Lecture 5 Mitochondria and Peroxisomes Exam Review

Wallace's description of the human cell (2007)

"The human cell is a symbiosis of two life forms, the nucleus-cytosol and the mitochondrion"

NIH definition of mitochondria (2009)

Structures within cells that convert the energy from food into a form that cells can use

Four major functions of mitochondria

Produce energy as ATP; sequester and release calcium for cellular signaling; store and release pro-apoptotic molecules; serve as a major source of reactive oxygen species (ROS) and associated cell signaling

Endosymbiotic origin of mitochondria

Mitochondria arose when an aerobic prokaryotic cell was engulfed by an anaerobic pre-eukaryotic cell; internal membranes formed, the surrounding membrane derived from the pre-eukaryotic cell was lost, resulting in mitochondria with a double membrane

Why do mitochondria have a double membrane?

Because they originated from an aerobic prokaryotic cell engulfed by a pre-eukaryotic cell, retaining both their own original membrane and one derived from the host

Outer mitochondrial membrane

The outer boundary of the mitochondrion; permeable to ions and small molecules

Inner mitochondrial membrane

The membrane inside the outer membrane; impermeable to ions (unlike the outer membrane) and folds into cristae

Cristae

Foldings of the inner mitochondrial membrane that increase surface area

Intermembrane space

The space between the outer and inner mitochondrial membranes; stores pro-apoptotic molecules like cytochrome C, AIF, and SMAC/DIABLO

Mitochondrial matrix

The space inside the inner mitochondrial membrane, where the citric acid cycle occurs

How is mitochondrial DNA inherited?

Maternally inherited

How many proteins are coded by mitochondrial DNA vs. the full mitochondrial proteome?

Mitochondrial DNA codes for only 20-30 proteins, while the full mitochondrial proteome consists of about 2,000 proteins

Where are most mitochondrial proteins made?

Most are made in the cytoplasm (cytosol) and imported into the mitochondria; only a few proteins are made using the mitochondria's own protein synthesis machinery

Mitochondrial protein import: binding step

A precursor protein with a signal sequence binds to an import receptor protein on the outer mitochondrial membrane

Mitochondrial protein import: translocation step

The protein moves through protein translocators in the outer and inner membranes into the matrix, where the signal peptide is cleaved to yield the mature mitochondrial protein

Do all mitochondrial proteins have an obvious targeting sequence?

No — many mitochondrial proteins lack any obviously recognizable mito-targeting sequence, and proteins targeted to non-matrix compartments use different targeting sequences

Diseases/conditions linked to mitochondrial dysfunction

Type II diabetes, cardiomyopathy, aging, cancer, Parkinson's disease, Alzheimer's disease, Huntington's disease

What percentage of ATP production occurs via oxidative phosphorylation in mitochondria?

More than 95% of ATP production occurs by oxidative phosphorylation during the respiratory (electron transport) chain

How does mitochondrial density/organization vary by cell type?

It depends on cellular energy needs — e.g., cardiac muscle mitochondria are densely packed between myofibrils of the contractile apparatus, and sperm tail mitochondria wrap around the flagellar core to power motility

Can mitochondria form networks?

Yes, mitochondria can form elongated tubular networks (continuous mitochondria) rather than existing only as separate discrete organelles

Key molecules in cellular energy metabolism

Glucose and fatty acids, pyruvate, acetyl-CoA and NADH, ATP

Role of glycolysis in cellular energy metabolism

Converts glucose (via glucose-6-phosphate) to pyruvate in the cytosol, generating some ATP directly

Role of pyruvate dehydrogenase (PDH)

Converts pyruvate into acetyl-CoA, linking glycolysis to the citric acid cycle

Role of fatty acid beta-oxidation

Breaks down fatty acids (transported in via CPT-1 and FAT/CD36) into acetyl-CoA for use in the citric acid cycle

CPT-1

Carnitine palmitoyltransferase 1; involved in transporting fatty acids into the mitochondrion for beta-oxidation

FAT

Fatty acid transporter/CD36; transports fatty acids into the cell for use in metabolism

MCT

Monocarboxylic acid transporter; transports lactate out of the cell

Step 1 of mitochondrial ATP production

Acetyl-CoA (from pyruvate or fatty acids) is used in the citric acid cycle

Step 2 of mitochondrial ATP production

NADH is produced by the citric acid cycle

Step 3 of mitochondrial ATP production

NADH donates an electron and proton to the respiratory (electron transport) chain

Step 4 of mitochondrial ATP production

The electron travels through the respiratory chain toward Complex IV, coordinated with proton exit from the matrix

Step 5 of mitochondrial ATP production

A membrane potential is generated as a result of proton efflux

Step 6 of mitochondrial ATP production

ATP is produced by ATP synthase when protons flow back into the matrix

What percentage of cellular oxygen usage occurs via this mitochondrial electron transport process?

