Unit 5: Mitochondria and Chloroplasts

I. Orientation — Semiautonomous Energy-Converting Organelles

Mitochondria and chloroplasts are membrane-bound organelles that convert energy through electron transport, chemiosmosis, and ATP synthesis. Their bacterial-like features support the endosymbiotic theory: mitochondria arose from an alphaproteobacterium, while chloroplasts arose from a cyanobacterium, although most ancestral genes were transferred to the nucleus.

• Shared principle: Both organelles generate a proton gradient across an internal membrane and use ATP synthase to convert that gradient into ATP.
• Endosymbiotic characteristics: Both contain circular DNA, bacterial-type ribosomes, double membranes, and divide by a process resembling binary fission.
• Genomic dependence: Neither organelle is genetically autonomous; most of its proteins are encoded in the nuclear genome and imported after translation in the cytosol.
• Compartmentalization: Specific membranes and aqueous spaces separate electron transport, metabolite processing, genome expression, and protein targeting.
• Protein-targeting convention: Nuclear-encoded proteins generally contain targeting information in their amino-acid sequences, often as cleavable N-terminal transit peptides or presequences.

II. Mitochondria — Architecture, Bioenergetics, and Protein Distribution

Mitochondria are dynamic organelles specialized mainly for oxidative phosphorylation, although they also participate in metabolism, calcium regulation, apoptosis, and biosynthetic pathways. Their internal organization allows electron transport and ATP production to occur efficiently.

A. Organization and function of mitochondria

The organization of mitochondria creates distinct compartments whose membranes and enzymes support energy conversion.

• Outer membrane: This smooth boundary contains porins, or voltage-dependent anion channels, allowing many metabolites and ions smaller than approximately 5 kDa to cross relatively freely.
• Intermembrane space: This compartment contains cytochrome c and is chemically similar to the cytosol for small solutes because of outer-membrane porins.
• Inner membrane: This highly selective membrane contains respiratory complexes I–IV, ATP synthase, transporters, and cardiolipin; it is folded into cristae to increase surface area.
• Matrix: The innermost compartment contains mitochondrial DNA, mitochondrial ribosomes, enzymes of the citric acid cycle, and enzymes for fatty-acid β-oxidation.
• Chemiosmotic function: Electrons from NADH and FADH₂ pass through the respiratory chain, and complexes I, III, and IV pump H⁺ from the matrix into the intermembrane space.
• ATP synthesis: The proton-motive force drives H⁺ through the F₀ portion of ATP synthase; rotation of F₀ induces catalytic changes in F₁ that convert ADP + Pi into ATP.
• Other roles: Mitochondria regulate programmed cell death by releasing cytochrome c, buffer Ca²⁺, and synthesize compounds including heme intermediates and steroid precursors.

B. Mitochondrial genome

The mitochondrial genome is a compact, maternally inherited genetic system that retains a small set of genes essential for oxidative phosphorylation.

• Genome structure: Human mitochondrial DNA is a circular molecule of approximately 16.6 kb and is present in multiple copies per mitochondrion.
• Encoded products: Human mtDNA encodes 13 oxidative-phosphorylation polypeptides, 22 transfer RNAs, and 2 ribosomal RNAs; nearly all other mitochondrial proteins are nuclear encoded.
• Gene expression: Mitochondrial transcription produces polycistronic RNAs, which are processed and translated by mitochondrial ribosomes in the matrix or near the inner membrane.
• Genetic code: Mitochondria use a modified genetic code in some organisms; for example, human mitochondrial UGA specifies tryptophan rather than serving as a stop codon.
• Inheritance: Human mtDNA is usually transmitted through the egg cytoplasm, producing predominantly maternal inheritance.
• Heteroplasmy: A cell may contain normal and mutant mtDNA molecules simultaneously. The phenotype depends partly on the proportion of mutant genomes and the energetic threshold of each tissue.
• Mutation consequences: Mutations in mtDNA can impair respiratory complexes, especially in energy-demanding tissues such as muscle, brain, and heart.

C. Import of mitochondrial inner membrane proteins

Most inner-membrane proteins are synthesized on cytosolic ribosomes and imported through coordinated translocons in the mitochondrial envelope.

• Targeting signal: A matrix-directed precursor commonly contains an N-terminal amphipathic, positively charged presequence lacking acidic residues; it is recognized by receptors of the TOM complex in the outer membrane.
• Outer-membrane entry: The TOM40 channel transports the unfolded precursor into the intermembrane space.
• Inner-membrane routes: The precursor may use the TIM23 pathway, the TIM22 pathway, or an insertion route involving the outer-membrane SAM machinery and inner-membrane Oxa1.
• TIM23 pathway: Presequences guide proteins through TOM and TIM23; a matrix-targeting sequence can be cleaved, while a hydrophobic stop-transfer sequence arrests translocation and anchors the protein in the inner membrane.
• TIM22 pathway: Multi-pass carrier proteins, such as the ADP/ATP translocase, use internal targeting signals and are inserted by the TIM22 carrier translocase.
• Energy requirements: Import depends on cytosolic chaperones that prevent aggregation, ATP-dependent mitochondrial chaperones, and the inner-membrane electrical potential, Δψ.
• Example: A carrier protein with several internal hydrophobic segments is kept soluble in the cytosol, passes through TOM, binds small Tim chaperones in the intermembrane space, and is inserted by TIM22.

D. Protein sorting to different mitochondrial compartments

Mitochondrial proteins reach the matrix, inner membrane, intermembrane space, or outer membrane through distinct signals and translocation mechanisms.

