Meeting 10 & 11

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Meeting 10 & 11
2012-04-14 13:39:51

Endocytosis & Protein Targeting
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  1. enocytosis/phagocytosis
    • when macrophage takes up bacteria/prey and takes it to the lysosome
    • -material targeted for degredation are targeted to the lysosome
    • -endocytosis: can also help cell acquire fat
  2. which coat proteins interact with transmembrane receptor?
    AP complex: interact with receptor, trigger assembly, and trigger endozytosis
  3. apolipoprotein B
    • -important for endocytosis...
    • ....
  4. receptor-mediated endocytosis
    process by which a specific receptor on the cell surface binds tightly to an extracellular macro-molecular ligand that it recognizes; the plasma-membrane region containing the receptor-ligand bound complex then buds inward and pinches off, becoming a transport vesicle
  5. lipoproteins
    particle that has a shell composed of proteins (apolipoproteins) and a cholesterol-containing phsopholipid monolayer

    low-density lipoprotiein (LDL): transports cholesterol via blood; composed of shell which is phospholipid monolayer + apoB-100 protein & inside is made up of cholesterol esters
  6. LDL binds and releases from receptor because of:
    changes in pH
  7. Endocytic pathway for internatlizing LDL
    1) LDL receptors bind to apoB protein in the outer layer of
  8. Fe2+ Transport: Transferrin ---> Apotransferrin
  9. vesicles know where to go becuase of:
    • Rab proteins; ex. Rab5 is a marker for early endosomes
    • Rab4: fast recycling loop
    • Rab11: essental for recylcing from late endosome
    • Rab7: targets degredation?

    -Rab's allow fusion of vesicle to a specific target membrane that has specific receptors for Rab
  10. ESCRT Complex + other Proteins
    -Early Sorting Complex Required for Transport; required for the recognition and sorting of ubiquitin-modified cargo proteins into the internal vesicles; ubiquitin tags are recognized by the ESCRT complexes, which bind sequentially and work in concert to pass along the cargo proteins from one complex to the next

    -Vps4: an ATPase that dissassembles the ESCRT complexes after the vesicle's been pinched off and returns to them to floating around in the cytoplasm

    • -proteins that are usually tagged with ubiquitin & recognized by ESCRT: Hrs, which helps load NON-ubiquitinated proteins into cargo buds
    • -the HIV protein GAG can immitate its action when tagged with ubiquitin, thereby promoting the transfer of HIV DNA throughout cells using vesicle transport
  11. Hrs
    carries ubiquitin; helps ubiquitinate cargo protein that will go inside; also, protein that recruits ESCRT, which contains Tsg101 that recognizes ubiquitin on Hrs
  12. autophagy
    catabolic process involving the degradation of a cell's own components through the lysosomal machinery; mechanism by which a starving cell reallocates nutrients from unnecessary to more-essential processes
  13. Nuclear Localization Sequences (NLSs)
    signal in proteins to be sent to the nucleus; such proteins are are fully formed in the cytosol, & specific locations in the nuclear envelope (nuclear pores) are how they're transported inside

    -usually Lys enriched
  14. Nuclear Pores
    • -very large
    • -40-80x bigger than the ribsome
    • -there's radial symmetry; repetition of various units forming the 'crown' (view from cytoplasm)
    • -view from inside nucleus is different; looks more like a basket

    -structure is polarized; two different sides look very different
  15. pyruvate kinase
    • should be naturally found in the cytoplasm; important for breakdown of pyruvate
    • -if you attach an NLS to pyruvate kinase (something usually found in cytoplasm) and stain for it it'll be found in the NUCLEUS
  16. digitonin
    • treating cells with this permeabilizes the plasma membrane; just makes the cell leak; many of the cytoplasmic components are gone
    • -if you add lysate and look at nuclear protein you can see it (if you don't add it, nuclear

    -tells us: there are components in the CYTOSOL required for nuclear transport
  17. importins
    • type of protein that moves other proteins into the nucleus by binding to the NLS (nuclear localization signal); has 2 subunits, α & β
    • -β can bind & transport cargo by themselves or can form heterodimers w/ α
    • -in the heterodimer, β interactis with the pore complex & α acts as anadaptor protein to bind NLS on the cargo
  18. GAP is cytosolic:
    GEF is on the nucleic side of things

    GAP (GTPase-Activating Proteins): regulatory proteins who bind to activated G-proteins and stimulate GTPase activity = results in a stop of a signaling event

    GEF (Guanine nucleotide exchange factor): activate GTPase by stimulating release of GDP to allow binding of GTP
  19. one key difference:
    • -proteins are fully synthesized before/WHEN they're
    • -mitochondria/chloroplasts are not synthesized de novo: they replicate
    • -this has reprecussions for protein targeting; proteins targeted to either of these organelles are targeted to fully formed organelles
  20. mtDNA
    contains only a few genes; is a small chromosome that encodes a couple of proteins & machinery mitochondrion needs for transLATION (less so for transcription)

    -examples include ribosomal RNA, small tRNAs, couple proteins (Cytochrome oxidase 1 & 2: belong to the last complex & reoxidize cytochrome + reduces oxygen)
  21. trypsin is a protease
    proteins that are taken up by mitochondrion will be protected from trypsin degeneration if inside mito; simply left in make-shift cytoplasm, they'll be degraded