98% of oxygen usage

ATP synthase (forward/synthesis mode)

Uses the energy of the proton-motive force (protons flowing back into the matrix) to synthesize ATP from ADP and phosphate

ATP synthase (reverse/hydrolysis mode)

Can run in reverse — hydrolyzing ATP to pump protons out and increase the membrane potential, since ATP synthesis is a reversible process

Proton-motive force

The combined electrochemical driving force pulling H+ back into the mitochondrial matrix, composed of a force due to membrane potential (ΔV) and a smaller force due to the pH gradient (ΔpH)

Who proposed the chemiosmotic theory, and when?

Dr. Peter Mitchell, published in Nature in 1961 ("Coupling of Phosphorylation to Electron and Hydrogen Transfer by a Chemi-Osmotic Type of Mechanism")

Oxidation of NADH (in oxidative phosphorylation)

Movement of electrons from NADH to oxygen, which results in generation of the membrane potential

What is oxidative phosphorylation?

The coupling of oxidation (electron transport generating membrane potential) to phosphorylation (using that membrane potential to attach phosphate to ADP, producing ATP)

Three things mitochondrial membrane potential contributes to

Ion transport, heat generation, and membrane leak (proton movement into the matrix not coupled to ATP production)

Membrane leak (proton leak)

Movement of protons into the mitochondrial matrix that is not coupled to ATP production; a normal part of physiology contributing to heat, metabolism, and ROS regulation, though dysregulation can be protective or damaging

Calcium uniporter (mitochondrial)

A mitochondrial ion channel that regulates rates of energy metabolism by allowing calcium uptake into the matrix

KATP channel (mitochondrial)

A mitochondrial ion channel proposed to be involved in mitochondrial volume regulation

PTP (Permeability Transition Pore)

A large pathological pore/channel involved in cell death; opens during calcium overload and leads to loss of the membrane potential

NCX (Sodium-calcium exchanger)

A mitochondrial transporter that helps recycle calcium

UCP (Uncoupling protein)

A mitochondrial protein involved in heat generation by uncoupling the proton gradient from ATP production

VDAC

Voltage-dependent anion channel; provides a passage for metabolites across the outer mitochondrial membrane

MAC (Mitochondrial Apoptotic Channel)

An outer membrane channel whose opening allows release of pro-apoptotic molecules into the cytoplasm, triggering apoptosis

Apoptosis and mitochondria

Pro-apoptotic molecules (Cytochrome C, AIF, SMAC/DIABLO) are stored in the intermembrane space; their release into the cytoplasm via MAC opening is required to execute the apoptotic cascade

TOM and TIM complexes

Protein complexes in the outer (TOM) and inner (TIM) mitochondrial membranes involved in protein import

Why must mitochondria typically be intact to study their function?

Because the integrity of the membrane is required for accurate measurement of mitochondrial activity

What can be measured in intact mitochondria to investigate function?

NADH levels (imaging), membrane potential (imaging), ATP levels (imaging), oxygen consumption (respirometry), and channel currents (electrophysiology)

TMRM

A dye used to measure mitochondrial membrane potential via imaging

What effect does the flavonoid Quercetin have on neurons in culture?

Addition of Quercetin to neuronal culture leads to an increase in mitochondrial membrane potential

Oligomycin (in respirometry)

A compound that inhibits ATP synthase, used to assess ATP-linked respiration during mitochondrial function testing

FCCP (in respirometry)

An uncoupling agent used to reveal maximal respiration capacity by collapsing the proton gradient

Rotenone (in respirometry)

A Complex I inhibitor used in mitochondrial respirometry assays

Antimycin (in respirometry)

A Complex III inhibitor used in mitochondrial respirometry assays, often paired with rotenone to shut down mitochondrial respiration and reveal non-mitochondrial oxygen consumption

Spare respiratory capacity

The extra capacity mitochondria have to produce ATP beyond basal respiration, revealed by uncouplers like FCCP; flavonoids (E+Q) have been shown to increase this capacity and protect the brain from stroke damage

What happens to a cardiac cell during massive mitochondrial depolarization?

ATP is no longer produced, leading to cell contraction from lack of ATP and cell death within a few minutes

Peroxisomes

Membrane-bound organelles (single membrane) that break down alcohol, toxins, and fatty acids, and are involved in lipid biosynthesis

What energy-rich molecules result from peroxisomal fatty acid hydrolysis?

FADH2 and NADH

How does peroxisomal fatty acid oxidation differ from mitochondrial fatty acid oxidation?

Both break down fatty acyl-CoA through similar chemical steps producing FADH2/NADH, but peroxisomes use an oxidase step that generates and then breaks down H2O2 via catalase, rather than feeding electrons directly into a respiratory chain like mitochondria do

Mitochondrial ultrastructure components (summary)

Outer membrane, inner membrane, intermembrane space, matrix, and cristae