use ATP to do this
-cyotoplasmic chaperones also help to prevent aggregation –> misfolded proteins tend to aggregate from hydrophobic part and tend to bind to each other
different mechanisms to send the protein into the matrix, IM, and OM
-in the case of the matrix protein, they use TOM and TIM23 combo to drive protein into the matrix
-if you use SAM after TOM, it will lead to sorting into the outer membrane
-if you use another TIM22 after TOM, the protein can go to the inner membrane
mitochondrial protein import and disease
-if you cannot properly form mitochondria and cannot import proteins that are necessary, you will create defective mitochondria
Ex. TIM22 is important for IM sorting- you can have issues with progressive blindness and if you mutate another TIM important for matrix protein import, can lead to heart disease
mitochondrial lipids
-most lipids in cells are made in the ER
-lipid used to generate mitochondria first produce in the ER and need to be imported into mitochondria –> lipid transport process happens at physical contact site between ER and mitochondria interact
-connection between two membranes allow the lipid to transport between them and lipid generated in the ER can be transported to another membrane and can be bidirectional
cardiolipin, a unique phospholipid, is made through steps controlled by several enzymes in the mitochondrial IM and required for the activity of many IM protein complexes
-precursor of CL is phosphatidic acid
-PA is transported to OM and IM using contact site between ER and OM
-once the PA is in the IM, several steps occur to create cardiolipin and some gets transported to the OM
-CL is a unique phospholipid –> usually phospholipids have one head and two tails that are hydrophobic but CL is a dimer of phospholipid with four tails and one head
-if you don’t have CL, you reduce the amount of functional super complexes and reduce effective ATP production
CL and disease
-defect in enzyme for creating CL can lead to X-linked disease in heart
-young boys have growth defects, neutropenia, and develop severe cardiomyopathy
mitochondria in different cell types can have different shapes and functions
-hepatocytes in the liver tend to show round structures but if you look at the pancreatic acinar cells that secrete many enzymes, elongated mitochondria
-if you look at the heart muscles, mitochondria are sitting next to myofibrils to provide ATP effectively
-if you look at sperm, mitochondria are enriched at the base of flagellum and provide ATP for sperm to swim
-if you look at apical epithelial cells in the gut, you can see many mitochondria are enriched at the apical surface
-in neurons, longer cells are mitochondria that can go up and down the axon
-another type of movement is diffusion and division- they can fuse and divide to control their site and morphology
mitochondria fusion and division control #, size, and shape of mitochondria
-WT yeast mitochondria have tubular structures that looks like human mitochondria but if you block mitochondrial fusion, mitochondria now continue to divide and create small structures
-these mitochondria aren’t happy since they lose mtDNA and create empty mitochondria that are defective
-if you block mitochondrial division, you keep fusing mitochondria and create big fishnet structures –> now mitochondria are hard to move in the cells
-GTPases control the fusion and division processes associated with mitochondria- DRPI assemble onto the surface of mitochondria and create wraparound structure –> they use GTP hydrolysis to have the filaments cut the mitochondria and be fused back again by fusing OM with IM using another set of GTPases
mitochondrial dynamics and quality control
-fusing helps to mix mitochondrial contents and exchange between different mitochondria to maintain homeostasis
-division is part of quality control to remove damaged mitochondria to be degraded
-once you reach certain size, mitochondria divide to keep size down
-defect in GTPases are linked to human diseases in parts like eyes and sensory neurons
origin of mitochondria (endosymbiotic theory)
idea is that cells that did not have intercellular organelles engulfed bacteria and used parts of their activities to make energy ATP –> this may be why you see two membranes in mitochondria (one from bacteria and other is host cell plasma membrane)
peroxisomes look very different from mitochondria
-many peroxisomes, like many mitochondria, in each cell and they only have a single membrane
-do not contain DNA
peroxisome functions
-function in breaking down lipids in a process called beta oxidation to extract energy from lipids
-they can also make lipid plasmalogen, a major lipid in myelin sheaths
-create bile acids in liver that takes up hydrophobic molecules in the body
peroxisomes contain a number of oxidases
-during beta oxidation process, peroxisomes also create ROS and they have defense mechanisms
-they also have enzymes called catalase, which convert toxic reactive oxygen species hydrogen peroxide into water and oxygen
-peroxisomes can protect toxic ROS generated by themselves by making nontoxic molecules
-system can help with mitochondria- peroxisomes use this to detoxify the mitochondria and generated hydrogen peroxide and break it down into water and oxygen
peroxisomal import machinery
-peroxisomes do not have DNA –> all the peroxisome proteins need to be imported
-precursor proteins have their own version of targeting sequences- 3 amino acids are important and sufficient to target protein to peroxisome and its receptors called PTS
-most peroxisomal matrix proteins carry a C-terminal import signal (PTS1) consisting of ser-lys-leu (SKL)
-another signal (PTS2) is located at the N-terminus and only a few proteins have this
-PTS1 can be recognized by receptor –> in this case, receptor is in the cytosol (not on the surface of peroxisomes) and complex can be targeted to another protein on the surface of peroxisomes then handed to transporters
-peroxisomes have big pores and proteins don’t have to be unfolded and the chaperones aren’t necessary
-depending on precursor type of protein you want to import, the pore can be small or big using assembly of subunits of pore complex
differences between peroxisomes and mitochondria
- mitochondria have tiny pores so the proteins need to be unfolded and need chaperones, while peroxisomes have big pores and the proteins do not have to be unfolded
- mitochondria have mtDNA to make some proteins, while peroxisomes have to import all of their proteins
- mitochondria need existing mitochondria to be created and peroxisomes can be generated de novo
- mitochondria has two membranes and peroxisomes have one
peroxisomal assembly
de novo biogenesis:
-peroxisomes can be generated de novo because they can be generated by the ER but you cannot make mitochondria de novo (need existing mitochondria)
-it has been shown that the ER can create tiny precursor vesicles or peroxisomes that are not mature and mini version of peroxisome coming out of ER
growth and division:
-you import other components into precursor peroxisomes and make mature versions of peroxisomes
-once they reach a certain size they divide and control the size and increase the # for cells to divide
peroxisomal diseases
- lack of enzyme functional protein in peroxisomes
- defect in peroxisomal protein import (Zellwegger syndrome)
-can create peroxisomes but they aren’t fully functional- empty and cannot import functional components
mitochondria and peroxisomes: protein import
-signals on imported protein
-signal recognition (receptors)
-translocation across membranes via pores (translocons)
-sorting to soluble or membrane compartment
