Lecture #3 - Mitochondria and Peroxisomes Flashcards

Question

Pore in outer membrane

Answer 1

Porin creates pores in the outer membrane Pore = allows small molecules (Ex. ATP + ADP + phosphates + sugars) to freely diffuse in/out across the outer membrane (don’t need a specific transporter) - EXCEPTION = need trasnpoorters for proteins Porin = type of beta barel protein - Beta barel protein = also found in bacterial membrane (found in bacteria membrane + mitochondria membrane = links mitocondria and bacteria in orgin)

Answer 2

Intermebrane space = between the the outer and inner membrane Intermembrane space has very few proteins but does have Cytochrome C

Answer 3

Cytochrome C --> protein in the ETC machinery (moves between complexes 3 and 4) Cyt C = controls life AND death of cells - Life - Cyc C normally functions as a protein SU of ETC complex 4 --> part of the Oxydative Phosphorylation machinery that creates the H+ gradient and actually creates ATP = importnt for cells to live - Death – when cells decide to die through apoptosis --> Cyt C is released from complex 4 and goes to the cytosol -> in the Cytosol Cyt C binds to regulator of apoptosis and induces apoptosis = cell dies Depending on the condition of the cells Cyt C will function in ATP production or cell death

Answer 4

Inner membrane has Oxidative Phosphorylation machinery - Inner membrane Cardiolipin --> important for mitocondria function Inner membrane = has big membrane area because of Cristae --> Area allows for a lot of transporters + oxidative phsophorylation machinery

Answer 5

ETC complex (Oxidatative phosphorylation machinery) - found on the inner membrane Mitocondria inner membrane carries the machinery for oxidative phosphorylation (ATP production) 5 complexes (made of of many proteins)

Answer 6

Complex 5 = ATP synthase --> uses the gradient to create ATP from ADP and phosphate - Once ATP is made (it will be in the matrix) --> ATP is transported to the intermembrane spoace and to the cytosol (goes to the cytosol through porins in the outer membrane) Complex 1-4 = used to generate H+ gradient by pumping protons from the matrix to the intermembrabe space (Pumps H+ using NADH and FADH2 as teh energy source (because pumping against the gradient)) - Transporters transport ATP and ADP through the inner membrane using the H+ gradient

Answer 7

Matrix = inside inner membrane Matrix has mtDNA TCA cycle = happens in the matrix (has the enzymes for the TCA cycle) - TCA = uses pyruvate as primary sugar source to create higher energy compounds (high energy compouds = makes NADH and FADH2) --> NADH and FADH2 = used to create proton gradient in ETC - Pyruvate = sugar produced in glycolysis

Answer 8

mtDNA is different from nuclear DNA 1. mtDNA uses different codons - Example – Nuclear DNA uses AUA as isoleucine BUT mtDNA uses AUA for methionin - Patterns are similar to the pattern of codons found in bacteria (Again suggest the link between mitochondria and and bacteria) 2. Structure of mtDNA is different from nuclear DNA (Nuclear DNA = linear ; mtDNA is circular (similar to bacteria) )

Answer 9

mtDNA has 13 protein coding genes - ALL genes code for SU for different ETC complexes - Different species have different numbers of genes in mtDNA MtDNA also codes for rRNA and rTNA for protein syntehsis for genes encoded in mtDNA (Have translation in matrix)

Answer 10

mtDNA tends to have more mutations at an increased rate compared to nuclear DNA Have more mutations because mtDNA have more ROS that can damage the DNA in the mitocondria AND because mitochondria have less efficient repair mechanisms for DNA damage

Answer 11

Many diseases are caused by mutations in mtDNA (many diseases affect the mitochondria because of mutations in mtDNA) Example: 1 . 1 – Leber’s hereditary optic neuropathy (LHON) - Mutations in genes ND1,4, and 6 (SU in complex 1) 2. Myoclonic epilepsy with Ragged Red fiberes (affects muscles) - Mutate the tRNA = creates defects in protein synthesis in the mitocondria 3. 3 – Neuropathy, ataxia, and retinitus pigmentosa (affects nervous system) - Mutations in compelx 5 (ATP synthase) These are types of disease caused by mtDNA defects BUT many mitochondria proteins are ALSO encoded in the nuclear genome (mutations in nuclear genoms encoding mitocondrial proteins can also lead to mitocondrial diseases)

