Gene expression



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Gene expression

  • Gene expression

    • Transcription
    • Translation
  • Functional proteins: post-translational modifications

    • Phosphorylation
    • Glycosylation
  • Sorting: targeting to appropriate organelles

  • Modulation by extracellular signals

  • Degradation



Sorting and Secretory pathways

  • diverged in trans-Golgi

    • Constitutive pathway
      • many soluble proteins
    • Regulated pathway
      • stored in secretory vesicles
      • active transport from cytosol to vesicles
      • complexed with macromolecules (e.g. proteglycans)


Regulated secretory pathway

  • In response to extracellular stimuli: hormone, transmitters, digestive enzymes

  • stored in secretory vesicles

  • active transport from cytosol to vesicles

  • complexed with macromolecules (e.g. proteglycans) to reach high concentration





Three pathways in Golgi

  • to lysosome

    • with mannose-6-phosphate, via late endosome to lysosome
  • to secretory pathways





“Roadmap” for traffic

  • Transport

    • Gated transport: cytosol and nucleus via nuclear pore complexes
    • Membrane transport: via membrane-bound translocators; unfolded
    • Vesicle transport: vesicles
  • Sorting signals



Membrane transport

  • Membrane-bound translocators

  • Unfolding of protein to be transported

  • Passing through a topologically distinct space: cytosol to ER; cytosol to mitochondria



Equivalent space for transport

  • Cycles of budding and fusion permits transport of molecules



Sorting signals

  • Signal sequences

    • 15-60 aa
    • Removed by signal peptidase when reaching the target
  • Signal patch



Sorting signals



Signal sequences

  • Function: specify the direction for destination

  • for initial transfer to the ER: with a signal sequence at N-terminus; consisting of 5-10 hydrophobic aa

    • Go forward Golgi: most proteins
    • Return to ER (ER residents): with a specific sequence of 4 aa at C-terminus
    • Go to mitochondria: positively charged amino acids alternate with hydrophobic ones
    • Go to peroxisome: with a signal peptide of 3 characteristic at C terminus


Signal sequences



Experiments to demonstrate “zip code”

  • Swap the signal sequence:

  • e.g.: adding the ER signal sequences to a cytosolic protein results in the retention of this protein in ER

  • Hyprophobicilty: maybe a determing factor of signal sequences



Günter Blobel



Effect of misdirected protein





Overview of protein sorting



Protein sorting



Glycosylation in ER



Lysosomal protein: phosphorylation of mannose



Sorting in Golgi

  • Export

    • Lysosome
    • Secretion
      • Constitute
      • Regulated
  • Retain in Golgi

    • transmembrane


Transport to plasma membrane

  • TGN

  • Apical domain

  • Basolateral domain



Endocytosis

  • Phagocytosis (cell eating)

  • Pinocytosis (cell drinking)

    • Up-take of fluids or macromolecules in small vesicles
    • Receptor-mediated endocytosis
    • Common among eukaryotic cells


Phagocytosis

  • Pseudopodia

  • Phagosome

  • Phagolysosome



Receptor-mediated endocytosis



LDL: low density lipoprotein

  • Core

    • ~1500 Cholesterol ester
  • Coat

    • ~500 cholesterol
    • ~ 800 phospholipid
    • 1 Apoprotein B100


LDL receptor

  • MS Brown, JL Goldstein

  • Familial hypercholesterolemia

  • LDL-binding domain

  • Internalization signal



LDL receptor

  • Internalization signal: Tyr



Clathrin-coated pits



Lysosomal proteins in clathrin-coated pits



Sorting in early endosome

  • Acidic ph by H+ pump for dissociation of proteins



Recycling of synaptic vesicles



Protein sorting by transcytosis



Lysosomal system

  • For membrane-bound proteins and proteins taken by endocytosis

  • Multiple acid proteases (cathepsins) and other hydrolases

  • Studied with weak bases to inhibit lysosomal acidification or lysosomal inhibitors (E64)




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