Structure and Function of Mitochondria, Chloroplasts, and Peroxisomes (F24) PDF
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The University of Texas at Rio Grande Valley
2024
Tobias Weinrich, PhD
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These lecture notes describe the structure, function, and protein import processes of mitochondria, chloroplasts, and peroxisomes. They cover topics like oxidative metabolism, ATP synthesis, and biogenesis. The content is suitable for an undergraduate-level biology course at the University of Texas Rio Grande Valley.
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1 STRUCTURE AND FUNCTION OF MITOCHONDRIA, CHLOROPLASTS AND PEROXISOMES 10/26/2024 Tobias Weinrich, PhD School of Integrative Biological and Chemical Sciences 2 Student Learning Outcomes ▪ Reading guid...
1 STRUCTURE AND FUNCTION OF MITOCHONDRIA, CHLOROPLASTS AND PEROXISOMES 10/26/2024 Tobias Weinrich, PhD School of Integrative Biological and Chemical Sciences 2 Student Learning Outcomes ▪ Reading guide learning outcomes ▪ Compare the structural and functional organization of chloroplasts with mitochondria. ▪ Contrast chloroplast and mitochondrial genomes. ▪ Summarize the mechanisms of protein import into mitochondria and chloroplasts. ▪ Explain the role of the proton gradient in transport across the mitochondrial membrane. ▪ Explain mitochondrial oxidative metabolism - including pyruvate oxidation, Krebs cycle, electron transport chain, and ATP synthesis. ▪ Explain the role of chlorophyll in harvesting energy from sunlight. ▪ Describe the mechanisms used for generation of ATP and NADPH in chloroplasts. ▪ Summarize the reactions of the Calvin cycle. ▪ Summarize the structure and function of peroxisomes. ▪ Describe the pathways responsible for peroxisomes biogenesis. 3 Lecture Structure 1. Mitochondria and Chloroplasts – shared properties 2. Mitochondria A. Structure and composition B. Lipid and Protein import C. Function 3. Chloroplast A. Structure and composition B. Protein import C. Function 4. Peroxisomes A. Structure and function B. Protein import 4 1. Mitochondria and Chloroplasts – shared properties ▪ Double membrane organelles ▪ Endosymbiotic origin ▪ Semi-autonomous organelles ▪ Own DNA and ribosomes ▪ Independent replication of nucleus ▪ Energy conversion ▪ ATP synthesis – ATP synthase 5 2.A. Mitochondria A. Structure and Composition ▪ Present in all eukaryotes ▪ Small organelles (0.5–3 μm long) ▪ Numbers – vary (cellular requirements) ▪ Dynamic in shape 6 2.A. Mitochondria – Compartments A. Structure and composition - Compartments Two membranes and two inner compartments: ▪ Outer membrane ▪ 6% prot ▪ Intermembrane space ▪ 6% prot ▪ Inner membrane ▪ Cristae ▪ 21% prot ▪ Matrix ▪ 67% prot 7 2.A. Mitochondria – Biogenesis A. Structure and composition - Biogenesis ▪ Preexisting mitochondria ▪ Independent of cellular division Dynamic Tubular network ▪ Fusion ▪ Fission 8 2.A. Mitochondria – Genome A. Structure and composition – Mitochondrial Genome ▪ Circular - multiple copies per organelle ▪ Genome size – variable ▪ Humans – 16 kb ▪ Yeast – 80 kb ▪ Plants – 200 kb ▪ Exceptions to genetic code ▪ Genes: ▪ 13 proteins ▪ 22 tRNA ▪ rRNA ▪ Most proteins are encoded in nuclear genes imported into mitochondria 9 2.B. Mitochondria – Lipid and Protein Import B. Lipid and