1	Mitochondria: genetics and transport
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3	How do you get the protein through the outer membrane? First, protein must have a signal sequence that can be recognized. Specific Tom receptors recognize import proteins: Tom70, Tom22, and Tom20. Different proteins bind to different Tom receptors. Second, it binds to a “chaperone” that uncoils it so it can get through the channel. (Hsp70) Then it needs guidance (Tom guiding proteins 5, 6, and 7) through the General Import Pore (GIP)
4	How do you get a protein into the inner membrane or matrix? Mitochondria can’t make all of their own proteins. How do the remaining proteins and subunits get in? Both membranes contain complexes of receptor and translocator proteins that promote passage of membrane and matrix proteins. Translocator Outer Membrane (Tom proteins) Translocator Inner membrane proteins (TIM proteins)
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6	What makes the protein move through the pore? Mitochondria are pumping electrons from matrix! This creates a net negative charge in the matrix. Protein to be imported has a concentration of positive charges on the end that enters the pore. The negative charges in the matrix “attract” the positive charges on the protein to be imported.
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8	Simplified entry view
9	Electron transport chain
10	Tom-Tim overview
11	What happens to worn out mitochondria?
12	What can mitochondrial DNA encode? Human mtDNA is 16,569 bp Encodes a number of mitochondrial proteins Subunits 1, 2, and 3 of cytochrome oxidase Subunits 6, 8,9 of the F_o ATPase Apocytochrome b subunit of CoQH2-Cytochrome C reductase Seven NADH-CoQ reductase subunits
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17	Mitochondrial Inheritance 99.99% of mtDNA is maternal 100 mitochondria in sperm + 100,000 in egg. Most of sperm tail does not get in. Eventually sperm mitochondria are deleted or diluted out. Thus, any mutations in mitochondrial DNA are inherited from the mother.
18	Maternal inheritance
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20	Mitochondrial bottleneck Start with large number of oogonium which divide and form primary oocyte. Then, these become apoptotic and by birth, less than half survive. Thus, the concentration of an oocyte with mutant mtDNA has increased. By maturation, there are significant reductions and even fewer at the end of puberty. Thus, surviving mutant mtDNA has concentrated even more, making chances of passing on the mutant strand greater.
21	Bottleneck
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24	Segregation of mitochondrial DNA… Review focuses on genetic advice given to mother with maternally inherited mtDNA disease. Poulton and Marchington have pointed out that there are thousands of copies of mtDNA/nucleated cell. Most of us are homoplasmic (all mtDNA is identical). mtDNA may be heteroplasmic (contain both normal and mutant) in patients with mitochondrial diseases mtDNA encodes subunits of the respiratory chain.
25	Background… mtDNA may affect different tissues unequally Those tissues requiring the most ATP (like muscle) are affected most profoundly. Accumulation of mutant mtDNAs may increase in a tissue (seen by biopsy) Mitochondria replicate even faster as ATP drops. Decreased after muscle damage and repair. Why? Satellite cells used for muscle replacement contained low mutant mtDNA.
26	D-loop mutations in Mitochondrial DNA… mtDNA replication begins with synthesis of RNA primer Light strand promoter is on the displacement loop (D-loop) of mtDNA Primer is cleaved at “conserved sequence blocks (CSB)” by RNAse. DNA is then polymerized by mtDNA polymerase gamma D-Loop is the main cis-regulatory region of the mtDNA. What happens if it is mutated?
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28	Is mtDNA involved in its own deletion? Many clinical cases linked to nuclear DNA and its mitochondrial proteins. This paper describes a link to mtDNA itself and maternal inheritance. A patient and her mother both have defective muscle. Used immunocytochemistry to show absence of cytochrome C oxidase (COX) in some fibers. Figure 4: PCR shows less abundant mtDNA in muscle, leukocytes, kidney, skin fibroblasts Figure 5: Used Denaturing gradient gel electrophoresis (DGGE) to screen the sequence of mtDNA. Found 13 sequence variations in the D-loop region. Further analysis of the mutations found 2 mutations in mtDNA Ruled out nuclear DNA products
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32	How would you differentiate? Disease due to Mitochondrial gene mutation for cytochrome oxidase? D-Loop mutation? Disease due to mutation of nuclear gene?
33	How does mitochondrial architecture dictate function? Architecture of inner membrane complexes allow electron flow and functioning of ATP synthase. Architecture of Inner membrane sets up charges that attract entry of matrix and Inner membrane proteins.
34	How does mitochondrial architecture determine function? Matrix contains DNA, RNA, ribosomes which encode and translate the code for key proteins. Outer membrane architecture recognizes mitochondrial proteins and translocates them to appropriate space/membrane Inner membrane architecture continues translocation of proteins to matrix or Inner membrane.