8. Organelles: Mitochondria

Protein import into mitochondria

- Fully translated in cytosol - Bound by chaperone Hsp 70, prevents folding - Imported into mitochondria: -- Mitochondrial signal sequence -- post translational translocation

How does Electrochemical proton gradient drive ATP synthase?

- Protons flow down gradient through ATP synthase - This rotates ATP synthase (converted to mechanical energy) - Mechanical energy drives ATP synthesis from ADP + Pi

Where is ATP synthase located?

ATP synthase — embedded in inner mito membrane

Pyruvate & fatty acid broken down to what in the mitochondria?

Acetyl CoA

What is Acetyl CoA metabolized by?

Acetyl CoA metabolised by TCA reducing NAD+ to NADH

What protects from Reactive Oxygen Species damage?

Antioxidants

Electrochemical proton gradient

As electrons move along respiratory chain, energy is stored as electrochemical proton gradient across inner membrane Drives ATP synthesis

Pro-apoptotic bcl-2 proteins

BAD, BAX

simple summary of intrinsic pathway of apoptosis

BAX translocate into mito mebrane as a respinse to cell damage/stress BAX create pores in mito membrane Cytochrome C is released through these pores Cytochrome C activates caspase cascade apoptosis occurs

What is essential for inner membrane Impermeability?

Cardiolipin

What occurs in the outer membrane?

Cardiolipin synthesis Lipid modification

Mitochondria: Major functions

Cell's power plants ATP production (Aerobic respiration) Also; Cell signaling & apoptosis

Mitochondrial pore formation & Apoptosis steps

Pro-apoptotic bcl-2 proteins (BAD, BAX) translocate from cytosol to mitochondrial membrane in response to cell damage/stress & signalling ↓ Formation of pores in outer membrane (BAX) ↓ Release pro-apoptotic factors (cytochrome C) into cytosol ↓ Activation caspase cascade ↓ Apoptosis

Glycolysis & Mitochondria

Pyruvate (from glycolysis) & fatty acids enter mitochondrion & are broken down to Acetyl CoA in matrix

Reactive Oxygen Species can inactivate what? what is the result of the inactivation?

ROS can inactivate ETC e- acceptors: further build-up of ROS

What diseases are Reactive Oxygen Species implicated in?

ROS implicated in degenerative diseases(Alzheimer), cancer & aging

Shape of mitochondria in skeletal muscle

Skeletal muscle: rows, nestled between myofibrils

Shape of mitochondria in sperm

Sperm: wrapped around flagellum

Summary of Energy-producing reactions of the Mitochondrion

TCA cycle (creates CO2 + NADH) Electron transport chain uses energised electrons from NADH to pump protons into intermembrane space Protons fall down electrochemical gradient through ATP synthase, making ATP Inner membrane very impermeable to hold ion gradient

Import proteins of the mitochondria

TOM & TIM

Electron transport & ATP synthesis: summary

Transfer of electrons to electron acceptors in the inner membrane results in pumping of protons out of mitochondrial matrix to the intermembrane space, which return to power ATP synthase ultimately ½ O2 will take e- and become water

TIM

Translocase of Inner Membrane requires electrochemical gradient

TOM

Translocase of Outer Membrane - Hsp70+protein binds to TOM - TOM recognises mt signal sequence

What is driven by Mitochondrial gradient?

Transport processes driven by the gradient used to bring essential components in and export essential products out

Shape of mitochondria in Steroid secreting cells

Tubular cristae

What does Thermogenin (uncoupling protein) uncouple?

Uncouples respiration from ATP synthesis

Bcl-2 family

can be pro or anti apoptotic Bcl-2 is blocking release of cytochrome C

What protects peroxisomes from Reactive Oxygen Species?

catalase

What complexes are involved in Electron transport ?

complex I complex III complex VI

What does the TCA (citric acid/Krebs) cycle produce?

creates CO2 + NADH

Protons flow (up/down) gradient through ATP synthase

down gradient

What occurs in the matrix?

enzymes of oxidative metabolism - Pyruvate oxidation - Fatty acid oxidation - TCA cycle (Citric acid cycle; krebs cycle)

Apoptosis: Extrensic pathway

extrinsic pathway == death ligand and receptor

two Antioxidants?

glutathione peroxidase superoxide dismutase

Cardiolipin synthesis

super important for inner membrane but actually made in outer membrane

Mitochondria protein import

the Mitochondria is not self sufficient - most proteins must be imported in - Majority of proteins encoded by nuclear genes

Thermogenin

uncoupling protein allows protons to "leak" from cytoplasm to matrix uncouples gradient: uncoupling —> bypassing ATP synthesis, so no making of ATP

What is driven by the voltage gradient?

voltage gradient drives ADP-ATP exchange

oxidative phosphorylation

— making of ATP — in the presence of Oxygen (no other way)

Caspase cascade

→ cell death in both pathways, the enzyme that actually mediates apoptosis

Function of ATP synthase

Couples oxidation to phosphorylation mechanical rotation leads to phosphorylation energy transformation from mechanical to biochemical energy Transmembrane H+ carrier Head: ATPase Subunits bind to ADP + Pi (in the matrix)

Function of BAX

Formation of pores in outer membrane; which allows release of pro-apoptotic factors (cytochrome C)

What can diffuse across the outer membrane?