• Matrix targeting: An N-terminal presequence carries the protein through TOM and TIM23; mitochondrial processing peptidase often removes the presequence after matrix entry.
• Inner-membrane targeting: A hydrophobic stop-transfer sequence can laterally exit TIM23 into the inner membrane, or multiple internal signals can direct a carrier through TIM22.
• Intermembrane-space targeting: Some proteins contain cysteine motifs, such as twin Cx₉C sequences, that are oxidized and trapped by the MIA machinery rather than transported into the matrix.
• Outer-membrane targeting: β-barrel proteins first cross TOM and are folded into the outer membrane by the SAM complex; tail-anchored proteins can use specialized insertion pathways.
• Sorting logic: The final destination depends on signal location, hydrophobicity, cleavage, folding, and the order in which translocons interact with the precursor.
• Quality control: Import defects can trigger proteases and chaperones that remove stalled or misfolded proteins, preserving membrane function.

III. Chloroplasts — Photosynthetic Architecture and Protein Trafficking

Chloroplasts are plastids specialized for oxygenic photosynthesis and carbon assimilation. Their internal membranes separate light capture from carbon fixation and provide extensive surfaces for electron transport.

A. Structure and function of chloroplasts

Chloroplast structure reflects the sequence of photosynthesis: light energy is captured in thylakoids, while carbon fixation occurs in the surrounding stroma.

• Envelope: The outer and inner envelope membranes enclose the chloroplast; the intermembrane space lies between them, and selective transporters control metabolite exchange with the cytosol.
• Stroma: This aqueous compartment contains chloroplast DNA, 70S ribosomes, enzymes of the Calvin–Benson cycle, starch granules, and metabolic pathways for fatty acids and amino acids.
• Thylakoid system: Flattened thylakoid sacs form interconnected membranes suspended in the stroma. Stacked regions are called grana, while unstacked regions are stroma lamellae.
• Photosystems: Photosystem II oxidizes water and releases O₂; photosystem I transfers high-energy electrons to NADP⁺, producing NADPH.
• Electron transport: Electrons move from water through plastoquinone, cytochrome b₆f, plastocyanin, and the photosystems. Cytochrome b₆f contributes to H⁺ accumulation in the thylakoid lumen.
• ATP production: Light-driven electron transport establishes a proton gradient with high H⁺ concentration in the lumen; ATP synthase allows H⁺ to return to the stroma and synthesizes ATP.
• Carbon fixation: Rubisco catalyzes the addition of CO₂ to ribulose-1,5-bisphosphate in the stroma. ATP and NADPH then support production of carbohydrate during the Calvin–Benson cycle.
• Additional functions: Chloroplasts synthesize lipids, pigments, amino acids, and some hormones; they also participate in redox signaling and immune responses.

B. Chloroplast genome

The chloroplast genome preserves bacterial-like genetic features but works together with the nuclear genome to build a functional photosynthetic organelle.

• Genome structure: A typical chloroplast genome is a circular DNA molecule of about 120–160 kb, often organized into large and small single-copy regions separated by inverted repeats.
• Encoded products: Plastid DNA encodes selected subunits of photosystems, cytochrome b₆f, ATP synthase, ribosomal RNAs, transfer RNAs, and some ribosomal proteins.
• Gene expression: Chloroplast genes are transcribed and translated within the stroma using bacterial-type RNA polymerases and 70S ribosomes, although nuclear-encoded factors control much of this expression.
• Maternal inheritance: In many flowering plants, chloroplasts are inherited maternally because the egg contributes most of the cytoplasm; inheritance patterns can differ among species.
• Genome reduction: Endosymbiotic gene transfer moved many ancestral plastid genes to the nucleus, explaining why chloroplasts depend heavily on imported proteins.
• Functional coordination: Nuclear and chloroplast genomes must coordinate expression of protein subunits; for example, photosynthetic complexes contain products from both genomes.
• Genome variation: Plastid DNA can occur in multiple copies and may show heteroplasmy, producing mixtures of normal and altered genomes within a cell or organism.

C. Import and sorting of chloroplast proteins

Chloroplast proteins encoded by nuclear DNA are synthesized in the cytosol and directed first across the envelope before being sorted to the stroma, thylakoid, inner envelope, or intermembrane space.

• Transit peptide: A chloroplast precursor usually carries an N-terminal transit peptide enriched in hydroxylated and basic residues but lacking a strongly hydrophobic signal peptide; it is removed after import.
• Envelope entry: The TOC complex in the outer membrane recognizes the precursor, and the TIC machinery in the inner membrane transfers it into the stroma.
• Stromal delivery: Stromal proteins are released after transit-peptide cleavage by stromal processing peptidase and then fold with the assistance of chaperones such as Hsp70 and chaperonins.
• Thylakoid targeting: Proteins destined for thylakoids acquire additional signals that use one of four routes:
  • Sec pathway: Transports soluble proteins across the thylakoid membrane using a Sec-dependent signal and the proton gradient.
  • Tat pathway: Transports folded proteins containing twin-arginine motifs, powered by the thylakoid ΔpH.
  • SRP pathway: Directs light-harvesting and other membrane proteins to the thylakoid membrane through signal-recognition-particle components.
  • Spontaneous or assisted insertion: Some proteins insert through local hydrophobic sequences or accessory insertion factors.
• Envelope sorting: Inner-envelope proteins may retain transmembrane segments or use specific envelope translocases after crossing TOC/TIC; intermembrane-space proteins can be released through specialized envelope routes.
• Signal removal: Sequential cleavage is common: a stromal processing peptidase removes the chloroplast transit peptide, and a thylakoid processing peptidase may remove a second signal.
• Example: A nuclear-encoded thylakoid lumen protein first crosses TOC/TIC, then uses a lumen-targeting sequence and the Tat pathway to cross the thylakoid membrane in a folded state.