D103 Mitochondria (ALS 8, Video 14)

oxidative phosphorylation

AAs, simple sugars, fatty acids → high energy electron carriers (NADH and FADH2) → ATP

• electrons move down the ETC, leading to the establishment of a electrochemical proton gradient that is then used to generate ATP 

• ATP syn requires a tight, proton impermeable inner mito membrane 

• ATP syn is able to convert chem energy into mech energy 

atp production by mito

food breakdown, citric acid cycle, electron transport chain, atp synthase

food breakdown in mito

occurs in cytosol

via glycolysis

citric acid cycle in mito 

occurs in mito matrix 

• acetyl coa is converted into high energy electron carriers (NADH) 

etc in mito

occurs in inner membrane of mito

• electrons move between the complexes of the electron transport chain (arranged in order of increasing redox potential)

• last electron acceptor: O2, which is reduced to H2O

• electron movement generates a steep electrochemical gradient for protons (membrane potential + pH gradient)

atp synthase in mito

occurs in inner mito membrane

• uses steep electrochemical proton gradient to generate ATP

• has a catalytic domain

• hydrolysis of ADP → ATP causes conf change

what does atp production by mitos depend on 

specialized inner membrane 

• highly permeable ions

• high protein content (76% of the weight is protein, such as transporters) 

• cardiolipin 

• cristae arrangement generates proton sink 

cardiolipin

diphosphatidyl glycerol with 4 fatty acid chains

• controls membrane permeability

• promotes high curvature of the membrame

mito outer membrane and atp production

porins make this membrane permeable to small molecules and proteins (>5kDa)

• slightly basic pH in cytosol

mito behavior

highly dynamic

• fission and fusion

mito fission

dynamin-1 gets activated with GTP, then constricts to separate them

defective: elongated pieces bc it cannot break apart

mito fusion 

regulated by GTPase and requires 2 gtp inputs 

1. gtp input to fuse the outer membranes 

2. gtp input to fuse inner membrane

defective: small fragments bc it cannot fuse together to make bigger parts 

what determines mito organization

contact sites with the ER 

• ER wraps and determines where GTPase pinches it off 

mitochondria and dna

mito contain their own dna

• mito dna is organized as clusters (=nucleoids)

• clusters are anchored to the inner membrane

• 10-1000s nucleoids/cells

significance of mito DNA

humans: 37 genes which encode 13 mito proteins

other organisms: varying size of the mito genome

• ex: yeast = 80,000 bp; plants = 200,000 bp

mito dna vs nuclear dna 

mito dna has dense gene packing = no introns 

• does not replicate in parallel ot nuclear DNA during s-phase (as needed) 

• uses a different genetic code (4 out of 64 codons are different) 

• no proofreading and DNA repair mech (mutation rate is 1000x higher than for nDNA)

endosymbiont theory

primordial eukaryotic cells without ability to use oxygen

• colonized by aerobic bacteria

• gene traensfer of 1000+ genes to nucleus

• 13 structural proteins encoded by bacterial/mito DNA

supportive evidence for the mito endosymbiont theory

• double membrane that surrounds the mito matrix

• circular dna

• transcription and translation machinery of mitos are similar to those of bacteria

how are mito inherited

maternal inheritance

• egg contributes the majority of cytoplasm to the zygote; mitochondria are contained in the cytopalsm

• a mutation in mtDNA is only transmitted from the motherl it is passed on to all offspring

symptoms of leigh syndrome

• normal at birth, but progressive loss of mental and motile ability

• brain lesions that are detectable by mri

• caused by at least 26 mutations that affect mito energy production

causes of leigh syndrome

mutations in genes that encode for complex i subunits 

• complex i: 39 subunits (mDNA), 7 subunits (mtDNA) 

nuclear dna mutations leigh’s syndrome (autosomal recessive inheritance) + mt dna mutation leigh’s syndrome (maternal inheritance) → progressive degeneration of the CNS 

sibling inheritance of mito

siblings may have different mito and a different degree of severity of a mito disease

• the offspring from a mother can display different severities of a mito disease phenotype

what happens where inside mitochondria 

A = cytosol 

B = tca cycle enzymes 

D = porins (gives permeability) 

F = high conc of atp-syntahse 

you have identified a compound that makes the inner mitochondrial membrane permeable to ions. which of the following describes the most direct consequences of incubating your cells with this compound?

proton gradient and atp synthase become uncoupled

you have learned that mito and er form membrane contact sites. which of the following experiments would be allow you to determine if the er controls the morphology of mito

alter er organization and observe mitochondria morphology by immunofluorescence microscopy

mito constantly undergo fusion and fission, processes regulated by the gtoases Drp1 and Mfn1/2. A researcher knocks down Drp1 in cultured cells and stains mito wiht a fluorescent dye. the average mito length was measured from confocal micrographs. based on the data, which conclusion is best supported?  

Drp1 promotes mitochondrial fission 

you have identified a novel protein (x) thru a functional screen. since you dont have an antibody, you tag the cDNA of this protein with GFP. In colocalization studies in which you also use an antibody to the TOM complex (together with the correct secondary antibody with a red fluorophore. which of these conclusions is best supported by result?

x localizes to mito

where do mitochondria come from; which of the following facts provides strong support for the endosymbioent theory for the origin of mitochondria?

mitochondrial dna is circular

who in this family has differnet mitochondrial dna 

maternal, so not in the father