Answer 12

Have specific pattern of inheritance of mtDNA because mtDNA is only inherited in the oocyte (inherited from the mom) - Get from mom because mitocondria and mtDNA are degraded once fertiization and fusion between the oocyte and sperm happens --> only maintain the mtDNA and mitocondira derived from the oocyte - MEANS mutations in oocyte can be inherited BUT mutation in mtDNA in sperm is not = have a maternal inheritance pattern Pedigre – shows 4 chidlren ate affected --> Son – does not pass to offspring BUT the daughteer can pass to offspring

Answer 13

2nd factor for mtDNA associated disease = mtDNA associated diseases are not 100% penetrance - Not 100% penetrance because have lots of mtDNA (100s) in each cell --> depending on the ratio of mutant DNA to WT mtDNA the symptoms/outcomes can be different Ex – even of the mom is affected --> the ratio between mutant mtDNA and WT mtDNA can chnage in each individual - Each tissue can have diffrent ratio + the ratio can chnage over time - MEANs not all offspring will have the disease even though the mom has a mutation (depending on the amount of mutant mtDNA you may or may not have the symptims)

Answer 14

Found that need over 60% of the mitocdonria to be mutated to see defected --> shows that the WT copy of mtDNA can be dominant - Explains why there is heterogeneity in inheritance of mitocondrial diseases

Answer 15

Answer – False --> Cells often contain more than one type of mtDNA (called heteroplasmy). So, the ratio of mutated mtDNA to wild-type dictates whether you will have disease or not. Note, the ratio can change in different individuals, in different tissues of the same individual, and over time!

Answer 16

1000 genomes project study --> found 90% of healthy participants have at least 1 mtDNA mutation - 20% of healthy participates have mtDNA mutations implicated in diseases - Most people have disease causing mutation in mtDNA

Answer 17

Done by replacement of diseased mtDNA and mitocondira with WT mitocondria Do mitocondrial transfer in human occytes --> Take nucleus from affected oocyte and transport the nucleus to a healthy donor that has had the nuclear DNA removed = replace the affected with mitochondria with healthy mitochondria and still have the parents nuclear DNA = remove the disease causing mutation The successful replacement of mtDNA in human oocytes and genration of blastocytes and embryonic stem cell line have been reported

Answer 18

Answer – Yes --> human cells can lose mtDNA WHY - mtDNA encodes only some OXPHOS proteins (need mtDNA for Oxphos) --> BUT As long as cells have another way to make ATP, they don’t need mtDNA. - Can make ATP in other ways (ex. glcyolysis) to bypass the requirement for oxidative phosphorylation - IF you culture human cells and add glucose and other metabolites --> you can force the mitocondria to lose mitocondrial DNA (cells can live in culture)

Answer 19

Yeast cells grown on glucose can make all the ATP they need (ferment) and can live without any mtDNA. These mutants still have MITOCHONDRIA (and would die without them)! - Still need mitochondria because oxidative phosphorylation is not the only function of mitochondria (have other pathways that you need mitocodnira for) = need mitochondria but don’t need mtDNA

Answer 20

How do you get cells with mitocodnira without mtDNA --> cells don’t need mitochondria to replicate (mtDNA only codes for oxidtaive phosphorylation machinery) -- need the nucleaus to make mitochondria or mitochondria proteins = cells make mitochondria without mtDNA (make empty mitochondria) BUT they can’t do oxidative phosphorylation without mitochondria IN BODY – you couldn’t lose mtDNA but in certain experimental condition you can force cells to lose mtDNA

Answer 21

Answer – 100 lb NOTE – glycolysis occurs in the cytosol ; In cancer – ATP is mainly made by glycolysis