protein import LIPID ▪ Cardiolipin – synthesized by mitochondria ▪ Other lipids – phospholipid exchange protein ▪ Import from ER PROTEIN ▪ 13 proteins synthesized by mitochondria ▪ 99% of proteins encoded by nuclear genes ▪ Import from cytosol as precursor proteins: ▪ OMM (outer mitochondrial membrane) ▪ Intermembrane space ▪ IMM (inner mitochondrial membrane) ▪ Matrix 10 2.B. Mitochondria – Protein Import PROTEIN import from cytosol 1. Matrix proteins ▪ Synthesized in cytosol ▪ Sorting sequence - presequence (N-ter, rich in basic amino acids) ▪ Translocated as precursor protein - unfolded polypeptide ▪ Cytosolic receptor: Tom import receptor ▪ Tom complex – Translocase of the outer mitochondrial membrane ▪ Cytosolic Hsp70 – ATP hydrolysis ▪ Tim complex – Translocase of the inner mitochondrial membrane ▪ Mitochondrial Hsp70 – ATP hydrolysis ▪ Proton motive-force is also required ▪ Sequence is cleaved by MPP (matrix processing peptidase) 11 2.B. Mitochondria – Protein Import PROTEIN import from cytosol 2. Outer membrane and intermembrane space ▪ Synthesized in cytosol as precursor (unfolded) – no presequence i. Translocation through Tom ii.Intermembrane space chaperons Protein folded – intermembrane space protein Protein unfolded – outer membrane protein ▪ Outer membrane - Porins – β-barrel ▪ Insertion into OMM by SAM complex OUTER MEMBRANE PROTEIN FOLDED INTERMEMBRANE SPACE PROTEIN 12 2.B. Mitochondria – Protein Import PROTEIN import from cytosol 3. Inner Mitochondrial Membrane ▪ Synthesized in cytosol as precursor (unfolded) ▪ Multiple pathways ▪ Hydrophobic region – inner mitochondrial membrane ▪ Single pass or multipass ▪ Some mitochondrial DNA proteins are inserted into IMM 13 2.C. Mitochondria – Function A. Mitochondrial Oxidative metabolism ▪ Pyruvate, fatty acids → acetyl-CoA ▪ Krebs cycle/Citric acid cycle (acetyl-CoA → NADH + CO2) ▪ Electron transport chain Oxidative ▪ ATP synthesis phosphorylation 14 2.C. Mitochondria – Function A. Mitochondrial Oxidative metabolism i. Electron transport chain (ETC) – Energy Derived from Oxidation Is Stored as an Electrochemical Gradient ▪ NADH and FADH2 donate their high energy electrons to the ETC ▪ Complex I (NADH), II (FADH2), III, and IV ▪ Inner mitochondrial membrane ▪ As electrons shuttle from complex to complex, protons are pumped to the intermembrane space ▪ O2 is the final electron acceptor → H2O ▪ Electron transport chain 15 2.C. Mitochondria – Function A. Mitochondrial Oxidative metabolism i. Electron transport chain (ETC) – Energy Derived from Oxidation Is Stored as an Electrochemical Gradient ATP synthesis Active transport (through IMM) Heat production Protein translocation Electrochemical Gradient 16 2.C. Mitochondria – Function A. Mitochondrial Oxidative metabolism ii. ATP synthesis – chemiosmotic coupling ATP Synthase – two major parts connected by rod/stalk ▪ Rotor in inner mitochondrial membrane (F0) ▪ Knob in matrix stabilized by stator anchored in membrane (F1) ▪ F-type ATPase in reverse 4 H+ 1 ATP 1 NADH (H+) 2.5 ATP 1 FADH2 (2 H+) 1.5 ATP 1 Glucose 30 ATP 10 NADH 2 FADH2 2 GTP 17 2.C. Mitochondria – Function A. Oxidative metabolism B. Transport across mitochondrial membranes Dependent on energy status AND proton-motive force ▪ ATP / ADP antiport ▪ Pyruvate – H+ symport ▪ Phosphate – H+ symport 18 2.C. Mitochondria – Function