    • -no co-translational translocation
    • -mitochondria has to be energized from (1) ATP or (2) proton-motive force
  22. Mitochondrial Matrix Signal Sequence for Proteins Encoded in the Nucleus
    • •Not identical but they share common motifs
    • •need a matrix-targeting sequences
    • •found at N-terminus of protein
    • •are 20–50 amino acids in length (short)
    • •contain hydrophobic aas, basic a.a.s, hydroxylated a.a.s; tend to lack acidic amino acids
    • •usually take the shape of an amphipathic α-helix
  23. Steps for Getting Mitochondrial Proteins into the Mt:
    • 1) protein must have already been translated (are moved POST- translationally)
    • 2) proteins MUST be unfolded to enter the matrix (this is done by chaperones [Hsc70] in the cytosol); uses ATP b/c folding is spontaneous, so must counteract
    • 3) outer mt membrane has receptor: Tom 20/22 (translocon outer membrane) is a receptor; recognizes signal sequence
    • -attracts Tom40, a general import pore; has a beta-strand barrel structure (this is indicative of mt's bacterial origin)

    -unsure where energy to PUSH protein through translocon comes from...(b/c already translated)

    • 4) Tim (translocon on INNER mt membrane); comes in close contact with Tom ['contact site']; protein goes through both ~at the same time
    • 5) binding of Hsc70 to protein INSIDE matrix helps with import in matrix
    • 6) matrix protease cuts uptake-targeting sequence
  24. How Proteins are Targeted to Inner Membrane
    have signals in primary sequence, directs them to inner membrane via 3 paths

    (A) this type of protein has matrix-targeting sequence (cleaved by matrix protease) & hydrophobic STS (stop transfer sequence; which sticks when passing through Tim channel)

    (B) this type of protein has matrix-targeting sequence (cleaved by matrix protease) + sequences recognized by Oxa1 (inner-membrane protein)

    (C) this type of protein DOESN'T have a matrix-targeting sequence, but instead has multiple internal mt targeting sequences, which are (hydrophobic) and arranged in the inner membrane by Tim complexes
  25. How Proteins are Targeted to Intermembrane Space
    (A) this type of protein has matrix-targeting sequence that's 1st cleaved by matrix protease & then an intermembrane-space-targeting sequence is cleaved 2nd by proteases in intermembrane space

    (B) this type of protein only has a targeting sequence for general import port (Tom40)/intermembrane space, so it stays there
  26. How Proteins are Targeted to Outer Membrane
    this type of protein (ex. Tom40) has the simplest path; comes from the outside & only need to be put at that membrane; first interact with general import pore (Tom 40), then SAM [sorting & assembly machinery] complex; they have a short matrix-targeting signal (?) & a (hydrophobic) stop-transfer/outer-membrane localization sequence

    • -NEITHER signal sequence is cleaved
    • -mechanism/energy source/how they interact with SAM is unclear
  27. Chloroplast DNA
    • -is circular; proteins it encodes are made in stroma (inside inner membrane but outside thylakoid), but most are encoded by nucleus
    • -an example of protein that has some subunits encoded in chloroplast & some in nucleus = RUBISCO; has 8 large subunits made in chloroplast, & 8 small ones made in nucleus
  28. How Proteins are Targeted to Thylakoids
    1) SRP-dependent pathway (plastocyanin): similar to how proteins are targeted to mt, proteins have both a stromal-import & thylakoid-targeting sequence; 1st go through Toc (outer membrane) then Tic (inner membrane); once in stroma import sequence is cleaved, & SRP binds to thylk-targeting sequence to import through SecY protein's in lumen

    2) pH pathway (metal binding proteins): same as above except once protein's in stroma & import sequence has been cleaved, metal ions are added to unfolded protein, thereby folding them; need (a) 2 argenine residues (RR) & (b) pH gradient for subsequent transport of protein into inner lumen.....argenines are cleaved once in lumen
  29. Plastocyanin
    copper-containing protein involved in electron-transfer (now you know it's found in the lumen of the thylakoid)
  30. peroxisome
    • •single membraned
    • •have no DNA
    • •Compartment of Oxidation; whenever there’s oxidative
    • stress/destroy things via oxidation
    • •2 O2 + 2 S → 2 H2O2 + 2 S(ox)
    • •Contains catalase: 2 H2O2 → 2 H2O + O2
    • •has peroxisomal signal sequences
    • –PTS1: located at the C-terminus Ser-Lys-Leu (SKL)
    • –PTS2: N-terminus ???

    -where oxidation occurs, b/c they create hydrogen peroxide

    catabolize fatty acid chains, branched chain fatty acids (+ D-amino acids, polyamines, biosynthesis of plasmalogens, etherphospholipids) & necessary for normal brain and lung function
  31. How Proteins are Targeted to Peroxisomes
    • 1) proteins are fully folded in cytosol; PTS1 is found at the C-terminus (like KDEL, different from practically everything else)
    • 2) Pex5, a soluble receptor floating in cytosol recognizes PTS1 (again, different mechanism, NOT a transmembrane receptor)
    • 3) Pex5 interacts with a receptor (Pex 14) on peroxisome membrane; subsequently transported in through another membrane protein (Pex10/12/2)
    • 4) in the matrix Pex5 dissociates and is transported back to the cytosol via same Pex10/12/2 complex