Lec 13 - Mitochondria and Peroxisomes

What are the four mitochondrial sub-compartments

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

Are the outer and inner mitochondrial membranes each a lipid bilayer

Yes, each is a separate lipid bilayer.

What is the function of cristae in the inner mitochondrial membrane

They increase surface area for oxidative phosphorylation.

What is the intermembrane space composition relative to cytosol

It is similar to cytosol and contains enzymes that use matrix ATP to phosphorylate nucleotides.

What key processes occur in the mitochondrial matrix

Oxidation of pyruvate and fatty acids, the TCA cycle, and housing mtDNA, mitoribosomes, and tRNAs.

What is the permeability feature of the outer mitochondrial membrane

It is permeable to ions and small hydrophilic molecules via porins.

Why must the inner mitochondrial membrane be impermeable to ions

To maintain the proton gradient required for ATP synthase function.

Where are pyruvate and fatty acid transporters located

In the inner mitochondrial membrane.

What are the approximate pH values in mitochondrial compartments

Intermembrane space ~7, matrix ~8.

What drives ATP synthesis in mitochondria

The electrochemical gradient of H+ across the inner membrane drives ATP synthase.

What transporter exchanges ATP and ADP across the inner membrane

The adenine nucleotide translocator.

Why is ATP export from the matrix favored

ATP has −4 charge vs ADP −3, and the matrix is more negatively charged

How is inorganic phosphate imported into the matrix

Via a phosphate transporter that imports H2PO4− in exchange for OH−, driven by the proton gradient.

What ensures directionality of metabolite transport across the inner membrane

Both the electrical and chemical components of the proton motive force.

What percentage of the inner mitochondrial membrane is cardiolipin

Approximately 20%.

What is cardiolipin’s structural hallmark

Double phospholipid with two phosphate headgroups and four fatty acyl chains.

What types of fatty acyl chains are typical in mature cardiolipin

Predominantly unsaturated fatty acyl chains.

How does cardiolipin shape affect membranes

Its conical shape promotes negative curvature, supporting cristae formation.

How does cardiolipin improve oxidative phosphorylation efficiency

Its double phosphates create elevated negative charge that traps protons, restricting leak and maintaining matrix pH.

From which precursor and location does cardiolipin biosynthesis begin

From ER-derived phosphatidic acid, then remodeled within mitochondrial membranes.

Why is the proton gradient essential for ATP production

It provides the energy for ATP synthase to phosphorylate ADP.

What happens when the proton gradient is dissipated

ATP generation falls and energy is released as heat (e.g., brown fat thermogenesis).

What is the size and form of human mtDNA

Circular, double-stranded, ~16.5 kb.

How many genes are encoded by mtDNA, and of what types

37 genes: 13 proteins (ETC complexes I, III, IV, V), 2 rRNAs, and 22 tRNAs.

How many copies of mtDNA are present per mitochondrion

Approximately 2–10 copies per mitochondrion.

How many mitochondria can be present per cell

Hundreds per cell.

Do mitochondria have robust DNA repair

No; limited repair leads to mutation accumulation with age.

What is the evolutionary origin of mitochondria

Endosymbiosis of alpha-proteobacteria into ancestral eukaryotes.

How are mtDNA transcripts organized

Both heavy and light strands are transcribed as polycistronic RNAs then processed and polyadenylated.

What distinguishes the heavy strand of mtDNA

Enriched in guanine, making it biochemically heavier than the light strand.

How can mitochondria translate proteins with only 22 tRNAs

Via “extreme wobble” where U in the tRNA anticodon pairs with any base in the third codon position.

Is the mitochondrial genetic code identical to the universal code

No; some codons are reassigned in mitochondria.

What is replicative segregation in mitochondrial genetics

Random replication and distribution of mtDNA among mitochondria and random partitioning into daughter cells.

Define homoplasmy and heteroplasmy.

Homoplasmy: pure mutant or normal mtDNA; heteroplasmy: mixture of mutant and normal mtDNA.

What is the threshold effect in mitochondrial disease expression

Symptoms occur when mutant mitochondria exceed a tissue-specific threshold.

How is mtDNA inherited

Almost exclusively maternally; sperm mtDNA is eliminated.

