Premed · Premed · Cell Biology
Lecture 14: Mitochondria and Chloroplasts: Structure and Biogenesis
Cell Biology
Learning Objectives
By the end of this lecture, students will be able to:
- Describe the structure of mitochondria and chloroplasts in detail
- Explain the endosymbiotic theory and the evidence supporting it
- Describe oxidative phosphorylation and the chemiosmotic mechanism
- Explain how proteins are imported into mitochondria and chloroplasts
- Describe mitochondrial dynamics (fusion, fission) and their significance
Lecture Content
I. Mitochondrial Structure
Mitochondria are double-membrane-bound organelles approximately 0.5 to 1 micrometer in diameter and 1 to 10 micrometers in length. They are highly dynamic, constantly undergoing fusion, fission, and movement along the cytoskeleton. The number of mitochondria per cell varies widely, ranging from hundreds to thousands, with the highest densities in metabolically active cells such as cardiac muscle cells and hepatocytes.
The outer membrane is permeable to molecules smaller than approximately 5 kDa through porins (specifically VDAC, the voltage-dependent anion channel). It also houses the TOM complex (Translocase of the Outer Membrane) for protein import and is the site of fatty acid elongation and phospholipid transfer. The intermembrane space (IMS) has a composition similar to the cytosol for small molecules but contains important signaling proteins including cytochrome c and pro-apoptotic factors such as Smac/DIABLO, AIF, and endonuclease G.
The inner membrane is the functional heart of the mitochondrion. It is highly folded into cristae that greatly increase its surface area. Unlike the outer membrane, it is impermeable to most ions and small molecules, requiring specific transporters for passage. It contains the electron transport chain complexes and ATP synthase, and is uniquely rich in cardiolipin (diphosphatidylglycerol), a lipid important for ETC function. The TIM complexes (TIM23 and TIM22) reside in the inner membrane for protein import. Cristae are connected to the inner boundary membrane through narrow tubular openings maintained by the MICOS complex (Mitochondrial Contact Site and Cristae Organizing System). The dynamin-related GTPase OPA1 shapes cristae structure, and mutations in OPA1 cause autosomal dominant optic atrophy.
The matrix contains the mitochondrial genome (mtDNA), mitochondrial ribosomes (55S), and tRNAs. It houses the enzymes for the TCA cycle, fatty acid beta-oxidation, and amino acid catabolism, as well as mitochondrial chaperones including Hsp60 and mtHsp70.
II. The Endosymbiotic Theory
The endosymbiotic theory, proposed by Lynn Margulis in 1967, holds that mitochondria and chloroplasts originated from ancient prokaryotes that were engulfed by ancestral eukaryotic cells and subsequently evolved into obligate endosymbionts. The evidence supporting this theory is compelling and multifaceted.
The double membrane is consistent with an engulfment event, with the inner membrane deriving from the ancestral prokaryote and the outer from the host cell. Mitochondria possess their own DNA, which is circular and double-stranded, similar to bacterial chromosomes. Human mtDNA is 16,569 base pairs and encodes 13 proteins, 22 tRNAs, and 2 rRNAs. Unlike nuclear DNA, mtDNA lacks histones and is instead associated with the protein TFAM. Mitochondrial ribosomes are 55S, more similar to bacterial 70S ribosomes than to eukaryotic 80S ribosomes. They are sensitive to bacterial ribosome inhibitors such as chloramphenicol and erythromycin but not to cycloheximide, which inhibits eukaryotic ribosomes. Mitochondria divide by binary fission, independently of the cell cycle. Phylogenetic analysis reveals that mitochondrial genes are most closely related to those of alpha-proteobacteria, while chloroplast genes trace their ancestry to cyanobacteria.
Over evolutionary time, most mitochondrial genes have been transferred to the nuclear genome. Human mtDNA encodes only 37 genes, but approximately 1,500 proteins function in mitochondria. The vast majority are encoded by nuclear DNA, synthesized on cytoplasmic ribosomes, and imported into the organelle.