Answer 22

Although mitochondria contain their own genome the majority if mitocondria proteins are encoded in the nucleus Human mtDNA codes for 13 proteins BUT Proteomic studies shoe that mammalial mitochondria have 1500 proteins = means that most mitochondria proteins are encoded in the nuclear genome and then are imported into the mitocondria

Answer 23

Multi-subunit complexes often contain SU encoded in the mtDNA and other SU coded in the nucleus (oxphox machinery uses mtDNA and nuclear DNA) Example – Complex 1 --> 7 mtDNA encoded SU ; 39 nuclear encoded SU - Some proteins are generated in the matrix and inserted into the inner membrane ; some proteins are made in the cytosol and transported to the mitochondria and inserted into the inner membrane --> Biogenesis depends upon the import of nuclear encoded SU and their coordinated assembly with mitocondria encoded SU - Import and assmbly = highly regulated process

Answer 24

Overall- DNA is made to RNA in the nucleus --> RNA is exported to the cytosol --> RNA is tranlted to the precurosor protein --> precursor crosses the outer and inner membrane to get to the matrix

Answer 25

1. Precurosr proteins need to find the mitochondria (taregting) - Have a targeting signal that allows the precursor protein to go ti the mitochondria outer membrane 2. Once the precursor protein reach the mitocondria it crosss one or both mitochondria membranes (translocation) - Have pores and transporters to help the proteins cross 3. Once the proteins get in they must sort themselves into the outer membrane vs. Inner membrane vs. Inter membrane space Vs. Matrix 4. Assemble with other SU (some encoded by mtDNA) (find protein complex)

Answer 26

1. Precursor protein binds to cytoplasmic chaperones 2. Precursor binds to receptors on mitochondrial surface 3. 3 and 4 - precursor is translocated across the outer membrane via TOM complex - Precursor crosses the outer membrane through pores 4 and 5 - precursor translocates across the inner membrane via TIM complex 5. precursor is ‘pulled’ through TOM-TIM complexes by matrix chaperone (PAM or Hsc70) complex 6. presequence is removed by processing protease in the matrix 7. Imported protein folds into 3D structure with help of matrix chaperones

Answer 27

Precursor protein is targeted to the outer membrane --> Have a receptor that recognizes the targeting sequence on the precursor protein Targeting sequences helps the protein find the mitocondria because the targeting sequence interacts with the receptor proteins (identifications of the receptor and precursor occurs through protein-protein interactions) Once recognized the protein can go through the outer and inner membrane to the matrix

Answer 28

Many mitochondria proteins carry N-termial preseqeucnes that are cleaved off after their import - Presenquences in different proteins differ in primary amino acid sequence - Amphipathic helix = reocgnzied by important receptors on mitochondria

Answer 29

Targeting sequences of precursor protein = called the pre-sequnece - In the preseqeucne the Amino Acids are not important BUT it needs to form a unique 3D structure 3D structure = alpha helix (on side is hydrophobic and one side is positively charged) --> pattern is important for being recognized by receptor proteins All presequences contain: 1. Several basic residues 2. No Acidic resideues 3. No long stretches of hydrophobic aa’s 4. Common 3D structure

Answer 30

Uses TOM and TIM machinery to corss the membranes - TOM and TIM = have pores for proteins to go through TOM = translocase outer membarne (Pink in image) TIM = translocase inner membrane (yellow in image) Chanels do NOT function alone --> Have many proteins that help (example – receptor proteins) - Once a precursor is recognized by a receptor they can go through the pore (different receptor recognizes different seq)

Answer 31

The TOM and TIM complexes form large aqueous pores in the outer and inner membrane Image – negative stain of purified TOM complexed by EM - Have 3 holes in TOM - Size of pore = small (Diamter of holes = 2-4 nm)

Answer 32

Imported proteins cross the membrane via the holes in an unfolded state Holes in TOM and TIM are small = protein going through the pore is unfolded - Unfolded state = helped by chaparones by inhibited the folding of teh protein

