Unit 5: Mitochondria and Chloroplasts - Subjective Questions

Mitochondria are double-membrane-bound organelles responsible mainly for aerobic energy production.

• Outer membrane: Contains porins and is relatively permeable to small molecules and ions. It also contains receptors involved in protein import.
• Intermembrane space: Lies between the outer and inner membranes. It contains proteins such as cytochrome and factors involved in apoptosis.
• Inner membrane: Highly selective and folded into structures called cristae, which increase the surface area for oxidative phosphorylation. It contains the electron transport chain complexes, ATP synthase, and transport proteins.
• Matrix: The innermost compartment contains mitochondrial DNA, ribosomes, enzymes of the citric acid cycle, and enzymes for fatty acid oxidation.

This compartmental organization allows electron transport, proton pumping, ATP synthesis, and metabolic reactions to occur efficiently in distinct regions.

Mitochondria perform several essential functions:

• ATP production: Electrons from NADH and FADH pass through the respiratory chain, creating a proton gradient used by ATP synthase to produce ATP.
• Citric acid cycle: Enzymes in the matrix oxidize acetyl-CoA and generate NADH, FADH, and GTP or ATP.
• Fatty acid oxidation: Fatty acids are broken down by -oxidation to produce acetyl-CoA and reduced electron carriers.
• Calcium storage: Mitochondria help regulate cytosolic calcium concentrations.
• Apoptosis: Release of cytochrome from mitochondria can activate caspases and initiate programmed cell death.
• Biosynthesis: Mitochondria contribute to the synthesis of heme, steroid precursors, and certain amino acids.

Thus, mitochondria function as metabolic, signaling, and regulatory centers rather than merely as ATP-producing organelles.

The mitochondrial genome is a small, usually circular DNA molecule located in the mitochondrial matrix in structures called nucleoids.

• It is generally double-stranded and circular.
• It occurs in multiple copies per mitochondrion and multiple mitochondria per cell.
• It encodes a limited number of proteins, mainly components of the respiratory chain, as well as mitochondrial rRNAs and tRNAs.
• Most mitochondrial proteins are encoded by nuclear genes and imported after synthesis in the cytosol.
• Mitochondrial DNA is usually inherited maternally in animals.
• It has limited DNA repair and lacks the extensive noncoding and regulatory organization characteristic of nuclear chromosomes.
• Mitochondrial genes are often transcribed as polycistronic RNA molecules.

These features support the view that mitochondria evolved from an ancestral bacterial endosymbiont.

The endosymbiotic theory proposes that mitochondria originated when an ancestral eukaryotic cell engulfed an aerobic bacterium. Instead of being digested, the bacterium established a mutually beneficial relationship and eventually became an organelle.

Evidence includes:

• Mitochondria contain their own circular DNA, resembling bacterial chromosomes.
• They possess 70S-like ribosomes, which are more similar to bacterial ribosomes than to cytosolic eukaryotic ribosomes.
• Mitochondria divide by a process resembling binary fission.
• They have a double membrane, consistent with engulfment by a host cell.
• Their size is comparable to that of many bacteria.
• Several mitochondrial genes and proteins show evolutionary relationships with bacterial genes.
• Mitochondrial gene expression includes bacterial-like features, such as polycistronic transcription.

Although most ancestral genes were transferred to the nucleus, mitochondria retained genes essential for their specialized functions.

Most mitochondrial proteins are encoded by nuclear genes, synthesized on free cytosolic ribosomes, and transported into mitochondria after translation.

• A precursor protein usually contains an N-terminal mitochondrial targeting sequence enriched in positively charged and hydroxylated amino acids.
• The targeting sequence forms an amphipathic -helix and lacks a long stretch of acidic residues.
• Cytosolic chaperones maintain the precursor in an unfolded or import-competent state.
• The precursor first binds receptors of the TOM complex in the outer mitochondrial membrane.
• It then passes through the TOM channel.
• Depending on its destination, it is transferred to the TIM complexes of the inner membrane or to other sorting pathways.
• After translocation, the targeting sequence is commonly removed by a matrix processing peptidase.
• Chaperones and folding enzymes assist in final maturation.

This pathway ensures that proteins reach the correct mitochondrial compartment.

Mitochondrial inner-membrane proteins use several import mechanisms depending on their targeting signals and topology.

• Presequence pathway: Proteins with an N-terminal presequence pass through the TOM complex and are transferred to the TIM23 complex. Some are released laterally into the inner membrane.
• Stop-transfer mechanism: A hydrophobic stop-transfer sequence prevents complete passage through TIM23 and causes the protein to become embedded in the inner membrane.
• Conservative sorting pathway: Certain proteins are completely imported into the matrix and then inserted into the inner membrane by the OXA translocase.
• Carrier pathway: Multipass carrier proteins lack cleavable presequences and contain internal targeting signals. They are recognized by TOM receptors, chaperoned across the intermembrane space, and inserted by the TIM22 complex.

The electrochemical membrane potential across the inner membrane is important for the movement of positively charged presequences through TIM23.