III. Oxidative Phosphorylation
The electron transport chain (ETC) consists of a series of protein complexes in the inner membrane that transfer electrons from NADH and FADH2 to molecular oxygen while pumping protons from the matrix to the IMS. Complex I (NADH dehydrogenase) accepts electrons from NADH and transfers them to ubiquinone while pumping 4 H+. Complex II (succinate dehydrogenase), which also functions in the TCA cycle to convert succinate to fumarate, transfers electrons from FADH2 to ubiquinone without pumping protons. Ubiquinone (coenzyme Q) is a mobile electron carrier in the lipid bilayer that transfers electrons to Complex III (cytochrome bc1), which passes them to the mobile carrier cytochrome c in the IMS while pumping 4 H+. Complex IV (cytochrome c oxidase) completes the chain by transferring electrons to molecular oxygen, forming water (O2 + 4H+ + 4e- -> 2H2O) and pumping 2 H+. In total, 10 H+ are pumped per NADH and 6 H+ per FADH2.
The chemiosmotic hypothesis, proposed by Peter Mitchell (Nobel Prize, 1978), explains how the ETC drives ATP synthesis. Proton pumping creates a proton-motive force across the inner membrane, consisting of two components: a pH gradient (delta-pH) and a membrane potential (delta-psi, approximately 180 mV, inside negative). Protons flow back into the matrix through ATP synthase (Complex V), and this flow drives ATP synthesis.
ATP synthase is a remarkable rotary molecular machine. Its F0 subunit is embedded in the membrane and contains a proton channel with a c-ring. The F1 subunit projects into the matrix and contains the catalytic alpha3-beta3 hexamer connected to the c-ring by the gamma stalk. Proton flow through F0 rotates the c-ring and gamma subunit, and the rotation of gamma within F1 induces conformational changes in the beta subunits that drive ATP synthesis through the binding change mechanism, elucidated by Paul Boyer (Nobel Prize, 1997). Each complete 360-degree rotation produces 3 ATP molecules. Approximately 3 to 4 protons are consumed per ATP, yielding roughly 30 to 32 ATP per glucose molecule from complete oxidation.
Uncoupling dissipates the proton gradient without producing ATP. Uncoupling protein 1 (UCP1) in brown adipose tissue allows protons to flow back across the inner membrane, generating heat for thermogenesis. Chemical uncouplers such as DNP and FCCP carry protons across the membrane, collapsing the gradient.
<image>Oxidative phosphorylation and the electron transport chain. Panel A: Cross-section of the inner mitochondrial membrane showing Complexes I-IV arranged in sequence with electron flow from NADH/FADH2 to O2 (blue arrows for electrons, red arrows for H+ pumping from matrix to IMS). Ubiquinone and cytochrome c shown as mobile carriers. ATP synthase (Complex V) shown with H+ flowing back into the matrix driving ATP synthesis. Panel B: Detailed structure of ATP synthase showing the F0 rotor (c-ring) in the membrane, the gamma stalk, and the F1 catalytic head (alpha3-beta3) in the matrix. Arrows indicate rotation direction. Panel C: Energy diagram showing the free energy drop at each complex as electrons move from NADH (high energy) to O2 (low energy).</image>
IV. Protein Import into Mitochondria
Approximately 99 percent of mitochondrial proteins are encoded by nuclear DNA and must be imported after synthesis in the cytoplasm. The matrix-targeting sequence (MTS) is a positively charged, amphipathic alpha-helix at the N-terminus that directs proteins to the mitochondrial matrix. It is cleaved by the mitochondrial processing peptidase (MPP) after import. Internal targeting signals direct proteins to the IMS, inner membrane, or outer membrane.
The import machinery consists of several coordinated complexes. The TOM complex on the outer membrane serves as the entry gate, with Tom40 forming the beta-barrel translocation pore and Tom20 and Tom22 acting as receptors that recognize the MTS. The TIM23 complex in the inner membrane imports matrix-destined proteins and some inner membrane proteins. Import through TIM23 is driven by the membrane potential (delta-psi), which exerts an electrophoretic pull on the positively charged MTS, and by mtHsp70 (part of the PAM motor), which acts as a molecular ratchet by binding the emerging polypeptide and preventing backsliding. The TIM22 complex imports multi-pass inner membrane proteins such as the ADP/ATP translocase, using small TIM chaperones in the IMS as intermediaries. The SAM complex inserts beta-barrel proteins (such as VDAC and Tom40 itself) into the outer membrane. The MIA pathway imports small IMS proteins with twin-CXnC motifs through an oxidative folding mechanism. Importantly, proteins are generally imported in an unfolded state, with cytoplasmic Hsp70 chaperones keeping them unfolded until import.
