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2.26.2010 2.26.2010 Document Transcript

  • 2.26.2010<br />
    • Peroxisomes – Slide 14
    • Oxidation of long-chain fatty-acids produces H2O2
    • Decompose/Detoxify H2O2
    • Catalse generates H20 and O2 from H202
    • Can utilize organic compounds as a source of H to make water and then leave that dehydrogenated compound as a left-over.
    • Free ribsomes make proteins for ribosomes that are translated into vesicles and are now peroxisomes
    • Growth = molecules transported into vesicle
    • Can undergo fission
    • Characterized as an isolated organelle
    • Double material to undergo fission
    • Can form de novo (from the beginning) from the ER.
    • Budding of ER and in are the proteins for peroxisomal functions.
    • Mitochondria
    • Outer membrane brings components from cytosol through inter membrane space
    • Review citric acid cycle
    • Beware of process that produce NADH and FADH2
    • Pyruvate acetyl coa
    • Fatty acids to acetyl coa through beta oxidation
    • Were likely incorporated in eukaryotic cells through phagocytic process
    • Can replicate itself in cell
    • Autophagy after 10 days
    • F1 is ATP generating site
    • There are 3 beta subunits and 3 alpha subunits in ATP synthase
    ----------------------------------------------------------------------------------------------------------------------------------<br />----Protein Trafficking----<br />
    • Slide 2
    • All have signal sequence that tell where unit should end up.
    • Slide 3
    • Signal sequences are strands of amino acids found in the unfolded proteins at Amino or Carboxy terminus. Or in middle of protein
    • Recognized in an unfolded state
    • Signal patches are signal sequences that function once when they are brought together and the protein has begun to fold
    • Near amino terminus,
    • Hydrophobic amino acids that have some basic positively charged amino acids, followed by hydrophobic amino acids and more basic positively charged amino acids.
    • Signal sequence for target
    • Lys-Asp-Glu- provide signal to transport protein into the lumen of the ER
    • Import into nucleus signal are found in the middleish
    • Ser-lys-leu is speciic for targeting peroxisomes. (SKL)
    • KDEL sequence = ER retention signal at carboxy terminus
    • Slide 4
    • Mechanisms to bring things back to the ER
    • Some proteins will be constitutively secreted and others are regulatory proteins.
    • Slide 5
    • Anterograde transport from rER to and through golgi occurs by two methods
    • Vesicular tranport
    • Only vesicles will bring them back through retro. transport
    • Cisternal Maturation model
    • Maturation of tubular clusters that bud off ER.
    • Maturation of whole cisternae that allows for a transition state
    • Tubular cluster matures into the cis golgi, medial, trans golgi
    • Anterograde
    • Retrograde
    • Budding of small vesicles off of trans golgi network, through golgi, and into the ER
    • Slide 6
    • Pulse-chase experiments
    • Radio-labeled amino acids to label newly generated proteins w/in the lumen of the ER for about 30 minutes
    • Pulse:
    • Specific period when radio-labeled amino acids were ‘bathed’ onto cells
    • Used pancreatic endocrine cells constantly secrete hormones.
    • Chase
    • Unlabeled amino acids added
    • For a different time period, let the cell rest then fix and analyze where the radio signal is.
    • Slide 11 – what proteins are involved in each step?
    • Mutation experiments for KO proteins
    • Yeast mutagenesis experiments.
    • Mutant yeast population then try to figure out why they are mutants, characterize the mutation, group them, figure out why they have that mutation
    • Normal secretion from yeast is a conserved process so you can irradiate yeast
    • Invertase – developed antibodies to the protein and look where invertase is found, when expressed, and what is its role?
    • ability to secrete = invertase
    • Slide 11
    • We ca change signal sequences for different proteins.
    • Cytosolic sequence and KDEL sequence maintained in the ER.
    • Insert ER sequence for cytosolic proteins
    • Slide 13
    • SRP recognizes ER signal sequence near N terminus.
    • Sequence reognition peptide that recognizes ER SS near N terminus
    • Trans-locon is made of SRP receptor, pore protein, ribosome receptor protein, and a signal peptidase
    • Pore allows protein to enter ER as being translated
    • Rib. Receptor binds to large subunit of ribosomal protein to be held during translation
    • Signal peptidase cuts off signal sequence from protein as being generated.
    • GTP is hydrolyzied in this process for energy
    • Translation wont take place until GTP is hydrolyzed
    • SRP and SRP receptor when hydrolyzed, SRP signal sequence is released and you can begin to translate protein into lumen of the ER
    • From there, sequences will determine where protein goes, secreted, lysosomes, ER etc.