Mitochondrial protein sorting depends on targeting signals and translocation machinery.

• Matrix proteins: Usually possess cleavable N-terminal presequences. They pass through TOM and TIM23 and are processed in the matrix.
• Inner-membrane proteins: May use presequences and the TIM23 stop-transfer pathway, internal targeting signals and TIM22, or matrix import followed by OXA-mediated insertion.
• Intermembrane-space proteins: Some proteins pass through TOM and are released by stop-transfer signals. Others use the mitochondrial intermembrane space assembly pathway, in which cysteine-containing proteins are oxidized and stabilized by the disulfide relay system.
• Outer-membrane proteins: The beta-barrel proteins are transported through TOM and inserted by the SAM complex. Many alpha-helical outer-membrane proteins use targeting signals recognized by TOM receptors and are inserted by the mitochondrial import machinery.

Thus, distinct targeting information directs proteins to specific compartments.

A mitochondrial targeting sequence is a peptide signal that directs a newly synthesized protein to mitochondria.

• It is commonly located at the N-terminus of a precursor protein.
• It is enriched in positively charged residues such as arginine and lysine.
• It contains few or no acidic residues.
• It can form an amphipathic -helix with a hydrophobic face and a positively charged face.
• It is recognized by receptors of the TOM complex.
• The membrane potential across the inner membrane helps drive positively charged presequences toward the matrix.
• After import, the presequence is often cleaved by a matrix processing peptidase.

Unlike many secretory pathway signal peptides, mitochondrial targeting sequences are generally not strongly hydrophobic and do not usually form a permanent transmembrane segment.

The TOM and TIM complexes cooperate to import mitochondrial proteins.

The TOM complex acts as the universal entry gate, whereas TIM23 and TIM22 determine the subsequent route based on the targeting information of the precursor protein.

Mitochondrial chaperones assist proteins before, during, and after import.

• Cytosolic chaperones such as Hsp70 bind newly synthesized precursor proteins and prevent premature folding or aggregation.
• At the mitochondrial surface, chaperones help deliver precursors to TOM receptors.
• In the matrix, mitochondrial Hsp70 provides a pulling force for translocation through TIM23 and prevents imported proteins from aggregating.
• Hsp60 and related chaperonins help newly imported proteins fold into their functional conformations.
• Chaperones in the intermembrane space maintain hydrophobic carrier proteins in an import-competent state and guide them to the TIM22 pathway.
• Chaperones also assist the assembly of multisubunit respiratory complexes.

Without these proteins, hydrophobic or incompletely folded precursors could aggregate and disrupt mitochondrial membranes.

Chloroplasts are double-membrane-bound plastids found mainly in plant and algal cells.

• Outer membrane: Relatively permeable to small molecules and contains proteins involved in exchange and protein import.
• Intermembrane space: Separates the outer and inner envelope membranes.
• Inner membrane: Selectively controls movement into and out of the stroma and contains protein-import machinery.
• Stroma: The aqueous interior containing chloroplast DNA, ribosomes, enzymes of the Calvin cycle, and enzymes for biosynthetic pathways.
• Thylakoids: Flattened internal membrane sacs containing chlorophyll, photosystems, electron carriers, and ATP synthase.
• Grana: Stacks of thylakoids that increase membrane surface area.
• Stroma lamellae: Membranes connecting different grana.

The thylakoid membrane carries out light-dependent reactions, whereas the stroma is the primary site of carbon fixation.

Photosynthesis occurs in two coordinated stages.

Light-dependent reactions:

• Occur in the thylakoid membrane.
• Chlorophyll absorbs light energy in photosystems II and I.
• Photosystem II splits water, releasing oxygen, electrons, and protons.
• Electrons pass through an electron transport chain that pumps protons into the thylakoid lumen.
• The proton gradient drives ATP synthesis by ATP synthase.
• Photosystem I helps reduce NADP to NADPH.

Calvin cycle:

• Occurs in the stroma.
• Rubisco fixes carbon dioxide to ribulose bisphosphate.
• The resulting molecules are reduced using ATP and NADPH.
• Triose phosphates are produced, while some molecules regenerate the carbon dioxide acceptor.

Therefore, light reactions provide ATP and NADPH, which power carbon fixation and sugar production.

The chloroplast genome, or plastome, is located in the stroma and is usually a circular DNA molecule.

• It occurs in multiple copies within each chloroplast.
• Many plastomes contain large single-copy and small single-copy regions separated by inverted repeats.
• The genome encodes some ribosomal RNAs, transfer RNAs, and proteins required for photosynthesis and gene expression.
• Genes encode components of photosystems, the cytochrome complex, ATP synthase, and the large subunit of Rubisco in many plants.
• Chloroplast ribosomes and transcription machinery have several bacterial-like features.
• Most chloroplast proteins are encoded by nuclear genes and imported from the cytosol.
• Chloroplast genes can be transcribed as polycistronic units and may undergo RNA processing, editing, and intron removal.

The chloroplast genome is therefore only a partial genetic system, dependent on extensive cooperation with the nuclear genome.

