Quiz 3 Material Cytoskeleton MFs Ac1n Polymeriza1on8Based Mo1lity MFs Ac1n Motor Protein8Based Mo1lity MTs Ac1n8Myosin Systems MTs MT Motors Mitosis Mitosis Cytokinesis Cilia IFs Mitochondria Structure Genome Origins Mitochondria Protein Import Mitochondria Structure Genome Origins Mitochondria energy conserva1on organelle converts energy from food into major energy currency ATP 2 membranes 2 compartments outer membrane derived from phagocy1c membrane intermembrane space inner membrane includes cristae derived from PM of 1st mitochondria matrix space enclosed by cristae Outer Membrane contains porin channel8forming protein pore formed by Ibarrels permeable to molecules 5 kDa Intermembrane Space b tw inner outer membranes chemically iden1cal to cytoplasm Inner Membrane creates PMF for ATP genera1on folded into cristae increases surface area has large quan11es of cardiolipin a di8phospha1dylglycerol PG decreases permeability of membrane to ions membrane 3 general func1ons 1 ETC 2 ATP synthase located here 3 transporters carriers for metabolites e g ATP phosphate ADP pyruvate contributes to establishment of membrane poten al across inner Matrix Func ons oxida1ve decarboxyla1on of pyruvate to acetyl CoA citric acid cycle enzymes located here 8oxida1on of FAs to acetyl CoA Mitochondrial Genome slide 1 of 2 small circular mul1ple copies human mitochondrial DNA mtDNA encodes ORI origin for mtDNA proteins rRNA tRNAs rRNA 12S 16S euks have 18S rRNA tRNA have 22 but need 30 for protein synthesis tRNA import known in yeast protozoans Mitochondrial Genome slide 2 of 2 proteins encoded by human mitochondrial genome NADH dehydrogenase 7 subunits cytochrome b 1 subunit cytochrome oxidase 3 subunits ATP synthase 2 subunits some subunits missing RNA Synthesis in Mitochondria mitochondrial RNA polymerase gene encoded in nucleus single subunit enzyme similar to phage RNA pols shows that it s nothing like a euk RNA pol bacterial RNA pol has mul1ple subunits mtDNA of Reclinomonas americana protozoan single8celled euk ancient mtDNA resembles proteobacterial ancestor 97 genes SecY Sec61 homolog use to help get proteins out into media mul18subunit RNA pol more like that of a modern bacterium NOT like a phage Mitochondrial Fission Fusion fusion mitofusins 1 2 promotes heteroplasmy defense against aging ssion dynamin8related protein DRP mitophagy apoptosis transport inheritance Interdependence of Mitochondria Nucleus Interdependence of Mitochondrion and Nucleus ATP produced in mitochondrion used in nucleus most mitochondrial proteins encoded in nucleus synthesized in cytoplasm imported into mitochondrion ATP Produced in mitochondrion Used in nucleus Most mitochondrial proteins Encoded in nucleus Synthesized in cytoplasm Imported into mitochondrion Lipid sources Sources of Macromolecules Lipids Sources of Macromolecules for Mitochondria for Mitochondria PHOSPHOLIPID EXCHANGE PROTEINS phospholipid exchange proteins 1 1 Lipids Obtained mainly from cytoplasm lipids obtained from PC and PS synthesized on ER cytoplasm and transferred to outer membrane of mitochondria transferred to outer 2 PS is DECARBOXYLATED TO PE membrane of mitochondria 3 CARDIOLIPIN is synthesized in 2 PS decarboxylated to PE 3 cardiolipin synthesized in Cardiolipin constitutes 20 PC PS synthesized on ER mitochondrion mitochondrion of total lipid Cardiolipin Synthase cons1tutes 20 of total lipid Inner membrane enzyme cardiolipin synthase inner membrane enzyme Substrates CDP substrates diacylglycerol and CDP8diacylglycerol CDP PG CDP8PG 4 pyruvate made in CDP cytidine diphosphate PG phosphatidylglycerol cytoplasm imported into mitochondrion 4 PYRUVATE Made in cytoplasm and imported to mitochondrion Modifications of Lipids Pyruvate Transporter Iden ed proteins in mice liver mitochondria CONSERVED in yeast ies humans MPC1 MPC2 proteomics yeast knockout muta1ons MPC1 MPC2 Pyruvate uptake defec1ve in isolated mutant mitochondria Put MPC1 MPC2 genes into bacteria that don t normally use pyruvate Recons1tute put yeast MPC1 MPC2 genes into bacteria Pyruvate uptake observed in bacteria 1 Mutants grow slower than WT in synthe1c dextrose 2 3 4 5 families w lac1c acidosis 1 Map gene Maps to region close to MPC1 2 3 4 5 Find muta1ons R97W L79H Test O2 consump1on by broblasts in pa1ents Put genes w human muta1ons into yeast Yeast w R97W L79H don t grow well Mitochondria Protein Import general features most proteins encoded in nucleus translated on cytosolic ribosomes imported post8transla1onally into mitochondria 2 kinds of mitochondrial signal pep1des matrix proteins MTS Matrix Targe1ng Signal inner membrane proteins internal hydrophobic signal pep1des 2 membranes 5 protein translocases Method Used to Study Protein Import in Mitochondria Method Used to Study Import of Proteins into Mitochondria Targeting Signals on Cargo Cytosolic Factors Receptors for Cargo Import routes and translocases Energy Requirements targe1ng signals on cargo cytosolic factors receptors for cargo import routes translocases energy requirements 1 Targe1ng sequence clipped upon entry into mitochondria 2 Any protein leR outside 3 4 mitochondria is chopped up by proteases 2 bands mitochondrial form mature signal sequence cleaved pre8cursor form s1ll has signal sequence CCCP prevents entry into mitochondria so really only see 1 pre8cursor form Comparing Mitochondria to ER co8transla1on import into ER vs post8transla1onal import into mitochondria 2 receptors in mitochondria 1 each for inner outer membranes vs only 1 membrane in ER
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