V. Chloroplast Structure and Function (Overview)
Chloroplasts are found exclusively in photosynthetic eukaryotes (plants and algae) and, like mitochondria, possess a double membrane. The outer membrane is permeable to small molecules, while the inner membrane contains specific transporters. Within the inner membrane lies the thylakoid membrane, which is the site of the light reactions of photosynthesis and houses photosystems I and II, the cytochrome b6f complex, and ATP synthase. Thylakoids can stack into grana, connected by stroma lamellae. The stroma, equivalent to the mitochondrial matrix, is the site of the Calvin cycle for carbon fixation. Chloroplast DNA is considerably larger than mtDNA (approximately 120 to 160 kb) and encodes about 100 proteins. Chloroplasts contain 70S ribosomes similar to those of bacteria.
Photosynthesis can be summarized in two phases: the light reactions (on the thylakoid membrane) split water to produce O2, NADPH, and ATP, while the Calvin cycle (in the stroma) uses ATP and NADPH to fix CO2 into the three-carbon sugar G3P, which is subsequently converted to glucose. Protein import into chloroplasts uses the TOC and TIC complexes, with a cleavable transit peptide analogous to the mitochondrial MTS.
VI. Mitochondrial Dynamics and Quality Control
Mitochondria are far from static organelles. They constantly undergo fusion and fission, processes that are essential for their function and quality control. Mitochondrial fusion is mediated by Mitofusin 1 and 2 (Mfn1/Mfn2, dynamin-related GTPases) for outer membrane fusion and by OPA1 for inner membrane fusion. Fusion allows content mixing and complementation of damaged components between mitochondria. Mutations in Mfn2 cause Charcot-Marie-Tooth type 2A peripheral neuropathy, and OPA1 mutations cause autosomal dominant optic atrophy.
Mitochondrial fission is driven by Drp1 (Dynamin-related protein 1), a cytoplasmic GTPase recruited to the outer membrane by adaptors including Fis1, Mff, and MiD49/51. Notably, ER-mitochondria contact sites mark the positions where fission will occur, with ER tubules wrapping around the mitochondrion to pre-constrict it before Drp1 assembly. Fission enables the segregation of damaged mitochondria for removal by mitophagy.
Mitophagy via the PINK1/Parkin pathway is the primary quality control mechanism for damaged mitochondria. In healthy mitochondria, PINK1 is imported and degraded by the PARL protease. When mitochondria are damaged and lose their membrane potential, PINK1 accumulates on the outer membrane. PINK1 phosphorylates ubiquitin and recruits Parkin (an E3 ubiquitin ligase) from the cytoplasm. Parkin ubiquitinates outer membrane proteins, and autophagy receptors (p62, OPTN, NDP52) bridge the ubiquitin chains to LC3 on the forming autophagosome. The damaged mitochondrion is engulfed and degraded. Mutations in PINK1 and Parkin cause autosomal recessive Parkinson's disease, highlighting the importance of mitochondrial quality control for neuronal health.
Mitochondria also play a central role in apoptosis through the release of cytochrome c from the IMS, which triggers apoptosome formation and caspase activation (discussed in detail in Lecture 22). Mitochondrial diseases result from mutations in mtDNA and exhibit distinctive genetic features: maternal inheritance, heteroplasmy (mixture of mutant and wild-type mtDNA in the same cell), and a threshold effect (symptoms appear when the proportion of mutant mtDNA exceeds a critical level). Important examples include MELAS (mitochondrial encephalomyopathy, lactic acidosis, and stroke-like episodes), MERRF (myoclonic epilepsy with ragged red fibers), Leber hereditary optic neuropathy (LHON), and Kearns-Sayre syndrome (caused by large mtDNA deletions).
<image>Mitochondrial dynamics: fusion, fission, and mitophagy. Panel A (Fusion): Two mitochondria approaching; Mfn1/2 on outer membranes tether and fuse the outer membranes; OPA1 mediates inner membrane fusion; result is one elongated mitochondrion with mixed contents. Panel B (Fission): ER tubule wrapping around a mitochondrion at the pre-constriction site; Drp1 rings recruited to the constriction site via Mff/Fis1 adaptors; GTP hydrolysis by Drp1 constricts and severs the mitochondrion into two. Panel C (Mitophagy): Damaged mitochondrion (depolarized) with PINK1 accumulating on the outer membrane; Parkin recruited, ubiquitinates outer membrane proteins; autophagy receptors (p62) link ubiquitinated mitochondrion to LC3 on the autophagosome; engulfment and fusion with lysosome for degradation.</image>