Chloroplasts are believed to have evolved from an ancestral cyanobacterium that was engulfed by a eukaryotic cell.

Supporting evidence includes:

• Chloroplasts contain their own circular DNA.
• They possess bacterial-type ribosomes and bacterial-like gene expression systems.
• They divide by a process resembling binary fission.
• Their double envelope is consistent with an engulfed prokaryotic cell.
• Their photosynthetic machinery is related to that of cyanobacteria.
• Chloroplast genomes contain genes with clear evolutionary relationships to cyanobacterial genes.
• Chloroplast size and internal organization resemble those of some free-living bacteria.

During evolution, many genes were transferred from the ancestral chloroplast to the host nucleus. Consequently, present-day chloroplasts require nuclear-encoded proteins for most of their activities.

Most chloroplast proteins are synthesized on free ribosomes in the cytosol and imported after translation.

• A precursor protein usually contains an N-terminal chloroplast transit peptide.
• Transit peptides are rich in hydroxylated and small uncharged residues and generally lack acidic residues. Unlike mitochondrial presequences, they do not usually form a strongly amphipathic helix.
• Cytosolic chaperones maintain the precursor in an import-competent state.
• The precursor is recognized by receptors of the TOC complex in the chloroplast outer membrane.
• It passes through the TOC channel and is transferred to the TIC complex in the inner membrane.
• ATP-dependent chaperones and stromal factors help pull the protein into the stroma.
• The transit peptide is cleaved by a stromal processing peptidase.
• The mature protein is then folded or directed to a further chloroplast compartment.

This pathway allows nuclear and chloroplast genomes to coordinate chloroplast biogenesis.

After entering the stroma, some proteins contain additional targeting signals that direct them to thylakoid compartments.

• Sec pathway: Transports largely unfolded proteins across the thylakoid membrane. It commonly uses a lumenal signal peptide and is powered by ATP.
• Tat pathway: Transports folded proteins across the thylakoid membrane. Its substrates contain a twin-arginine motif and passage is driven mainly by the proton gradient across the thylakoid membrane.
• SRP pathway: Directs certain chlorophyll-binding and photosynthetic membrane proteins to the thylakoid membrane. It involves a signal recognition particle and insertion machinery.
• Spontaneous insertion: Some highly hydrophobic proteins can insert into the thylakoid membrane without a large translocation complex.

Proteins destined for the thylakoid lumen are first imported into the stroma and then transported across the thylakoid membrane.

Both signals direct nuclear-encoded proteins to endosymbiotic organelles, but they differ in structure and recognition.

Both signals are generally cleavable and help maintain precursor proteins in an import-competent state.

Mitochondria and chloroplasts share several features because both originated through endosymbiosis, but their import systems have distinct characteristics.

• Both import most of their proteins from the cytosol.
• Both use N-terminal targeting signals that are recognized by receptors in the outer membrane.
• Mitochondria use the TOM complex followed by TIM complexes, whereas chloroplasts use TOC followed by TIC complexes.
• Mitochondrial proteins may be sorted to the matrix, inner membrane, outer membrane, or intermembrane space.
• Chloroplast proteins may be sorted to the stroma, thylakoid membrane, or thylakoid lumen.
• Mitochondrial inner-membrane import depends strongly on the electrical membrane potential.
• Chloroplast thylakoid sorting can use Sec, Tat, or SRP pathways.
• Both organelles contain their own genomes but rely mainly on nuclear genes for their proteomes.

Thus, the systems are evolutionarily related in principle but specialized for different organelle structures.

In both organelles, electron transport creates a proton electrochemical gradient across an energy-transducing membrane.

• In mitochondria, the respiratory chain pumps protons from the matrix into the intermembrane space.
• In chloroplasts, the light reactions pump protons from the stroma into the thylakoid lumen.
• This produces a proton-motive force consisting of a concentration gradient and an electrical gradient.
• Protons flow back through the membrane via the portion of ATP synthase.
• Proton movement causes rotation and conformational changes in the catalytic portion.
• ATP synthase uses these changes to convert ADP and inorganic phosphate into ATP:

The direction of proton flow and the compartment receiving protons differ, but the underlying chemiosmotic principle is similar in both organelles.

Disruption of the mitochondrial membrane potential would affect both protein import and ATP synthesis.

• The potential normally helps drive positively charged targeting sequences through the TIM23 complex.
• Loss of the potential would inhibit or greatly reduce import of many matrix and inner-membrane precursor proteins.
• Newly synthesized mitochondrial proteins could accumulate in the cytosol or at the mitochondrial surface.
• Assembly and maintenance of respiratory chain complexes would be impaired.
• Electron transport and oxidative phosphorylation would decrease.
• ATP production would fall, forcing the cell to rely more heavily on glycolysis.
• Ion and calcium homeostasis could be disturbed.
• Severe loss of potential may promote release of cytochrome and activation of apoptosis.

Therefore, the membrane potential is both an energy intermediate and an essential component of mitochondrial protein targeting.