A. Oxidative metabolism B. Transport across mitochondrial membranes C. Intermediate metabolism ▪ Provision of metabolites (precursors) for other metabolic pathways 19 2.C. Mitochondria – Function A. Oxidative metabolism B. Transport across mitochondrial membranes C. Intermediate metabolism D. Thermogenesis (heat production) ▪ Thermogenin – uncouples electron transport chain from ATP synthesis ▪ H+ channel ▪ Energy released from H+ gradient is converted into heat ▪ Brown fat 20 3. Chloroplasts A. Structure and Composition ▪ Plastids – heterogenous group of organelles ▪ Proplastid – immature plastid ▪ Present in algae and plants ▪ Large organelles (5–10 μm long) ▪ Numbers – vary 21 3.A. Chloroplasts - Compartments A. Structure and Composition ▪ Third membrane system: thylakoid membrane THREE MEMBRANES ▪ Thylakoids: network of flattened ▪ Chloroplast envelope – double discs membrane ▪ Grana: stack of thylakoid ▪ Outer: permeable (porins) ▪ Light absorption (chlorophyll) ▪ Inner: semipermeable/selective barrier ▪ Electron transport chain ▪ No folds ▪ ATP synthesis ▪ No ATP transporter 22 3.A. Chloroplasts – Compartments & Genome THREE INNER COMPARTMENTS ▪ Intermembrane space ▪ Stroma: ▪ Genome ▪ Carbon fixation process ▪ Thylakoid lumen/space – pH5 Genome: ▪ Circular DNA – multiple copies ▪ 100 – 200 kb ▪ 4 rRNA ▪ 30 tRNA ▪ 150 genes (~5% chloroplast proteins) 23 3.A. Chloroplasts – Biogenesis A. Structure and Composition - Biogenesis ▪ From proplastids in meristems ▪ During development ▪ From pre-existing chloroplasts ▪ Fission ▪ Fusion (not as common as in mitochondria) 24 3.B. Chloroplasts – Protein Import B. Protein Import ▪ 150 proteins synthesized by mitochondria ▪ 95% of proteins encoded by nuclear genes ▪ Import from cytosol and sorted to: CYTOSOL ▪ Outer envelope (precursor protein) ▪ Intermembrane space ▪ Inner envelope ▪ Stroma ▪ Thylakoid membrane ▪ Thylakoid lumen 25 3.B. Chloroplasts – Protein Import B. Protein Import 1. Stromal import ▪ Synthesized in cytosol ▪ Translocated as precursor protein (unfolded polypeptide) ▪ Signal sequence: transit peptide (N-ter) ▪ Cytosolic receptor: Hsp70 ▪ Toc complex – Translocase of the outer chloroplast membrane ▪ Hsp70 – GTP hydrolysis ▪ Tic complex – Translocase of the inner chloroplast membrane ▪ Hsp93 – ATP hydrolysis ▪ Sequence is cleaved by SPP (stromal processing peptidase) 26 3.B. Chloroplasts – Protein Import B. Protein Sorting 2. Sorting from stroma ▪ Chloroplast and nuclear encoded proteins a) Import thylakoid : ▪ Signal sequence: thylakoid target sequence (N-ter) ▪ Folded: ▪ pH dependent pathway (Tat) ▪ Preproteins (unfolded): ▪ Sec Pathway and SRP Pathway ▪ Thylakoid protein peptidase (TPP) – cleaves thylakoid target sequence 27 3.C. Chloroplasts – Function Photosynthesis Conversion of light energy into chemical energy Two linked stages: Active transport ▪ “Light capture” – light-reactions Protein translocation ATP synthesis ▪ Light absorption ▪ Electron transport chain ATP ▪ ATP synthesis ▪ “CO2 fixation”-light independent reactions ▪ Reduction of CO2 LIGHT CAPTURE CO2 FIXATION 28 3.C. Chloroplasts – Function Photosynthesis a) Light reactions – “light capture” ▪ Location: Thylakoid membrane ▪ Function: Converts radiant energy → chemical energy ▪ Pigment: Chlorophyll a ▪ Reactants: H2O + radiation energy ▪ Products: ATP and