Answer 33

Cytoplasmic chaperones (ex. Hsp70) = play a role in import + preventing premature folding (keep precorsor protein unfolded after translation) + prevent aggregation of proteins - Unfolded proteins tend to agregate because they have hydrophobic parts that would bind together BUT the chaparones bind to protein to prevent the agregation of proteins During tranlocation across the outer and inner memrbarens through TOM and TIM – chaperones keep the precursor straight so they go through unfolded

Answer 34

Matrix chaperones play an essential role in helping imported proteins fold and assemble in the matrix - Help protein form functional structure BOTH cytoplasmic and matrix chaperones use ATP

Answer 35

Once the protein is in the mitocondria it can go to different places - Mitocondria has different mechanisms to send proteins to the matrix vs. Inner membrane vs. Outter membrane Where the protein goes depends on the transporters in the outer and inner membrane - Matrix proteins = use TOM and TIM - Outer membrane = uses TOM and SAM - Inner membrane – use TIM22 and TOM

Answer 36

Defects in mitocondria import = get defective mitochondria = leads to human disease Example: 1. A defect in one of the subunits of the TIM22 complex --> causes X-linked deafness-dystonia syndrome (Mohr-Tranebjaerg syndrome) (neurologic disorder) 2. DCMA syndrome - A defect in hTimm14 (one of the subunits of the TIM23/PAM complex) --> cause a serious childhood cardiomyopathy (affects heart) These diseases show the impact on the nervous system and cardiovascular system

Answer 37

Most mitochondrial and cellular lipids = made in the Endoplasmic Reticulum (ER) - Exceptions = PE and CL - Lipids used to generate the mitocondria = made in the ER and need to be important into the mitocondria

Answer 38

Lipid transport = happens at the contact site between the RER and the mitochondria Lipids = get to the mitochondria at the physical connections between the ER and the mitochondria (called the MAMs --> mitocondiral associated ER membranes) - Have contact between the RER and the mitochondria (red circle in image)

Answer 39

PE (phosphatidly ethanolamine) = primarily made in the mitochondria inner membrane by decarboxylastion of phosphatidly serine (PS) - Phosphatidly serine = made in the ER - PS = imported from the ER and PE us exported to the ER via MAMs

Answer 40

IF want to connect 2 memebranes --> THEN want protein in mitochondria outer membrane and protein in ER --> the two proteins will interact - Image – protein complex in green – have 1 Su that is embded in the ER membrane and 2 SU that are embedded in the mitochondria outer membrane + have a lipid soluble protein between --> ALL form a complex = can physically connect ER and mitochondria membranes Connection between the 2 membranes allows lipid transport between the ER and the mitocondria membrane

Answer 41

We know proteins important for the transport of lipids from the outer membrane to the inner membrane (have mechanism to bring lipids from one membrane to the other) Can be bidirection (can go from inner to outer or outer to inner) After the lipid is in the inner membrane it is modified and can go back to the outer membrane and to the ER membrane

Answer 42

Cardiolipin – unique phospholipid made in the inner membrane of the mitochondria (mitocondria specific lipid ; unique to mitocondria) Cradiolipin = required for the activity of many Inner membrane protein complexes Cardiolipin = exmaple of ER/mitocondira contact – important role in lipid biosynthesis NOTE – cardiolipin = also is present in the plasma membrane of bacteria

Answer 43

Cradiolipin = generated in several steps controlled by enzymes in the mitochondria inner membrane Precursor of cardiolipin = phosphatidic acid (PA = phospholipid made in the ER) --> Phsphatidic acid = transported to the outer membrane then the inner mebrane using the contact between the ER and the outer membrane Once in the inner membrane --> PA is modified to make Cardiolipin --> most cradiolipin will stay in the inner membrane but some of it is transported to the outer membrane

Answer 44

Phospholipids usually have 1 heads and 2 tails (tails = hydrophobic) Cardiolipin = dimer of phospholipids --> have 4 tails with 1 head (big hydrophibic phospholipid)

Answer 45

Cardiolipid = important for connecting ETC complex together (Ex. complex 2 and 3) During ETC activity it is good if the complexes are next to each other and connected --> cardiolipin makes supercomplex containing multiple complexes (example – complex 2 and 30 If have no CL = reduce the function of complexes and reduce the effective ATP production in mitochondria