What processes enable sharing and distribution of mitochondrial contents

Fusion and fission with microtubule-motor positioning near high-energy demand sites.

Which organ systems are most affected by mitochondrial diseases and why

CNS, skeletal and cardiac muscle, eye, and liver due to high energy needs.

What common neurological manifestations occur in mitochondrial disease

Seizures, ataxia, migraines, developmental delay, and dementia.

What cardiac manifestations are typical in mitochondrial disease

Cardiomyopathy and conduction defects.

What skeletal muscle findings suggest mitochondrial myopathy

Weakness, exercise intolerance, cramps, and myopathy.

What ophthalmic findings can occur in mitochondrial disorders

Ptosis, external ophthalmoplegia, retinopathy, optic neuropathy, and cataracts.

What gastrointestinal problems may be seen in mitochondrial disease

Vomiting, chronic diarrhea, and pseudo-obstruction due to smooth muscle dysfunction.

Define MELAS and list hallmark features.

Mitochondrial encephalomyopathy, lactic acidosis, and stroke-like episodes; early onset with exercise intolerance, seizures, dementia, and cardiac issues.

Define Kearns–Sayre syndrome (KSS) and onset.

Onset <20 years; ophthalmoplegia, pigmentary retinopathy, heart block, and neurologic deficits.

Define Leigh syndrome and typical presentation.

Subacute necrotizing encephalomyopathy in infancy with brain abnormalities, seizures, feeding difficulties, and cardiac problems.

Define MERRF and classic features.

Myoclonus epilepsy with ragged-red fibers; epilepsy, ataxia, dementia, and muscle weakness.

Define LHON and typical course.

Leber’s hereditary optic neuropathy; unilateral optic neuropathy progressing to the other eye, typically age 10–30.

Why can two people with the same mtDNA mutation have different phenotypes

Differences in heteroplasmy levels and tissue distribution alter threshold expression.

How does aging influence mtDNA mutations and disease risk

Oxidative damage and limited repair increase mutations, impairing oxidative phosphorylation.

Approximately how many mitochondrial proteins are encoded by nuclear DNA

About 1500 proteins.

Where are nuclear-encoded mitochondrial proteins synthesized

On free cytosolic ribosomes.

What is a matrix targeting signal (MTS) and where is it located

An N-terminal, 20–35 aa, positively charged amphipathic helix directing proteins to the matrix.

Is the MTS (mitochondrial targetting sequence) removed after import

Yes, by matrix processing peptidase (MPP).

Must proteins be unfolded for mitochondrial import

Yes, at least partially; chaperones (cytosolic Hsp70 and mtHsp70) assist.

Which complexes mediate translocation across outer and inner mitochondrial membranes

TOM (outer) and TIM23 (inner) for matrix and many IMM proteins.

What energy sources drive matrix import

ATP in cytosol and matrix plus the inner membrane potential.

How do positive charges in the MTS facilitate import

They are electrophoretically pulled into the negatively charged matrix across the potential.

How are single-pass inner membrane proteins targeted

MTS plus an internal hydrophobic stop-transfer sequence enabling lateral insertion.

How are multi-pass inner membrane proteins imported

Internal targeting signals enter via TOM; Tim9/Tim10 chaperones deliver to TIM22 for insertion.

How are mtDNA-encoded IMM proteins inserted

Synthesized on mitoribosomes and inserted via the Oxa1 translocase.

How are single-pass outer membrane proteins inserted

Via Mim1 after TOM passage as needed.

How are beta-barrel outer membrane proteins assembled

TOM → Tim9/Tim10 chaperones → SAM complex for insertion.

How are intermembrane space proteins targeted and folded

They use internal cysteine-rich sequences and Mia40-mediated disulfide bonding.

What are the key targeting signals for major mitochondrial destinations

Matrix: N-terminal MTS (cleaved); IMM single-pass: MTS + stop-transfer; IMM multi-pass: internal hydrophobic signals; IMS: cysteine motifs with Mia40; OMM single-pass: Mim1; OMM beta-barrel: TOM→SAM.

How do lipids move from ER to mitochondria

By cytosolic phospholipid transfer proteins delivering to the OMM.

How are lipids transferred between OMM and IMM

By protein-dependent carriers and protein-independent mechanisms at contact sites.