NADPH + O2 b) Light-independent reactions – “CO2 fixation” ▪ Location: stroma (and cytosol) ▪ Function: CO2 reduction → sugars ▪ Enzyme: Rubisco ▪ Reactants: ATP, NADPH, CO2 ▪ Products: 3-phosphoglycerate → other organic molecules 29 3.C. Chloroplasts – Photosynthesis a) Light Reactions a.1.) Light Absorption ▪ Pigment (chlorophyll) absorbs energy in light → excited electron I. Transfer energy (resonance) II. Transfer electron (charge separation) III. Converting into heat (fluorescence) ▪ Photosystem: antenna complex + reaction center 30 3.C. Chloroplasts – Photosynthesis a.1.) Light Absorption - Photosystem ▪ Antenna complex ▪ Chlorophyll + other pigments ▪ Light absorption and transfer energy (resonance) ▪ Reaction center ▪ Two chlorophyll a molecules ▪ Donate: high energetic electron to acceptor in electron transport chain ▪ They must replenish with a low energetic electron ▪ PSI: plastocyanin ▪ PSII: H2O 31 3.C. Chloroplasts – Photosynthesis a.2.) Electron transport chain ▪ Photosystems ▪ Electron carriers – plastoquinone, plastocyanin, ferredoxin ▪ Flow of electrons: PSII – Q – cyt b6-f – pC – PSI – Fd – NADP+ → NADPH Linear ▪ Transfer of electrons → H+ gradient photosynthesis ▪ How are electrons replenished? Water photolysis at PSII 32 3.C. Chloroplasts – Photosynthesis a.2.) Electron transport chain ▪ Linear photosynthesis (Z scheme) → H+ gradient + NADPH ▪ Cyclical photosynthesis → H+ gradient only ▪ Ferredoxin transfers energy to Plastoquinone ▪ Electrons from PSI – Fd – Q – cyt b6-f 33 3.C. Chloroplasts – Photosynthesis a.3.) ATP synthesis ▪ ATP Synthase – thylakoid membrane ▪ Linear photosynthesis (Z scheme) → ATP + NADPH ▪ Cyclical photosynthesis → ATP only 34 3.C. Chloroplasts – Photosynthesis b) Light-independent reactions ▪ Reduction of CO2 → organic molecules ▪ Enzyme: ribulose 1,5-bisphosphate carboxylase/oxygenase (Rubisco) ▪ Uses ATP and NADPH produced during light phase ▪ Calvin cycle – three stages ▪ Carbon fixation ▪ Sugar Formation ▪ Regeneration of ribulose 1,5-bisphosphate ▪ Product: 3-phosphoglycareate/glyceraldehyde 3P 35 3.C. Chloroplasts – Photosynthesis ▪ Chloroplast and mitochondria cooperation ▪ Sugars produced by chloroplast: ▪ Storage as starchd ▪ Oxidized in mitochondria – ATP production ▪ ATP produced in photosynthesis cannot leave chloroplasts! 36 4.A Peroxisomes A. Structure and function: ▪ Small single membrane-bound (0.1–1 μm) ▪ No DNA – no ribosomes ▪ Rich in catalase Biogenesis: ▪ Fission (division) from pre-existing peroxisomes – all material is delivered from cytosol and ER ▪ De novo from ER precursor membranes + proteins (requires peroxins) 37 4.A Peroxisomes - Function A. Function: i. Oxidative metabolism (fatty acids, amino acids, methanol, toxins…) ▪ H2O2 production ▪ Catalase – breakdown of hydrogen peroxide ▪ EXAMPLE: Fatty acid β-oxidation (very long-chain fatty acids) ▪ H2O2 and Acetyl CoA destination → TCA ▪ No ATP production ii. Biosynthesis of lipids - plasmalogens (heart &brain) 38 4.B. Peroxisomes - Protein import B. Protein import Matrix: ▪ Synthesized in cytosol ▪ Translocated in folded conformation ▪ Signal sequence: PST1 (C-ter; majority) PST2 (N-ter; minority) ▪ Sequence is not cleaved ▪ Receptor: cytosolic receptor protein (peroxin) ▪ Translocation energy: ATP hydrolysis Membrane: a) Different signal sequence b) ER Pathway