Answer 46

Barth syndroe – X-liked disorder caused by a defcet in cardiolipin metabolsim - Young boys have growth defects + nuetropenia + cardiomyopathy - Shows the impact of mitocondria on energy demanidng organs (Ex. Brain and heart)

Answer 47

Mitocondria move within cells and talk by fusing and dividing

Answer 48

Mitocondria are in different shapes and different locations in different cell types Liver = round structure Pancratic acinar cells = elongated mitochondria Heart muscles = mitochondria sit next to the myofibrils (provides ATP for effective muscle contraction)

Answer 49

Sperm --> mitochondria are enriched at the base of flagella = give ATP for the sperm to swim Epithelial cells --> mitondira are enriched at the apical surface = used for taking up molecules from the apical surface

Answer 50

Mitochondria are often tethered to and move along the cytoskeleton Video – axons of nuerons --> see things moving - Longer things = mitochondria - Mitochondria can go in both directions – can go towards the synapse at the end of the axon or towards the cell body

Answer 51

Types of dynamics of mitochondria = fusion and division Regulated fusion and fission = controls the number + shape + size of mitocondria in the cell

Answer 52

IF you block fusion or division you can see changes in mitocondria Example (image): - WT mitochondria = have a tubular strctures - IF block fusion = mitochondria will continue to divide and make small structures = the mitocondria are not happy because they lose mtDNA because the cell is making so many mitochondria Make mitocondira beyond the copy number of mtDNA = make empty mitochondria = defective because mtDNA is important

Answer 53

If block mitochondria division = keep fusing the mitochondria together and get a big structure --> not good because now the mitochondria are hard to move in the cell because they are all connected

Answer 54

Fusion and division process = controlled by GTPases associated with the mitochondria Division - When mitocondira divide the Drp1 GTPase assembles into the surface of the mitocondria and wraps around --> Drp1 GTPase uses GTP hydrolysis -->the ring will squeeze and cut the mitocondria Fusion – Daughter mitochondria can fuse back together by fusing the oter membrane then the inner membrane (using another set of GTPases) - Uses Opa1 and Min1/2

Answer 55

Fusion helps mix mitochondrial contents and exchange information of material among mitocondria to maintain homeostasis Division = quality control for mitochondria (ex. mitochondria damaged by ROS) - Division = needed to remove damaged mitocondria --> part of the damaged mitocondria can be separated by mitocondrial division and the isolated mitocondria ca be targeted for degradation by autophagy - Mitocondria import makes mitocondria bigger = once the mitochondria get to be a certain size the mitochondria need to divide to maintain size and number in the cell (esocially during cell division) - Division is important for maintaining of function and the maintain of mitochondria number and size in each cell

Answer 56

Mutations in GTPase = linked to human disease - Diseases affect eyes or sesory/motor nuerons in spinal cords Fusions – Mutations in Opa1 and Mfn2 cause autosomal dominant optic atrophy (caused by Opa1) and Charco-marie-tooth neuropy (cuased by Mfn2) Division – Mutation in Drp1 leads to postnatal death with impaired brain development

Answer 57

Have similarity between mitocondria and bacteria (suggests that bacteria may be the origin of mitocondria during evolution) Similarities: 1. DNA is circular and shared codons + Prokayotic like tranlation system 2. Have porins in beta barel in both bacteria and mitochondrial membrane 3. Cardiolipin in mitocondira and also found in bacteria 4. 2 memebranes (1 memebrane is from bacteria and 1 memebrane is from the host cell plasma membrane) Idea – many yeas ago cells did not have organelles --> cells engulfed bacteria --> instead of killing the bacteria the cells used the bacteria to make ATP

Answer 58

Peroxisomes – surrounded by a single membrane and do NOT contain DNA Have many peroxisomes in the cell (Image – green dots = peroxisoms ) EM image – shows that peroxisimes have 1 membrane