What gives peroxisomes their name

They house oxidative enzymes that generate H2O2, sequestered to prevent damage.

How is H2O2 detoxified in peroxisomes

Catalase converts 2H2O2 → 2H2O + O2 or uses H2O2 to oxidize substrates.

Do peroxisomes contain DNA

No; they lack DNA and have a single membrane bilayer.

Approximately how many peroxisomes and enzymes do cells have

~500 per cell with >50 matrix enzymes.

Which fatty acids are primarily oxidized in peroxisomes

Very long-chain fatty acids (VLCFAs).

What is alpha-oxidation in peroxisomes and for what substrates

Alpha-oxidation of branched-chain fatty acids occurs only in peroxisomes to prepare for beta-oxidation.

What lipids are synthesized in peroxisomes

Plasmalogens plus contributions to cholesterol and dolichol; bile acids in liver.

Why are plasmalogens important

Major myelin phospholipids and “sacrificial oxidants” protecting membranes.

What is dolichol’s role and where is it synthesized

Carrier for N-linked glycosylation; synthesized in peroxisomes and ER.

From where do peroxisomal membrane proteins (peroxins) originate

From ER-derived membranes that fuse into mature peroxisomes.

Where are peroxisomal matrix proteins synthesized and in what folding state are they imported

Synthesized on cytosolic ribosomes and imported as fully folded proteins.

What is PTS1 and which receptor recognizes it

The C-terminal SKL signal recognized by Pex5.

Which peroxins form the docking/translocation site for matrix import

Pex13, Pex14, and Pex17.

How is Pex5 handled after cargo delivery

It is recycled for additional transport rounds.

What is PTS2 and which receptor recognizes it

An N-terminal targeting sequence recognized by Pex7.

What disorders result from peroxin mutations

Peroxisome biogenesis disorders due to defective assembly/import.

How does peroxin mutation severity relate to disease spectrum

Complete loss causes severe disease; partial loss yields milder Zellweger spectrum disorders.

What is the Zellweger spectrum, ordered by severity

Zellweger syndrome > Neonatal adrenoleukodystrophy > Infantile Refsum disease.

What are hallmark features of Zellweger syndrome

High forehead, low nasal bridge, micrognathia, severe hypotonia, hyporeflexia, hepatomegaly, seizures, psychomotor retardation, nystagmus.

What peroxisomal finding characterizes Zellweger fibroblasts

Peroxisomes are not detected but can be reconstituted by PXR1.

What defines RCDP type 1 clinically

Short limbs, small stature, cataracts, psychomotor disability, and failed plasmalogen synthesis.

What metabolic defect underlies X-linked adrenoleukodystrophy

Defective peroxisomal beta-oxidation of VLCFAs leading to demyelination.

Which receptor pathway defects map to PTS1 vs PTS2 diseases

Pex5 affects PTS1 import; Pex7 defects cause RCDP via impaired PTS2 import.

How does mitochondrial matrix import differ from nuclear import regarding folding state

Mitochondrial import needs partially unfolded proteins; nuclear import accepts fully folded proteins.

What is unique about peroxisomal matrix import vs mitochondrial import

Peroxisomes import fully folded proteins via PTS receptors, unlike mitochondria.

Which mitochondrial import pathway requires the membrane potential

TIM23-mediated translocation into the matrix.

Which organelle uses Oxa1 for membrane insertion and for which proteins

Mitochondria use Oxa1 to insert mtDNA-encoded inner membrane proteins.

What happens to pyruvate after cytosolic glycolysis regarding mitochondrial entry

It crosses outer membrane porins to the IMS, then uses an inner membrane transporter.

What other key metabolites are specifically transported into mitochondria

Fatty acids, phosphate, ADP, and ions via selective transporters.

What effect does a higher matrix pH have on import of positively charged presequences

The negative matrix potential attracts them, aiding import.

What are Hsp70 roles in mitochondrial import

Cytosolic Hsp70 maintains partial unfolding; mtHsp70 acts as import motor and assists folding.

What is a stop-transfer sequence

A hydrophobic alpha-helix that halts translocation to allow lateral membrane insertion.

What are Tim9/Tim10 roles

IMS chaperones that shield hydrophobic segments and deliver precursors to TIM22 or SAM.