BL 424 Chapter 11: Bioenergetics and Metabolism: Mitochondria, Chloroplasts, Peroxisomes In addition to involvement in protein sorting and transport, organelles are specialized compartments for metabolism: Mitochondria and chloroplasts are devoted to production of ATP. Proteins for organelles are mostly synthesized on free ribosomes in the cytosol and then directly transported; mitochondria and chloroplasts also have their own organelle transcription and protein synthesis. Student Learning outcomes: Proteins are the active players in most cell processes. 1*. Explain concisely the similarities and differences in structure and function of these organelles. 2*. Explain the process of transport of proteins to mitochondria and chloroplasts; to appreciate the signals on proteins that identify their destinations.. 3*. Describe the diverse protein complexes that read the protein’s signals and assist the transport: TOM, TIM, TOC, TIC complexes, chaperones, peptidases. 4*. Explain the essential metabolic functions of mitochondria and chloroplasts, (of oxidative phosphorylation and photosynthesis, respectively); note the importance of membrane compartments for formation of proton gradients and for function of the ATP synthase. 5. Describe the mitochondrial and chloroplast genomes, and that these genes are transcribed and translated within the organelle. 6. Recall that prokaryotes complete oxidative phosphorylation and photosynthesis by using their plasma membrane to generate the proton gradient. Important Figures: 2*, 4*, 5*, 6, 7*, 10*, 12, 13, 14, 15*, 16*, 17*, 18, 22, 25*, 33 Important Tables: 1, 2 1*. Mitochondria structure and organization. Mitochondria are critical for metabolic energy – for generation of ATP from the breakdown of carbohydrates and fatty acids. Mitochondria have a double membrane structure: (Fig. 11.1) Matrix (interior) has enzymes of citric acid (TCA) cycle to oxidize Acetyl CoA to CO2; also has DNA (Fig. 11.2) Inner membrane has numerous proteins involved in electron transport and oxidative phosphoyrlation (ATP). Outer membrane is permeable to small molecules Porins (channels) freely admit molecules < 1 kD. Recall that pyruvate is imported from cytosol, And converted to Acetyl CoA in matrix.Mitochondria have DNA genomes: mostly circular, reflecting endosymbiotic origins. Humans 16-kb; yeast 80-kb; plants > 200 kb. Encode rRNAs, tRNAs and some proteins involved in oxidative phosphorylation (about 13-32). Ribosomes are in the matrix; Translation in mitochondria has some special codon usage. Mutations of mitochondrial genes cause human disease: Mitochondria inherited from mother; Usually mix of normal and mutant – affect tissues with large ATP requirement (eye, brain) LHON (Leber’s Hereditary Optic Neuropathy) * Most mitochondrial proteins are encoded by the nuclear genome: (Figs. 4-7). About 1000 different proteins are synthesized on free ribosomes (cytoplasm) and then imported. Matrix proteins must traverse two membranes: Positively-charged N-terminal presequences (20-35 aa) target proteins for import; Presequences are removed by peptidase Matrix processing peptidase. Tom complex (translocase of outer membrane) includes receptors and channel proteins to to transport across first membrane. Tim complex (translocase of inner membrane) carries proteins into the matrix (Fig. 11.4). Chaperones assist the import, (Fig. 11.5, rachet) ATP hydrolysis powers the movement. Also electrical potential of membrane helps import +++ presequence into matrix (intermembrane space is more + charged H+ gradient) Proteins destined for insertion in membranes: (Fig. 11.6) Some transport proteins use Hsp90 and an Internal sequence for import and insertion. Lot of oxidative phosphorylation proteins in membranes Other proteins can have presequences and internal sequences (Fig. 11.7,8) to end up different places Phospholipids are carried to mitochondria from the ER by phospholipids transfer proteins. Ex. Cardiolipin (4 fatty acids) in inner mitochondrial membrane2. Mechanism of oxidative phosphorylation. Most energy (ATP) from oxidative metabolism comes from the transfer of electrons from NADH and FADH2 to a series of electron carriers in four complexes in the inner mitochondrial membrane. The transfer of electrons leads to proton transfer across the membrane (establishing a proton gradient – electrochemical gradient (Fig. 11.10*, 11.12*). Chemiosmotic coupling hypothesis (Peter Mitchell): (Fig. 11.12) A fifth protein complex, ATP synthase, couples ATP synthesis to the return of protons to the mitochondrial mateix (Fig. 11.13). 1 NADH -> 3ATP; 1 FADH2 -> 2 ATP. The electrochemical, proton gradient also drives transport of ATP, ADP and other metabolites into and out of the Mitrochondrial matrix (Fig. 11.14). (Outer membrane is permeable to small molecules). 3. Chloroplasts and other plastids Chloroplasts are larger organelles (5-10 um; Fig. 11.15), and have 3 membranes: the double membrane defines the organelle; the stroma is equivalent to the mitochondrial matrix. The internal thylakoid membrane is site of electron transport and chemiosmotic ATP synthesis; enzymes are located on outer (stromal) surface. **Fig. 11.16 compares chemiosmotic generation of ATP for mitochondrial and chloroplast compartments. [Note: prokaryotes use their plasma membrane for chemiosmotic ATP synthesis for photosynthesis or oxidative phosphorylation.] Chloroplast genomes are 120-160 kb, and contain about 150 genes; several plant chloroplast genomes have been sequenced. Encode 4 rRNAs, 30 tRNAs and 21 ribosomal proteins, plus ~30 proteins for photosynthesis (Table 2). Some proteins are synthesized in chloroplast.Import of proteins into chloroplast. About 95% of chloroplast proteins are encoded by nuclear genes; synthesized on free ribosomes, and imported; they must cross 2 or 3 membranes. The target sequence is an N-terminal (30-100 aa) Transit peptide which is bound by guidance complex and directed to the Toc complex (Translocation outer membrane chloroplast (Fig. 11.17). Import requires Hsp70 chaperones, ATP, GTP hydrolysis. The transit peptide is not positively charged, but the intermembrane space (and stroma) do not differ in charge. Transit across the inner membrane uses Tic complex of receptors and channels; ATP is required; the Hsp100 chaperone