Free diffusion of small molecules & ions

Mitochondrial Proteins are made where?

Fully translated in cytosol

Steps of protein import into Mitochondria

1. Hsp70+protein binds to TOM; TOM recognizes mito signal sequence 3. Precursor translocated across inner membrane via TIM; requires electrochemical gradient 4. Signal sequence removed by matrix protease 5. Protein spontaneously folds or helped by chaperone Hsp60 & ATP

Pyruvate oxidation

2 from splitting of glucose

Cardiolipin

20% inner membrane lipid "double phospholipid"; 4 tails; two glycerol backbone Made in mitochondria outer membrane, most others imported Impermeability to ions

Shape of mitochondria in hepatocytes

Hepatocyte: oval with numerous cristae

Mitochondria: Shape & distribution

Lamellar cristae: MOST cells

The Energy released from what used to generate ATP?

oxidation of nutrients Oxidative phosphorylation

What is driven by the pH gradient?

pH gradient drives pyruvate import and phosphate import

Mitochondria: Outer membrane

permeable to small molecules & ions (~5-10 kDa) biochemical reactions- primarily lipids (made)

What kind of translocation occurs?

post translational translocation

Cytochrome C

pro-apoptotic factors if Cytochrome C is released from mito — cell undergoes apoptosis

NAFH+

proton donor in ETC High energy electrons from NADH passed along electron transport chain to O2

What does the Electron transport generate?

proton gradient across inner membrane → drives ATP synthesis

What can Reactive Oxygen Species damage?

Damages protein, RNA, DNA (mtDNA: no histones & ↓ DNA repair)

How do mitochondria divide?

Divide by binary fission Replicate DNA & divide in response to energy needs of cell (not linked to cell cycle) so if you need more energy, mito will divide

the mother of all reductions

ETC (electron donation) —> O2 to H2O

What occurs in the inner membrane?

ETC & ATP synthase e- transport chain oxidative phosphorylation (ADP—>ATP)

What import complex is is the inner membrane?

ETC complexes

What drives ATP synthasis?

Electrochemical proton gradient

Mitochondria: Inner membrane

Impermeable to most molecules Cristae ↑ surface area ETC ATP synthase***

Outer membrane has what kind of receptors?

Import receptors for larger proteins

Inner membrane has what kind of receptors?

Import receptors for matrix proteins

What is the energy conversion catalysed by the mitochondrion?

Inner membrane changes chemical bond energy (NADH oxidation) into phosphate bond energy (in ATP) oxidative phosphorylation

Apoptosis: 2 different pathways

Intrinsic & extrinsic pathways

Is mitochondrial division related to the cell cycle?

NO; if you need more energy mito will divide

Mitochondria: Localization & Numbers

Near sites of high ATP requirement # varies in diff. tissues (1 - several thousand) Numbers & energy producing enzymes (TCA & ETC) increase with need (ie: regular exercise) example: tail of sperm, mito surround flagella

What produces heat in mito?

Oxidative metabolism (TCA, ETC) produces HEAT

Barth syndrome

X-linked cardiolipin synthesis disorder inner membrane becomes permeable Cardiomyopathy Generalised muscle weakness & chronic fatigue Neutropenia High mortality in infancy: Sudden infant death Infection Cardiac failure

Inner membrane permeability

impermeable to small, charged molecules

Brown adipose tissue

in newborns and babies fat with many specialised mitochondria uncoupling protein (UCP); thermogenin, in inner membrane forms channels through membrane

Where does glycolysis occur?

in the cytoplasm

Where is the site of ATP synthase?

inner membrane

Porins

integral proteins found in the outer membrane

In the inter membrane space the concentration of H+ ions is _______; so the pH is _______.

inter membrane space concentration of H+ ions is high, pH is low

Apoptosis: Intrinsic pathway

intrinsic pathway == think mitochondria **

Besides energy, what else is produced by oxidative phosphorylation in the mitochondria?

most endogenous Reactive Oxygen Species (ROS)

Mitochondria: Matrix

mtDNA mt ribosomes, tRNA, rRNA oxidative metabolism: production of ATP