Answer 59

1. Breakdown of lipids 2. Synthesis of lipids 3. Synethsis of bild acids (helps liver uptake fats) - Take up hydrophoboc molecules in the body (acts like a dtergents)

Answer 60

Beta oxidation of very long chain fatty acids (>C16) --> gets energy from lipids Beta oxidation process = can also make ROS (means there needs to be a defense mechanism)

Answer 61

Makes plasmalogen - Plasmalogen = an ether linked phospholipd abudnent in nuerons) --> Major lipid in the mylin sheeth in oligiodendrycytes in the brain Makes some cholestral

Answer 62

Peroxisomes contain a number of oxidases Peroxisomes = produce H2O2 (Type of toxic ROS) during lipid metabolsim BUT peroxisomes ALSO make catalase to degrade H2O2 - Beta oxidation and plamalogen producton = produces ROS Catalase = converts toxic ROS H2O2 into water and O2 – peroxisome can protects against the toxic ROS generated by the peroxisome itself

Answer 63

Sysem can ALSO help mitocondria Mitocondria and peroxisomes can interact --> peroxisomes will help detoxify the mitochondria generated ROS (turns H2O2 into H2O and O2) - Mitocondria ROS can be transported to the perosoxisomes through contact site and then catalse will make water and O2 IF you seperate the peroxisome and the mitochondria THEN the mitochondria accumulate more ROS

Answer 64

Peroxisomes have no DNA = they ned to import all of their proteins (all peroxisome proteins need to be imported)

Answer 65

Peroxisome precursor proteins have their own targeting signal (signal = PTS) - Signal has its own sequence and structure (3 Amino Acids is sufficient to target the protein to the peroxisome and the peroxisome receptor) Signals: 1. PTS1 - Most peroxisomal matrix proteins carry a C-terminal import signal (called PTS1), consisting of ser-lys-leu (SKL) 2. PTS2 - located at the N-terminus. - Only a few proteins have PTS2.

Answer 66

The PTS1 signal is recognized by a cytosolic protein, called Pex5, that targets the protein to the peroxisomal surface PTS1 is recognized by a receptor --> Receptor is in the Cytosol (NOT the peroxisome surface) --> receptor complexes with precursor protein --> complex can target protein to a protein to the peroxisome surface --> then will hand the protein to the translocase transporter in the membrane --> translocase will trasnport the protein

Answer 67

Peroxisomal proteins ca be imported in the folded state - Chaparones are not needed for peroxisome import because the proteins do not be to be unfolded - Depending on precursor protein you want to import the pore can be bigger or smaller using assembling of SU of the pore complex - Protein ologiomers can also be imported Mitocondrial translocation with TOM and TIM has small holes = the protein is unfolded during translation BUT in the peroxisome the pore can be big = means the protein does NOT need to be unfolded

Answer 68

Peroxisome membranes also come from ER Peroxisome proteins are inserted into teh ER memebrane --> ER can create small precurosrs vesicles/immature peroxisomes (make mini version of peroxisome coming from ER) --> THEN can import other components into the percursor form of peroxisome --> then make mature peroxisomes with all peroxisome proteins Once you reach a certain size – need to divide to maintain the number (increase the number) and size for cells to divide in the future MEANS that peroxisomes can be generated de novo

Answer 69

De novo means that when cells don’t have peroxisomes you can still make peroxisomes becasue peroxisomes can be made by ER Can't make mitocondria de novo because there is no way to make mitocondria from the ER = always need pre-existing mitochondria to make more mitochondria by importing lipids and proteins Mitocondria arise ONLY from growth and division of pre-existing organelles

Answer 70

Other proteins are added to the vesicles --> forming mature organelles Further import + growth and division of peroxisomes generate additional organelles

Answer 71

Type types of disease: 1. Lack of enzymes/functinoal proteins (defects in a single enyme or activity) - Example - X-linked adrenoleukodystrophy (ALD) 2. Defectes in perxisomal protein important - Make peroxisomes but they are not functional (have empty peroxisomes because you can’t import functional componetenst when you have defects in protein import - Zellweger syndrome Peroxisome disorders are often neurological due to the role they play in plasmalogen production